XXIV Fungal Genetics Conference at Asilomar

 

Poster Session Abstracts

 Please cite abstracts as follows: Fungal Genet. Newsl. 54 (Suppl):Abstract#.


Cell Biology (1-75)
Population and Evolutionary Biology (76-130)
Genomics and Proteomics (131-234)
Industrial Biology and Biotechnology (235-254)
Biochemistry and Secondary Metabolism (255-290)
Gene Regulation (291-356)
Developmental Biology (358-411)
Animal and Plant Pathogens; Fungal-Host Interactions (412-565)
Model systems (566-568)
Other (571-601)

 

Plenary session abstracts


CELL BIOLOGY

 

1. Metarhizium anisopliae perilipin homolog MPL1 regulates lipid metabolism, appressorial turgor pressure and virulence. Chengshu Wang and Raymond J. St. Leger. Department of Entomology, University of Maryland, College Park, MD, 20742, USA. stleger@umd.edu


Cells store lipids in droplets. Studies addressing the control of lipid-based energy homeostasis of mammals identified proteins of the PAT-domain family, such as perilipin that surround the lipid droplets. Factors that mediate lipid storage in fungi are still unknown. Here we describe a gene (Mpl1) in the insect pathogen M. anisopliae that has structural similarities to mammalian perilipins. Consistent with a role in lipid storage, Mpl1 is predominantly expressed when M. anisopliae is engaged in accumulating lipids and ectopically expressed GFP-tagged MPL1 localized to lipid droplets. Mutant M. anisopliae lacking MPL1 have thinner hyphae, fewer lipid droplets, particularly in appressoria and a decrease in total lipids. Mpl1 therefore acts in a perilipin-like manner with an evolutionary conserved function in lipid metabolism. However, animal and fungal proteins have also been selected to cope with different tasks. Thus, turgor generation by Mpl1 appressoria is dramatically reduced indicating that lipid droplets are required for solute accumulation. This was linked with reduced ability to breach insect cuticle so that Mpl1 is a pathogenicity determinant. Blast searches of fungal genomes revealed that perilipin homologs are found only in pezizomycotinal ascomycetes and occur as single copy genes. Expression of Mpl1 in yeast cells, a fungus that lacks a perilipin-like gene, blocked their ability to mobilize lipids during starvation conditions.


2. Characterization of AtmA and PrpA reveals a correlation between ROS and apical dominance in Aspergillus nidulans. Camile P. Semighini1, Gustavo H. Goldman2 and Steven D. Harris1. 1Plant Pathology, University of Nebraska Lincoln; 2 Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil Presenter’s e-mail: csemighini2@unl.edu


The DNA damage response (DDR) is a protective mechanism that ensures the maintenance of genomic integrity. DDR is composed of many conserved proteins such as the Ataxia telangiectasia mutated (ATM) kinase and the Poly (ADP)- ribose polymerases (PARP). We are using the filamentous fungus Aspergillus nidulans as a model system and we believe that certain aspects of the DDR in this organism may reflect the unique organization of fungal hyphae. We have characterized the function of the A. nidulans ATM (AtmA) and PARP (PrpA) homologues. Besides their expected roles in DDR, those homologues also make an unexpected contribution to hyphal morphogenesis. In the absence of DNA damage, the deltaatmA strain is defective in polarized hyphal growth while the deltaprpA+/- mutant produces an increased number of germ tubes per spore and displays an increase in the formation of lateral branches. Staining of a wild type strain with Nitro Blue Tetrazolium (NBT) revealed that a gradient of Reactive Oxygen Species (ROS) localize to the hyphal tip. NBT staining of deltaatmA and deltaprpA+/- strains correlated their hyphal morphology defect with disruption of ROS’ specific localization. Our results suggest that ROS gradient may enforce apical dominance.


3. Roles of Aspergillus nidulans cdc42 and rac1 homologues in polarized growth. Aleksandra Virag and Steven Harris. Plant Science Initiative, N235 Beadle Center, University of Nebraska, Lincoln, NE 68588. E-mail: avirag2@unlnotes.unl.edu


In Saccharomyces cerevisiae the Rho GTPase Cdc42 is required for polarity establishment and maintenance. In contrast, Cdc42 homologues in filamentous Euascomycetes and Basidiomycetes do not appear to be important for polarity establishment, but rather for polarity maintenance and the establishment of secondary polarity axes-branches. The latter fungi also possess another Rho GTPase, Rac1, which is important for polarization but is absent from S. cerevisiae. Here we utilized the model Euascomycete Aspergillus nidulans to characterize the connection between Cdc42 and Rac1, and their presumed downstream components and effectors. Potential effectors include putative polarisome components (SpaA and SepA), as well as proteins required for microfilament and microtubule organization. Results suggest that Cdc42 and Rac1 have overlapping functions in polarity establishment. Preliminary genetic observations have identified, in addition to SpaA, other downstream effectors such as two distinct paxillin homologues. Based on our results we propose that Cdc42 and Rac1 cooperate in polarity-related processes.


4. Pheromone receptor Bar2 of the homobasidiomycete Schizophyllum commune. Susann Jezewski* & Erika Kothe. Friedrich-Schiller-University, Dept. of Microbiology, Neugasse 25, 07743 Jena, Germany; E-mail: Susann.Jezewski@uni-jena.de


The tetrapolar mating system of Schizophyllum commune allows easy discrimination of mating types and is associated with two genetic systems, which contain the A and B genes. Each B locus codes for one pheromone receptor, belonging to the family of G protein coupled seven transmembrane domain receptors, and several pheromones, small lipopeptide ligands. The interaction of a pheromone receptor and non-self ligands results in nuclear migration and clamp cell fusion, which are part of sexual development. There is no induction of B gene dependent sexual development by pheromones of self-specificity, of which several are encoded within each locus. The recognition of pheromones by multi-specific pheromone receptors and the distinction of self and non-self are necessary for a compatible mating process. The function of the pheromone/receptor system implies the localization of the receptor protein in the tip of growing hyphae or in clamp cells of S. commune. The visualization of the Balpha2 receptor protein (Bar2) should be possible by a fusion of the intracellular C-terminus of Bar2 with fluorescent proteins, like GFP or DsRed. However, the transient process of clamp cell formation and the low expression of mating type genes make the determination of receptor molecules complicate. By means of real time PCR, a quantification of pheromone receptor mRNA will be possible in different mutant backgrounds under special conditions.


5. Does the single Dothideomycete MAT1-1-1 protein harbor domains for activities performed by multiple MAT1-1 proteins from Sordariomycetes? Banu Metin1, Shun-Wen Lu2, B. Gillian Turgeon3. 1Department of Food Engineering, Middle East Technical University, 06531 Ankara, Turkey, e102950@metu.edu.tr. 2Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA, swl2@cornell.edu 3Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA, bgt1@cornell.edu


In filamentous ascomycetes, mating type loci exist as two alternate forms, MAT1-1 and MAT1-2, defining two mating types. While MAT1-2 contains a single gene with an HMG box domain in all ascomycetes examined so far, MAT1-1 shows differences. In Sordariomycetes, the MAT1-1 idiomorph contains three genes: MAT1-1-1 encoding a protein with an alpha 1 domain, MAT1-1-2 encoding a protein with an HPG domain, and MAT1-1-3 encoding a protein with an HMG box domain. In contrast, in Dothideomycetes, the MAT1-1 idiomorph harbors a single gene, which has been believed to contain only the alpha 1 domain. Careful comparison of the MAT1-1-1 and MAT1-2-1 protein sequences, however, reveals conserved motifs, including one resembling the HMG box sequence, in both proteins. To determine if this second HMG motif is functionally important in both MAT1-1-1 and MAT1-2-1, point mutations were made in MAT1-1-1 (W152A, G160F, V186A, and Y198*) and MAT1-2-1 (W138A, I158A, H170E) sequences of Cochliobolus heterostrophus. These mutated genes are being used to transform the MAT-deleted C. heterostrophus strain C4-41.7 and the strains will be tested for mating competence. Effects of the mutations on mating ability will address the question of whether the single MAT1-1-1 proteins of Dothideomycetes have evolved to carry out all activities provided by multiple MAT1-1 proteins of Sordariomycetes.


6. Analysis of a protein complex required for septation in the filamentous ascomycete Ashbya gossypii. Andrea Walther and Juergen Wendland. Carlsberg Laboratory, Yeast Biology, DK-2500 Valby, Copenhagen, Denmark


Hyphae of filamentous fungi are compartmentalized in regular intervals by septation. Key steps for septation are (i) the selection of a septal site, (ii) the formation of protein complexes at this site and (iii) actin-myosin ring constriction, which is accompanied by chitin deposition. Previously we have analyzed the role of the A. gossypii BUD3 gene in septum formation. Here we present evidence that Bud3 belongs to a protein complex at septal sites that involves septins and Bud4. We have analyzed the function of the septin encoding gene CDC3 and the A. gossypii homolog of the Saccharomyces cerevisiae BUD4 gene. Deletion of CDC3 yields viable albeit slow growing mutants that exhibit a penetrant septation defect. Aberrant chitin deposition at random cortical positions is found in the cdc3 mutant bearing similarity to the septation defect found in bud3 strains. In contrast to cdc3 strains, bud3 hyphae also show correct septation. Mutant bud3 strains are able to sporulate while cdc3 strains do not form spores. Both, Cdc3 and Bud4 were localized at septal sites using GFP-tagged proteins. Using the S. cerevisiae two-hybrid system we established protein-protein interactions among these proteins. This establishes a network of proteins required for correct septum formation and further on may be used as a landmark to direct lateral branching.


7. Characterization of Protein O-mannosyltransferases in Aspergillus nidulans. Thanyanuch Kriangkripipat and Michelle Momany*. Department of Plant Biology, University of Georgia, Athens, Georgia 30602 USA tkriang@plantbio.uga.edu


Protein O-mannosyltransferases (PMTs) are found in bacteria, fungi, and animals but are not present in plants. In fungi, PMTs are divided into three subfamilies, PMT1, PMT2 and PMT4 and each species has 3-7 PMTs. Aspergillus nidulans possesses three PMTs, Pmt1, Pmt2 and Pmt4. Single pmt deletion mutants are viable. Each Δpmt mutant exhibits different phenotypes when characterized by growth at different temperatures, morphology and sensitivity to chemicals disturbing cell wall synthesis. Double mutants show additive phenotypes. The Δpmt1 mutant has hyphal tip lysis and produces aberrant conidiophores at 42OC. The Δpmt2 mutant cannot send out germ tubes at 42OC. The

Δpmt4 mutant has swollen hyphae and produces aberrant conidiophores at 42OC. The Δpmt1Δpmt2 double mutant is viable and has additive phenotypes of Δpmt1 mutant and Δpmt2 mutant. The Δpmt2Δpmt4 double mutant is viable but very sick and forms a microcolony only when an osmotic stabilizer is added to the medium. Lower temperatures and osmoticum can partially restore wildtype hyphal growth and conidiation of these Δpmt mutants except for the Δpmt2Δpmt4 double mutant. Our results suggest that protein O-mannosylation is important for cell wall integrity of A. nidulans.


8. The autophagy gene, atgA, is required for conidiophore development and starvation-induced hyphal growth in Aspergillus fumigatus. Daryl Richie, Michael Miley, David Askew . University of Cincinnati, Pathology and Laboratory Medicine


Autophagy is a highly conserved eukaryotic process in which cytoplasmic constituents are sequestered within a double membrane vesicle and delivered to the vacuole for the recycling of macromolecules. The purpose of this study was to determine the role of autophagy in the opportunistic human pathogen Aspergillus fumigatus. Autophagy was disrupted by deletion of the A. fumigatus atgA gene, which encodes the homolog of Saccharomyces cerevisiae Atg1p, a serine threonine kinase needed for induction of autophagy. The autophagy mutant showed abnormal conidiophore development and reduced conidiation, which could be corrected by supplementing the medium with additional nitrogen. When hyphal plugs of the autophagy mutant were transferred from rich medium to starvation medium they displayed reduced radial growth as compared to wild type. However, the addition zinc or manganese to starvation medium allowed the autophagy mutant to grow to the same degree as wild type. In rich medium, the autophagy mutant was hypersensitive to EDTA. However, the growth of the autophagy mutant in EDTA could also be restored to wild-type levels by the addition of excess zinc and manganese to the medium. Our results suggest that A. fumigatus uses autophagy to recycle intracellular stores of nitrogen to support optimal conidiation in a nitrogen limiting environment. Furthermore, our results suggest that autophagy in A. fumigatus allows for the recycling of limiting trace elements needed for optimal radial growth.


9. Cloning of the eln1 gene essential for stipe elongation of Coprinus cinereus. Hajime Muraguchi1, Naoki Takahashi1, Sonoe O. Yanagi1, Takashi Kamada2. 1Department of Biotechnology, Akita Prefectural University, Akita, Japan. 2Department of Biology, Okayama University, Okayama, Japan.

Coprinus cinereus fruit body exhibits remarkable stipe elongation in the final phase of development: the stipe elongates 8 times in one night. A dominant elongationless1-1 (eln1-1) mutant defective in stipe elongation was first induced from the dikaryon. A recessive elongationless1-2 (eln1-2) mutant was then induced from the monokaryotic fruiting strain, CopD5-12. During stipe elongation in the wild type, the cylindrical component cells of the stipe elongate with their diameter unchanged, resulting in slender stipe cells. In the mutants, however, the cylindrical cells increase in diameter and the rate of elongation is much reduced, resulting in globular stipe cells. Genetic analysis mapped the eln1 locus 0.3 cM apart from his5 on chromosome I. We transformed an eln1-2 mutant with BACs assigned near his5, and found that two BAC clones, s2F7 and s14B9, have the complementing activity. We then defined the genomic region of the eln1 gene within about 30 kb by testing subclones from s14B9 for their complementing activity. We are now trying to identify the eln1 gene by testing PCR-amplified fragments from the 30-kb subclone.


10. A Rho1 Homologue From Ustilago maydis, Uro1, is Required for Normal Cell Growth. Zhanyang Yu1, Cau D. Pham1, Scott E. Gold2, and Michael H. Perlin1. 1University of Louisville, Department of Biology, Program on Disease Evolution, Louisville, Kentucky, USA and 2University of Georgia, Department of Plant Pathology, Athens, Georgia, USA


In the pathogen of maize, U. maydis, a carefully orchestrated network of signaling is used to ensure that development proceeds in an orderly fashion. Uro1 is a homologue of the Schizosaccharomyces pombe and Saccharomyces cerevisiae Rho1 proteins, members of the family of Rho GTP-binding proteins that includes Cdc42 and Rac1. We have cloned and characterized the uro1 gene encoding this U. maydis homologue. Over- expression of either wild type or constitutively-active mutant Uro1 caused a slight reduction in mating efficiency. A diploid strain in which a single copy of uro1 was disrupted, similarly showed reduced “Fuz” reaction on charcoal agar relative to the wild type progenitor strain. This singly-disrupted diploid was unable to produce galls when injected into maize. To circumvent the likely lethality of disruption in haploid strains, the uro1 gene was placed under control of the crg promoter, so as to allow its expression in arabinose media or its shut down in glucose. When uro1 expression was shut off, cells failed to reproduce; however, they could recover if transferred to arabinose medium up to 72 h after the initial shut off by glucose. Several possible interactors of Uro1 were identified by yeast 2- hybrid, including Ump2 (an ammonium transporter), Smu1 (the Pak-like kinase involved in mating), a CDC24 homologue (a RhoGEF), and, quite interestingly, PTEN (phosphatase and tensin homologue deleted on chromosome 10), which acts as a tumor suppressor gene in mammalian cells. We propose that, among its several possible roles, Uro1 acts as an inhibitor of PTEN in U. maydis, and normally prevents the latter’s action to promote arrest of cell division and/or apoptosis sequelae. We will provide additional experimental data which address this hypothesis.


11. The Nuclear Migration Protein NUDF Associates with BNFA and NUDC at Spindle Pole Bodies in Aspergillus nidulans. Kerstin Helmstaedt, Karen Meng, Silke Busch, Özgür Bayram, Oliver Valerius and Gerhard H. Braus. Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany. Tel. +49-551-3919693, Fax +49-551-393820, khelmst@gwdg.de


In Aspergillus nidulans, nuclear division depends on microtubuli, the motor dynein and nuclear distribution proteins like NUDF. Applying tandem affinity purification, we isolated a unique NUDF-associated protein, which we named BNFA (Binding of NUDF). An A. nidulans bnfA deletion strain did not show a nud phenotype indicating that a protein with redundant function might exist. A GFP-BNFA fusion localized to spindle pole bodies (SPBs) throughout the cell cycle. This position was depended on NUDF, since in a nudF6 strain BNFA localized mainly to dots in the cytoplasm. In a yeast two-hybrid screen using BNFA as bait, we found that BNFA is a dimer and that a link might exist to the septation signalling pathway. In a candidate approach, we analysed the putative NUDC-NUDF interaction in A. nidulans. Although NUDC-GFP alone was localized to immobile dots at the cortex, we found a direct interaction between NUDF and NUDC in yeast two-hybrid experiments, which depended on NUDF's WD40 domain. Applying bimolecular fluorescence complementation microscopy, we showed that in vivo NUDF and NUDC interact also at spindle pole bodies throughout the cell cycle and at immobile dots at the cortex.


12. Regulation of hyphal morphogenesis and the DNA damage response by the Aspergillus nidulans ATM homolog, ATMA. Iran Malavazi1, Camile P. Semighini2, Marcia Regina von Zeska Kress1, Steven D. Harris2, Gustavo H. Goldman1. 1Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil; 2Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, USA.


Ataxia telangiectasia (A-T) is an inherited disorder characterized by progressive loss of motor function and susceptibility to cancer. The most prominent clinical feature observed in A-T patients is the degeneration of Purkinje motor neurons. Numerous studies have emphasized the role of the affected gene product, ATM, in the regulation of the DNA damage response. However, in Purkinje cells, the bulk of ATM localizes to the cytoplasm and may play a role in vesicle trafficking. The nature of this function, and its involvement in the pathology underlying A-T, still remain unknown. Here we characterize the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we find that AtmA is also required for polarized hyphal growth. We demonstrate that an atmA mutant has a severe defect in the control of nuclear proliferation and fails to generate a stable axis of hyphal polarity. Notably, cytoplasmic microtubules display aberrant cortical interactions at the hyphal tip. Our results suggest that AtmA regulates the function and/or localization of landmark proteins required for the formation of a polarity axis in A. nidulans. We propose that a similar function may contribute to the establishment of neuronal polarity. Financial Support: FAPESP and CNPq Brazil, Nebraska Research Foundation, USA


13. Endocytic Pathway in Mushroom Development: Role of Le.Rab7 and Interacting Proteins. M.T. Lee, Y.Y. Szeto, T.P. Ng, H.S. Kwan. The Chinese University of Hong Kong, Shatin, HKSAR

Endocytosis is a process through which substrates enter a cell without passing through the plasma membrane but via invagination of membrane and formation of intracellular vesicle. Rab7 regulates the trafficking between early and late endosomes as well as late endosomes to lysosomes in the endocytic pathway. We have isolated Le.Rab7, from Lentinula edodes, that is highly homologous to Rab7 (78% similarity). Two candidates, receptor for activated C kinase-1 (Le.RACK1) and Rab5 GTPase (Le.Rab5) were isolated as interacting partners of Le.Rab7 through in vivo and in vitro protein interaction assays. Le.Rab7 and Le.RACK1 express strongest in primordium, while Le.Rab5 expressed constitutively in all stages. In situ hybridization showed that the three transcripts were mainly localized at active growth regions of fruiting bodies. They strongly expressed at the outer region of trama cells and subhymenium in the hymenophore of gill tissue and at the prehymenophore in the ¡§eye organ¡¨ of young fruiting body. The protein localization of Le.Rab7 was also found in fruiting bodies. The existence of endocytosis in mycelium and gill tissue was shown by the internalization of endocytic dye, which co-localized with Le.Rab7 protein. It is the first report on the presence of endocytosis in basidiomycete. Le.Rab7, Le.Rab5 and Le.RACK1 may contribute to the production of basidiospores and the differentiation of mycelial cells to different cell types at the gill tissue.


14. Analysis of two cell end marker proteins, TeaA and TeaR, in Aspergillus nidulans. Norio Takeshita, Sven Konzack, Yuhei Higashitsuji and Reinhard Fischer. Applied Microbiology, University of Karlsruhe, Germany.


The interplay of the actin and the microtubule (MT) cytoskeleton in polarized growth of fungi has recently been revealed. In Schizosaccharomyces pombe, Tea1 is a key protein in this process. Tea1 is transported to the plus ends of MTs by the kinesin Tea2, and is delivered to cell ends by hitchhiking with the growing MTs. Mod5, which is posttranslationally modified by prenylation, anchors Tea1 at the cell ends. These three proteins were indentified by screening for polarity mutants. At the cell ends, Tea1 recruits formin which initiates actin assembly and the establishment of cell polarity. Tea1 and Tea2 homologues were identified in the Aspergillus nidulans genome (named TeaA and KipA), whereas Mod5 could not be identified due to sequence similarity. kipA mutants showed mislocalization of the Spitzenkorper and hence curved hyphae. GFP-KipA accumulated at the MT plus ends. teaA mutants showed a similar but not identical phenotype to kipA mutants. GFP-TeaA localized to one point in the apex of hyphal tips. To test whether the function of S. pombe Mod5 was conserved, we searched the A. nidulans genome for proteins with a C-terminal prenylation motif (CAAX). From 22 identified proteins one (536 amino acids) was likely to serve a TeaA-anchorage function. We named it TeaR. teaR mutants indeed displayed a phenotype similar to the kipA mutant. GFP-tagged TeaR localized to the membrane at hyphal tips. Their putative interactions will be analyzed.


15. The Aspergillus nidulans putative kinase KfsA plays a role in septation and is required for efficient asexual spore formation. Norio Takeshita, Anne Blumenstein, KayVienken and Reinhard Fischer Applied Microbiology, University of Karlsruhe, Germany


In eukaryotes cytokinesis has to be a well-controlled process to guaranty that each cell receives a nucleus. The process is well characterized in Saccharomyces cerevisiae, where a safeguard-machinery (MEN = mitotic exit network) has evolved to prevent cytokinesis before proper nuclear segregation. One important component is the kinase Kin4, which inhibits the MEN and prevents precautious cell division when the spindle position checkpoint is activated. In Aspergillus nidulans, nuclear division and cytokinesis are not coupled in hyphae, resulting in multinucleate compartments. In contrast, metulae, phialides and conidia are uninucleate. Here, we describe the role of a putative Kin4- related kinase, KfsA (kinase for septation) in the control of septum formation in A. nidulans. KfsA showed similarity to Kin4 only in the kinase domain but is conserved among filamentous fungi. kfsA mutants did not show any obvious phenotype in hyphal growth but produced less conidia. Conidiophores of kfsA mutants often contained several septa and a certain number of metulae harboured two nuclei and the corresponding phialides remained anucleate. The latter result suggests a septum-control mechanism of KfsA during nuclear distribution in the yeast-like growth of metulae, which guarantees that cytokinesis only occurs if both cells contain a nucleus. A GFP-KfsA fusion protein localized to septa in hyphae and the conidiophore and to the cortex of hyphae.


16. Nuclear anarchy: asynchronous nuclear division in Ashbya gossypii. Amy S. Gladfelter. Dartmouth College, Department of Biological Sciences amy.gladfelter@dartmouth.edu


Multinucleated cells are essential for disease processes such as tumorigenesis and fungal infection, but little is known about how the unique organization of multinucleated cells influences the control of mitosis. We have found that nuclei divide asynchronously in the filamentous ascomycete Ashbya gossypii. In these cells, neighboring nuclei are in different stages of the nuclear division cycle despite being only a few micrometers apart and bathed in the same cytoplasm. Similar asynchronous nuclear division also has been observed in multinucleated tumors and other filamentous fungi, but how independent nuclear behavior is established in the context of a common cytoplasm remains to be discovered. Using in vivo time-lapse microscopy, automated image analysis, and molecular genetic approaches we are currently testing two main hypotheses to determine when and how nuclear asynchrony develops: 1.) Nuclear asynchrony emerges in the G1/S transition due to the unequal distribution of cell cycle regulators among nuclei and 2.) The unequal localization of cell cycle regulators among nuclei arises due to differences in Spindle Pole Body age and/or Nuclear Pore Complexes between neighboring nuclei. With these studies we are identifying mechanisms regulating the G1/S transition and delineating how nuclear asymmetries are established in multinucleated cells.



17. CRE-1, a regulator of catabolite repression is involved in determining PKA-dependent polarity of N. crassa Carmit Ziv, Rena Gorovits and Oded Yarden. Dept. of Plant Pathology and Microbiology, The Otto Warburg Minerva Center for Agricultural Biotechnology Faculty of Agricultural, Food and Environmental Quality Sciences The Hebrew University of Jerusalem Rehovot 76100, Israel


Polar hyphal elongation is a complex event in fungal growth and is affected by multiple factors. The Neurospora crassa mcb gene encodes a regulatory subunit of the cAMP-dependent protein kinase A (PKA). We have identified a 19bp deletion at the 5' UTR of mcb temperature-sensitive allele which we propose impairs transcript integrity of the regulatory subunit and thus may result in increase PKA activity and a complete loss of growth polarity at restrictive temperatures. As a link between the PKA pathway and carbon source sensing has been established in Aspergillus and yeasts, we tested the effect of different carbon sources on mcb morphology. Surprisingly, when grown in the presence of fructose, but not in either glucose or sucrose, the typical bulbous mcb phenotype was completely suppressed. As glucose and fructose equally support N. crassa wild-type growth we concluded that the differential effect on mcb involves a regulatory, rather than a metabolic, mechanism. To determine the involvement of carbon catabolite repression (CCR) in determining PKA-dependent polarity, we inactivated a key CCR regulator, cre- 1. The delta-cre-1 strain showed reduced growth on glucose vs. fructose, in addition to altered hyphal morphology and impaired CCR, as evident by changes in inv and adh-1 expression and amylase and beta- gal activity. The deletion of cre-1 in an mcb background partially restored polarity when grown on glucose, indicating that downstream CRE-1-regulated elements can compensate for polarity defects caused by mis-regulated PKA activity. These results suggest a role for the CRE-1-mediated cellular response to carbon source in determining PKA- dependent polar growth in N. crassa.


18. Altering COT1 kinase phosphorylation sites affects its function in N. crassa Carmit Ziv, Rena Gorovits and Oded Yarden. Dept. of Plant Pathology and Microbiology, The Otto Warburg Minerva Center for Agricultural Biotechnology Faculty of Agricultural, Food and Environmental Quality Sciences The Hebrew University of Jerusalem Rehovot 76100, Israel


The Neurospora crassa COT1 kinase is the founding member of the conserved NDR protein kinase family. Defects in cot-1 confer a highly pleotropic phenotype typified by a restricted, colonial, growth. In vitro analysis of mammalian NDR demonstrated the involvement of conserved phosphorylation sites in the regulation of NDR kinase activity. Using antibodies against the phosphorylated human NDR protein we detected the presence of the two conserved phosphorylation sites in N. crassa COT1: Ser-419, a putative MOB2-stimulated autophosphorylated site and Thr- 591, a putative Ste20-like kinase-phosphorylated site required for NDR activity. To determine the significance of the phosphorylation states of COT1 on cell morphology in vivo, we analyzed strains harboring a 5xmyc::cot- 1 fusion allele at the cot-1 locus, in which these conserved regulatory residues have been substituted by either Glu or Ala to mimic the phosphorylated and unphosphorylated states, respectively. The resulting 4 strains (designated S419E, S419A, T591E and T519A) were viable, yet some presented altered morphology. The importance of Ser-419 was evident by a reduced growth rate of both S419E and S419A strains. In addition S419A exhibited a dramatic abnormal, hyper-branched, morphology and produced a ropy-like colony. Altering Thr-591 resulted in a milder effect; strain T591E showed reduced aerial hyphae and enhanced carotenoid production, while T591A displayed a reduced growth rate. Determining COT1 kinase activity levels, localization and interaction with additional proteins in these strains will help elucidate the in vivo role of phosphorylation in governing specific functions of COT1.


19. The BEM46-like protein is essential for hyphal growth from ascospores of Neurospora crassa and targeted to the ER. Mercker M, Alves S, Weiland N, and F. Kempken. Botanisches Institut, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, 24098 Kiel, Germany; fkempken@bot.uni-kiel.de


BEM46 proteins are evolutionary conserved members of the α/β-hydrolase super family. However, to date, their exact role remains unknown. Therefore, to better understand the cellular role of BEM46 and its homologs, we used the model organism Neurospora crassa in conjunction with bem46 RNAi, overexpression vectors, and repeat induced point (RIP) mutation analyses. These studies clearly demonstrated that BEM46 is required for cell-type- specific hyphal growth and indicate a role of BEM46 in maintaining polarity. For example, vegetative hyphae, perithecia, and ascospores develop normally, but hyphae germinating from ascospores exhibit a strong loss-of-polarity phenotype. Moreover, we found that the BEM46 protein is targeted to the ER, and localizes at or close to the plasma membrane. BEM46 thus being a new ER marker for filamentous fungi, and the first for the model organism Neurospora crassa. Thus our data suggest BEM46 plays a role in a signal transduction pathway that is involved in determining or maintaining cell-type-specific polarity. This finding also implies a higher degree of differentiation of fungal hyphae than currently expected.


20. Functional analysis of histidine-containing phosphotransmitter gene ypdA in Aspergillus nidulans. Natsuko Sato, Kentaro Furukawa, Tomonori Fujioka, Osamu Mizutani, and Keietsu Abe. Molecular and Cell Biology, Tohoku University, Sendai, Japan.


The high-osmolarity glycerol (HOG) response pathway responding to osmotic stimuli has been well studied in Saccharomyces cerevisiae. Sln1p-Ypd1p-Ssk1p proteins organize a two-component signalling (TCS) unit in the upstream of the HOG pathway, and negatively regulate the downstream Hog1p mitogen-activated protein kinase (MAPK) cascade. We previously revealed that a filamentous fungus Aspergillus nidulans possesses all counterparts of the components of S. cerevisiae HOG pathway. Deletion of Ypd1p, the TCS histidine-containing phosphotransmitter of S. cerevisiae, is known to cause lethality because of constitutive activation of Hog1 MAPK. While, S. cerevisiae possesses only one set of TCS unit consisted of Sln1p and Ypd1p, A. nidulans has been predicted to have 15 histidine kinases and some of them are thought to interact with the unique YpdA. Thus, the TCS pathway of A. nidulans might be more complex and robust than that of yeast . In addition, YPD1 is essential in S. cervisiae but not in Shizosaccharomyces pombe. In the present study, in order to examine in vivo functionality of A. nidulans ypdA gene, we constructed an ypdA delta strain conditionally expressing the ypdA gene under the control of A. nidulans alcA promoter and investigated its phenotypes under the ypdA-repressed condition. Downregulation of ypdA transcription caused sever growth inhibition. We observed a constitutive phosphorylation of HogA MAPK in A. nidulans ypdA delta. These results suggest that YpdA is a essential component of the upstream of A. nidulans HOG (AnHOG) pathway, and the growth inhibition caused by ypdA delta would be attributed to disorder of signalling through the AnHOG pathway.


21. Calcium plays an essential role in the toxicity of the Penicillium chrysogenum antifungal protein PAF in Aspergillus nidulans. Ulrike Binder1, Sairah Saeed2, Andrea Eigentler1, Diana Bartelt2, Florentine Marx1. 1Biocenter, Division of Molecular Biology, Innsbruck Medical University, Fritz-Pregl Strasse 3, A-6020 Innsbruck, Austria 2Dept. of Biological Sciences, Institute for Biotechnology, St. John's University, 8000 Utopia Parkway, Queens, NY 11439


The small, basic and cysteine-rich protein PAF is secreted by Penicillium chrysogenum and exhibits growth inhibitory activity against various members of filamentous ascomycetes including human and plant pathogens. We are interested in the elucidation of the mechanism of action of this antifungal protein. Previous investigations showed that PAF toxicity is related to plasma membrane hyperpolarization and the activation of ion channels. We hypothesize that PAF toxicity results at least in part in the perturbation of the calcium homeostasis. In this study we show, that minimal concentrations of calcium neutralize PAF activity. Furthermore, we detected a PAF–induced elevation of the intracellular calcium concentration in an Aspergillus nidulans strain that expresses the calcium sensitive photoprotein aequorin. This increase in intracellular calcium concentration was abrogated by the addition of the extracellular calcium chelator EGTA, which indicates that an influx of extracellular calcium is responsible for this effect. Verapamil, an L-type calcium channel blocker, positively influenced the growth of PAF–exposed A. nidulans. Further studies are presently carried out to characterize the role of calcium homeostasis in the toxicity of PAF in detail. This work is supported by the Austrian National Bank and the Austrian Science Foundation to F.M. and in part by NIGMS R15GM077345 to DB.


22. Functional Comparison of CDC42 and RAC1 in the Dimorphic Fungus Ustilago maydis. Andrea Hlubek and Michael Bölker. Fachbereich Biologie, Universität Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany; email: ahlubek@web.de


Cdc42 and Rac1 are small GTPases, which regulate various cellular processes like morphogenesis, vesicle trafficking, cytokinesis and cell polarity. Guanine nucleotide exchange factors (GEFs) convert the GTPases to their active form, GTPase activating proteins (GAPs) stimulate the GTPase activity, thereby leading to the formation of the inactive state. In the dimorphic fungus U. maydis the GTPases Cdc42 and Rac1 are highly similar but trigger distinct cellular responses. We could show that U. maydis Cdc42, but not Rac1, complements the lethal phenotype of the temperature sensitive cdc42-1 allele in S. cerevisiae. To define the regions that are responsible for the specificity of these proteins, we generated a set of chimeric proteins, which were checked for complementation of both U. maydis and S. cerevisiae mutants. These experiments demonstrate that the region between amino acids 41 and 56 is necessary and sufficient to determine the specificity of Cdc42 and Rac1. In vivo and in vitro experiments let us hypothesize that specificity is regulated by interaction with the GEFs, which we are currently characterizing. Another important spatial regulator of GTPases is the Guanine dissociation inhibitor RhoGDI (Rdi1), which is important for proper localization of GTPases and therefore necessary for distinct cellular responses. Interestingly, the deletion of rdi1 in U. maydis interferes with filament formation induced by Rac1 overexpression.


23. The Role of UncA, an Unc104-related kinesin motor protein, in Aspergillus nidulans. Nadine Zekert, Daniel Veith and Reinhard Fischer. Applied Microbiology, University of Karlsruhe, Germany.


We have previously shown that in Aspergillus nidulans conventional kinesin, KinA, is required for fast hyphal growth and the Kip2 family motor, KipA, for the establishment and maintenance of hyphal polarity. Here, we characterize UncA, an Unc-104 related motor protein. UncA (1631 amino acids) contains a motor domain, a forkhead association domain and a PH domain. A homologue of this motor was shown to be required for mitochondrial motility along microtubules in Neurospora crassa. However, in A. nidulans mitochondrial movement rather depends on actin. Deletion of uncA in A. nidulans caused slower hyphal extension with more branching, suggesting a role in polar growth. The fact that uncA deletion was not lethal suggested redundant functions of other kinesins. KinA was a good candidate for this, but surprisingly, a kinA-uncA double mutant was viable and displayed the same branching phenotype as the uncA mutant while its growth phenotype resemble the kinA mutant. UncA- GFP fusion displayed a spot-like distribution in the cytoplasm. The spots showed rapid bidirectional movement. The introduction of a rigor-mutation into uncA inhibited this movement and led to a rod-like staining pattern. Experiments to show whether these rods are microtubules or e.g. mitochondria are under way. A second Unc104-related motor was additionally found in the genome and named UncB (671 amino acids). The function will be characterized by deletion and subcellular localization studies.


24. Aspergillus fumigatus rasA: a non-essential Ras homolog that regulates germination, mitosis and hyphal morphology. Jarrod Fortwendel and Judith Rhodes. University of Cincinnati, Cincinnati, Ohio, USA.


Ras subfamily genes are known to control growth and differentiation in a wide variety of microorganisms. The majority of Ras homologs in pathogenic organisms act as molecular switches that regulate morphogenesis and, in some cases, virulence. The A. fumigatus genome contains two members of the prototypical Ras family of small GTPases, rasA and rasB. Of these Ras sub-family genes, rasA shares 98% homology to the essential rasA gene of Aspergillus nidulans. In this study, we report that deletion of A. fumigatus rasA caused delayed germination, nearly absent radial outgrowth, and decreased hyphal mass. Growth of the rasA deletion mutant on solid agar revealed an extremely compact colony morphology with markedly decreased conidiation. Although conidiation was reduced and delayed, the conidial viability is at wild-type levels. Growth in liquid media caused development of meandering hyphae with increased diameter, when compared to wild type. The rasA deletion mutant was resistant to protoplast preparation, even when using twice the levels of cell wall digestion enzymes that are sufficient for the wild type organism. Normal mitotic events in the mutant were also disturbed, as staining with propidium iodide revealed many small nuclei that were irregularly positioned throughout the hyphae. These data show that rasA is important to a wide range of cellular processes during the complex growth patterns of the important human pathogen A. fumigatus.


25. A PTS2 peroxisomal targeting sequence in ApsB is required for functioning of non-nuclear MTOCs in Aspergillus nidulans. Daniel Veith, Nadine Zekert, Rüdiger Suelmann and Reinhard Fischer. Applied Microbiology, University of Karlsruhe, Germany.


Peroxisomes are a diverse class of organelles involved in different physiological processes in eukaryotic cells. A special class of peroxisomes is represented by the fungal Woronin body, in which one protein, Hex1 in Neurospora crassa, forms a hexagonal crystal and serves a structural function in plugging septal pores after damage of hyphae. Here, we suggest a new class of peroxisomes, which is involved in microtubule formation. In Aspergillus nidulans, septum- associated microtubule-organizing centres (MTOCs) are polymerizing cytoplasmic microtubules in addition to MTOCs at the spindle pole bodies (SPBs). Previously, we identified a novel MTOC-associated protein in A. nidulans, ApsB, whose absence affected microtubule formation from septal MTOCs more than from SPBs, suggesting different organization of the two protein complexes. In a yeast-two-hybrid screening, we identified the peroxisomal protein HexA as an interacting protein of ApsB and discovered that ApsB is targeted to peroxisomes via a PTS2 peroxisomal targeting sequence or by a piggy-bag mechanism together with HexA. Peroxisomal localization of ApsB was necessary for normal function of septal MTOCs. ApsB translocated into Woronin bodies was not active, suggesting two distinct populations of septal peroxisomes, the Woronin bodies and the ones required for MTOC activity.


26. Evolution of the mechanisms that regulate cellular morphogenesis in fungi: a comparative genomics approach. Haoyu Si, Kaimei Xu, and Steven D. Harris*. Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, NE, 68588-0660. *Contact: Sharri1@unlnotes.unl.edu


The defining feature of fungal cells is polarized growth, whereby cell wall deposition is confined to a discrete location on the cell surface. The annotation of multiple fungal genome sequences has revealed that the signaling modules and morphogenetic machinery involved in polarized growth are largely conserved across the fungal kingdom. Nevertheless, fungal cells exhibit a diverse variety of shapes that are largely based on two growth patterns: hyphae and yeast. We suggest that these different patterns reflect variation in the mechanisms that spatially and temporally regulate cellular morphogenesis. To test our hypothesis, we are using a comparative genomics approach to examine the functional evolution of the regulatory modules that specify budding patterns in S. cerevisiae. The landmarks that specify the axial budding pattern (Bud3, Bud4, and Axl2) are weakly conserved in the Pezizamycotina. We are in the process of deleting and charactering these homologues in A. nidulans, Neurospora crassa, and Fusarium graminearum. Our preliminary results show that Bud3 and Bud4 are required for septum formation and, suggest that Bud3 may serve as a GEF (guanine nucleotide exchange factor) for the GTPase Rho4. We propose that the ancestral function of the axial markers may be the regulation of septin organization, which, upon the loss of hyphal growth in S. cerevisiae, allowed them to be co-opted into the module that regulates axial budding. By contrast, only two of the landmarks that specify the bipolar budding pattern (Rax1 and Rax2) are conserved in the Pezizamycotina. Moreover, our deletion analyses have failed to uncover an obvious role for these homologues in hyphal morphogenesis.


27. Survival following exposure to hydrogen peroxide (H2O 2) is developmental stage- dependent in Aspergillus nidulans. Sairah Saeed and Diana Bartelt. Dept. of Biological Sci., St. John’s University, Queens, NY 11439


Among the earliest antimicrobial defense mechanisms is the release of H2O2 formed during the respiratory burst into the phagosome during the degranulation process. Aspergillus sp. are opportunistic pathogens, causing a high rate of mortality in immunosuppressed patients. The ability to survive exposure to hydrogen peroxide is important in the onset of invasive Aspergillosis . We have found that survival of A. nidulans after exposure to hydrogen peroxide is dependent upon the stage of development, the density of the culture and the concentration of H2O 2. Exposure of 10 hr. mycelial cultures to 10 mM peroxide causes greater than 90 percent killing while greater than 90 percent of cells in an 18 culture at the same density survive. Stability of H2O 2 in the medium is also dependent upon the density of the culture, the stage of development and H2O 2 concentration. There appears to be a positive correlation between viability and the ability of the culture to destroy H2O 2. The induction of catalase and/or peroxidase activity following exposure to H2O 2 is examined. [Supported by NIGMS R15GM077345-01A1].


28. Genetic and molecular analysis of a temperature-sensitive mutant un-17 carrying a mutation in the poly (A) polymerase gene in Neurospora crassa. S. Tanaka, N. Takayanagi, K. Murasawa, C. Ishii, and H. Inoue. Lab. of Genet., Fac. of Sci., Saitama Univ., Saitama, Japan.


un-17 mutant was isolated as a temperature-sensitive (ts) mutant in Neurospora crassa. This mutant shows several interesting phenotypes. In restriction temperature (37 Co), the growth immediately stops and the cells are going to die. This death is suppressed by adding of cycloheximide or conditions repressing growth. Even in permissive temperature, it shows female sterility and is deficient in production of extracellular superoxide dismutase SOD4. To know the function of the un-17 gene, we tried to clone the gene. We mapped a position of the mutation to right of linkage group III, and searched a DNA fragment which complements the ts phenotype of un- 17 mutant from a N. crassa genome library. The cloned gene had homology to yeast poly (A) polymerase (PAP), and the PAP-homolog gene of un-17 had a mutation of one base substitution. New strains which express a recombinant PAP derived from either wild-type or un-17 strain were constructed and analyzed. The recombinant PAP of wild-type indicated the activity of PAP, and un-17 strains which were induced a constitutive expression of wild-type PAP recovered mutant phenotypes of original un-17 strains. These data suggest that un-17 encodes the essential PAP gene.


29. Two-component response regulators, ChSsk1p and ChSkn7p, additively regulate high-osmolarity adaptation and fungicide sensitivity in Cochliobolus heterostrophus. Kosuke Izumitsu. Kyoto University, Graduate School of Agriculture, Kyoto, Japan


Filamentous ascomycetous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two-component signaling systems. We previously reported that the fungi-unique group III histidine kinase regulates high-osmolarity adaptation and iprodione/fludioxonil fungicide sensitivity by controlling the phosphorylation of Hog1-type mitogen-activated protein kinase (MAPK) in filamentous ascomycetes. Here, we have characterized the response regulator genes ChSsk1 and ChSkn7 in the Southern corn leaf bright fungus Cochliobolus heterostrophus. Both Chssk1- and Chskn7-disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The ChSsk1/ChSkn7 double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1-type and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase. This study provides evidence that filamentous fungi have a unique two-component signaling system that is different from yeast and is responsible for high-osmolarity adaptation and fungicide sensitivity.


30. Functional topology of the growing hyphal tip. Michael Köhli1, Philipp Knechtle1, Virginie Galati1, Kamila Boudier1, Robert Roberson2 and Peter Philippsen1. 1 Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland. 2 School of Life Sciences, Arizona State University, Tempe AZ 85287, USA.


Growth of fungal hyphae depends on initial definition of a polarity axis, tip-directed intracellular transport and efficient polarized secretion. A complex protein network whose components are conserved in eukaryotes coordinates these processes. While molecular data have become available only recently, a hallmark of polarized growth in filamentous fungi was described long time ago: the Spitzenkörper, a vesicle-based, dynamic structure that is found in the tip of growing hyphae. In order to identify proteins colocalizing with the Spitzenkörper, we monitored GFP-fusions to 14 polarity factors of Ashbya gossypii. Interestingly, in several cases, a given GFP-fusion showed localization patterns that differed between hyphae of the same mycelium, even if these hyphae were spaced less than 50 µm apart from each other. We show that these differences in localization correlate with hyphal growth speed. In slow growing hyphae, the investigated polarity factors mostly localized to the apical cortex independently of the factor’s identity. Contrary, in fast growing hyphae, at least four functional zones defined by distinct sets of polarity factors were observed: the Spitzenkörper, the central apical cortex, the entire apical cortex and the extended apical cortex. The putative polarisome components AgSpa2, AgPea2 and AgBni1, the Rab-GTPase AgSec4 and the exocyst components AgSec3 and AgExo70 were found to localize to the Spitzenkörper. These findings suggest that the Spitzenkörper is directly involved in regulation of the actin cytoskeleton and secretion but not in polarity maintenance or establishment per se.


31. Variation in nuclear DNA content and chromosomes among parent isolates and progeny in Phytophthora infestans. Mursel Catal, Pavani Tumbalam, Willie Kirk, Gerard Adams. Department of Plant Pathology, Michigan State University, East Lansing, MI 48824.


The DNA content of nuclei of various isolates of P. infestans and progeny from controlled matings was assessed by laser flow cytometry. Variation in size of chromosomes among isolates was evaluated with Southern hybridizations, and some virulence genes were located on chromosome fragments. Progeny varied in virulence, race, mating type, cold tolerance and genetic markers. In each controlled mating a small proportion of progeny were unusual, showing secondary homothallism, loss of virulence, and the combined markers of both parents. Variation in DNA content of nuclei among parents and, more interestingly, among the unusual progeny are presented and discussed in relation to chromosome patterns and ploidy.


32. The Aspergillus nidulans nuclear pore complex protein An-Nup-2 plays a novel role in mitosis but is not essential for nuclear transport. Sarine Markossian and Stephen A. Osmani. The Ohio State University, Columbus, Ohio, USA. markossian.1@osu.edu


The nuclear pore complex (NPC) regulates nuclear trafficking and is composed of ~30 subunits called nucleoporins (Nups). In yeast, Nup2p has been shown to facilitate nuclear transport. Unlike yeast Nup2p, Aspergillus nidulans Nup2 (An-Nup2) localizes to chromatin during mitosis but to the NPC during interphase. This indicates An-Nup2 may play a role during mitosis. An-nup2 is essential and its deletion causes mitotic defects. We therefore speculate that the localization of An-Nup2 to mitotic chromatin is important for mitosis. To test this hypothesis, a domain study was performed to define the An-Nup2 domain responsible for its mitotic translocation to chromatin and an antibody was generated against An-Nup2. A domain spanning from aa 400 to aa 1200, which encompasses a basic stretch of amino acids, a coiled coil region, and two potential nuclear localization sequences (NLS), is sufficient to locate An-Nup2-GFP to the NPC during interphase and to DNA during mitosis. The An-Nup2 antibody was used for immunofluorescence and successfully stained the nuclear periphery during interphase and chromatin during mitosis confirming the An-Nup2-GFP localization. Most importantly, the heterokaryon rescue technique was used to define if nuclear transport and/or mitosis is defective without An-Nup2. An-Nup2 deleted cells were not deficient in nuclear transport of NLS-dsRed suggesting that the lethality caused by An-Nup2 deletion is due to mitotic defects and not nuclear transport defects. In conclusion, there is emerging evidence that the localization of An-Nup2 to mitotic chromatin is essential for mitosis although An-Nup2 may not be essential for nuclear transport.


33. Cell biology of the mode of action on Neurospora crassa of an antifungal agent from Hypocrea atroviridis. Verena Seidl1, 2, Christian P. Kubicek2 and Nick D. Read 1. 1Institute of Cell Biology, University of Edinburgh, UK, 2Institute of Chemical Engineering, Vienna University of Technology, Austria.


The well characterized model system, Neurospora crassa, was used as a host for the necrotrophic mycoparasite Hypocrea atroviridis (Trichoderma atroviride) to get new insights into the mechanisms underlying mycoparasitism by Hypocrea/Trichoderma spp. Although mycoparasitic Hypocrea/Trichoderma spp. have a wide host range, their interactions are mostly studied with plant pathogens, and many aspects of the mycoparasitic process are not well understood. Hypocrea/Trichoderma spp. produce highly effective antifungal compounds and antibiotics, but very little is known about their specific modes of action. H. atroviridis displayed antagonism against N. crassa in plate confrontation assays. Before making contact, N. crassa hyphae showed a severely stressed phenotype characterized by slow, bud-like growth and hyperbranching. Spore germination was also strongly inhibited. To characterize the stress response in more detail we are studying the effect of the inhibitory compound on various subcellular processes, including Spitzenkörper formation, endocytosis, membrane potential and mitochondrial activity. The inhibitory compound is constitutively secreted by H. atroviridis independent of the presence of a host. It was isolated and purified and could be shown to be a small, non-volatile organic molecule and it is currently being characterized by mass spectrometry.


34. Identification of novel genes involved in hyphal fusion in filamentous fungi. Alex Lichius and Nick D. Read. Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, UK. (A.Lichius@sms.ed.ac.uk)


Vegetative hyphal fusion is essential for the establishment of an interconnected colony network, and is a key feature of the filamentous fungi. A small number of proteins involved in regulating hyphal fusion have been identifed in Neurospora crassa. Hyphal fusion and appressorium formation in fungal pathogens share a number of features in common, including adhesion, cell swelling, secretion of cell wall degrading enzymes, penetration and MAP kinase signalling. We are asking the question: how much does the machinery involved in appressorium formation share in common with that involved in hyphal fusion? To answer this question and identify new proteins involved in these processes, we are screening 42 knockout mutants of genes in N. crassa which are orthologs of genes encoding proteins involved in appressorium formation and/or are pathogenicity determinants in the rice blast fungus, Magnaporthe oryzae. Processes in which these Magnaporthe proteins play a role include surface recognition, cell adhesion, appressorium induction, appressorium development, and host cell penetration. Conidial anastomosis tube (CAT) fusion is being used as simple and convenient experimental system for this genetic screen. We are also assessing hyphal fusion in mature colonies and use a wide range of live-cell analytical techniques to characterize the mutants. Acknowledgements: AL is funded by the School of Biological Sciences studentship of the University of Edinburgh.


35. A Cytoplasmic Nuclear Remnant is involved in the Segregation of Nucleolar proteins in Aspergillus nidulans. Leena Ukil, Colin De Souza, Hui-Lin Liu and Stephen A. Osmani, Department of Molecular Genetics, The Ohio State University, 484 W 12th Ave, Columbus, OH 43210, Email: leenaukil@yahoo.com


The nucleolus is a prominent nuclear structure whose mitotic segregation is poorly understood. During yeast mitosis the nucleolus segregates intact with rDNA. In contrast, during open mitosis the nucleolus is disassembled then reassembled. Aspergillus nidulans nuclei undergoes partially open mitosis, which is an evolutionary intermediate between open and closed mitosis. We therefore determined how A. nidulans nucleoli are segregated during mitosis. Unlike Saccharomyces cerevisiae, few A. nidulans nucleolar proteins segregate with DNA. Instead we have defined two patterns by which different nucleolar proteins segregate during mitosis: (1) Dispersal into the cytoplasm at the onset of mitosis but with some protein remaining bound to DNA, (2) A novel pattern in which nucleolar proteins remain in a nuclear remnant, distinct from daughter nuclei, before re-accumulating into daughter nucleoli during G1. Dual labeling of nucleolar proteins and nuclear envelope markers reveal that the nucleolar remnant is generated as a result of a double nuclear envelope fission event. This double fission occurs around a nucleolar protein mass during telophase. This mechanism generates two transport competent daughter nuclei and a very transient nucleolar remnant containing class 2 nucleolar proteins. This study indicates A. nidulans undergoes mitotic disassembly then reassembly of its nucleolus, as do higher eukaryotes, and that generation of daughter nuclei occurs via a double fission mechanism, not a single fission as occurs in yeasts. We suggest the novel mitotic nuclear remnant we have defined serves as a storage pool from which equal distribution of nucleolar proteins occur. It may also serve as a sink for unwanted cytoplasmic proteins or RNAs that gain access to nuclei during mitosis and may be a positional cue for the double fission.


36. Genetic dissection of phosphate acquisition in Neurospora crassa. Wayne K. Versaw, Yuan Jin, Lauren Baber and Kelly Guido. Dept of Biology, Texas A&M University, College Station, TX 77843


Depending on environmental conditions, N. crassa obtains phosphate via a constitutive, low-affinity uptake system or a phosphate-repressible, high-affinity uptake system. The high affinity system consists of two structurally unrelated transporters, PHO-4 and PHO-5. Homologous transporters with similar functional attributes have been well described in the yeast Saccharomyces cerevisiae. Low affinity phosphate transporters also have been described in yeast but based on mutant analyses, the single corresponding N. crassa homolog, PHO-6, does not appear to have a significant role in phosphate uptake. We describe a putative transporter, PHO-7, and evidence suggesting that PHO-7 constitutes the low-affinity phosphate transport system in N. crassa. PHO-7 represents a novel class of transport proteins that is conserved in filamentous fungi but is not represented in the yeasts.


37. The Aspergillus nidulans snxA1 and nimA5 mutations interact to affect mitotic spindle structure. Yulon Stewart, Ryan Day, Kirk Jackson, Michael Jackson, and Sarah Lea McGuire. Millsaps College, 1701 N. State St., Jackson, MS, 39210.


Both the nimA and snxA genes interact with nimXcdc2 to affect mitosis in Aspergillus nidulans. nimA affects the nuclear import of nimXcdc2, while the snxA1 mutation is a cold-sensitive suppressor of the nimX2cdc2 mutation. snxA1 leads to abnormal nuclear morphology at 17oC. To better understand effects of the snxA1 mutation on cells and the relationship between snxA and nimA, we generated strains expressing GFP-tubA (alpha-tubulin) and various combinations of snxA and nimA mutations. At 17oC snxA1/GFP-tubA cells had severe nuclear defects, thickened hyphae, abnormal spindle structures, and abnormal interphase microtubule arrays. Mitotic spindles were highly variable in length. Some spindles had no nuclei attached to them, while others were bifurcated or trifurcated and had fragmented, variably condensed nuclei along their lengths. Similar abnormal nuclei and spindle structures were observed when snxA1/nimA1/GFP-tubA cells and snxA1/nimA5/GFP-tubA cells were germinated at 32oC and upshifted to 44oC for 3 hours, suggesting that the effects of snxA1 on nimA are not allele-specific. After 3 hours at 44oC, 69% of snxA1/nimA1/GFP-tubA cells had abnormal nuclei, and 56% had abnormal spindles; similar results were obtained with snxA1/nimA5/GFP-tubA cells. Confocal microscopy of the abnormal spindles shows highly unusual spindle structures, which are more severe in cells carrying the snxA1/nimA5 double mutant; snxA1/nimA1 double mutant cells often have significantly shortened spindles. Efforts to clone the snxA gene are ongoing and should aid in the understanding of the interactions of the snxA and nimA genes in mitotic control. Supported by NIH R15GM55885 and NIH RR016476 from the MFGN INBRE program of the NCRR.


38. Withdrawn


39. Cell Biology of Biotrophic Blast Invasion by the Rice Blast Fungus Magnaporthe grisea Prasanna Knakanala1, Kirk Czymmek2, Barbara Valent1. 1Kansas State University, Manhattan, KS 2University of Delaware, Newark, DE


The filamentous ascomycete fungus, Magnaporthe grisea is a hemibiotroph that causes rice blast disease accounting for 60 million tons of grain loss annually. The blast fungus produces intracellular Invasive Hyphae (IH) to colonize the plant, but detailed cellular and molecular mechanisms by which this occurs were not studied. We applied live- cell imaging to characterize spatial and temporal development of IH and plant responses inside successively-invaded rice cells. Early loading experiments with the endocytotic tracker, FM4-64, showed dynamic plant membranes around IH. These hyphae showed remarkable plasticity and recruited plant cell components. IH exhibited pseudohyphal growth and were sealed in plant membrane, termed the Extra-Invasive Hyphal Membrane (EIHM). The fungus spent up to 12 hours in the first cell, often tightly packing it with IH. IH that moved into neighboring cells were biotrophic, although they were initially thinner and grew more rapidly. IH in neighboring cells were wrapped in EIHM with distinct membrane caps at the hyphal tips. Time-lapse imaging showed IH scanning plant cell walls before crossing them, and transmission electron microscopy showed crossing occurring at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Our studies have revealed insights into a novel hemibiotrophic strategy employed by the blast fungus. Analysis of biotrophic blast invasion will significantly contribute to characterization of secreted fungal effectors that impact normal plant processes. To further understand the molecular basis of this infection strategy we have employed laser microdissection to identify the effectors involved in these processes.


40. Identification of transcription factor regulated by OS-2 MAP kinase in Neurospora crassa. Azusa Shiozawa1, Shinpei Banno2, Kazuhiro Yamashita1, Setsuko Watanabe1, Fumiyasu Fukumori1, and Makoto Fujimura1. 1 Dept of Life Sciences, Toyo Univ, Gunma, Japan. 2PRRC., Toyo Univ, Gunma, Japan.


Expression of the genes for glycerol synthesis (gcy-1, gcy-3, and dak-1), catalase (ctt-1), and gluconeogenic key enzymes (fbp-1 and pck-1) are stimulated by osmotic stress and fludioxonil in OS-2 MAP kinase dependent manner in Neurospora crassa. To identify the transcriptional factor regulated by OS-2 MAP kinase, we isolated three disruptants for atf-1 (CREB-type), msn-1 (STRE-type), and nap-1 (AP-1-type) genes, which are orthologs of SKO1, MSN2/4 and YAP1 of budding yeast, respectively. The msn-1 disruptant stimulated aerial hyphae on agar medium and showed increased resistance to oxidative stress such as menadione and t-butyl hydroperoxide. In contrast, the nap-1 disruptant was sensitive to oxidative stress. No gene disruption including atf-1 affected sensitivity to osmotic stress and fludioxonil. However, fludioxonil-induced activation of the OS-2-dependent genes was almost thoroughly cancelled in the atf-1 mutant but not in the msn-1 and nap-1 mutants. The atf-1 mutant grew well on the agar medium, but no progeny with atf-1 was obtained when the atf-1 mutant was crossed with the wild-type strain. These data suggested that the transcription factor ATF-1 may acts downstream of OS-2 MAP kinase, and probably plays an important role in the viability of ascospore.


41. Expression of the glucose-repressible grg-1 gene is stimulated by osmotic stress and fludioxonil in OS-2 MAP kinase dependent manner. Setsuko Watanabe1 , Shinpei Banno2 , Azusa Shiozawa1 , Noriyuki Ochiai3 , Makoto Kimura3 , Makoto Fujimura1. 1Dept of Life Sciences , Toyo University , Gunma , Japapan. 2PRRC., Toyo University , Gunma , Japapan. 3Env.Mol.Biol., RIKEN , Yokohama , Japan.


Two-component histidine pathway consisting of OS-1 (histidine kinase) and OS-2 (MAP kinase) plays an important role in osmotic regulation in Neurospora crassa. The cAMP-PKA pathway, comprised of MCB/HAH (regulatory subunit) and PKAC-1 (catalytic subunit), participates in the switching of conidiation and filamentation probably in response to carbon sources. The hah;os-2 double mutant was more sensitive to osmotic stress than the os-2 mutant. In contrast, the pkac-1 mutation partially suppressed osmotic sensitivity of the os-2 mutant, suggesting crosstalk between these pathways. Furthermore, we found that expression of the grg-1 (glucose- repressible gene) gene was regulated under the OS pathway. Real-time PCR analysis demonstrated that transcription of the grg-1 gene was stimulated by osmotic stress and fludioxonil in the wild-type strain but not in the os-2 mutant. The GFP fluorescence intensity of the strain with the Pgrg-1-sgfp fused gene was significantly increased by osmotic stress and fludioxonil. There are several elements in the promoter region of the grg-1 gene such as CRE (cAMP-Responsive Element) and STRE (Stress Responsive Element). Promoter analysis is currently being undertaken.


42. Withdrawn


43. Analysis of DNA damage checkpoint genes in Neurospora crassa. Mitiyoshi Wakabayashi and Suuitu Tanaka. Saitama Univ. Regulation Biology, Saitama, Japan.

Checkpoint is an important mechanism for both DNA repair and chromosome maintenance. This mechanism has been studied in many organisms including yeast and human cells. In Neurospora crassa, various DNA repair genes have been studied in, but any studies concerning the DNA damage checkpoint had not been done. Recently, the clock gene prd-4 in Neurospora was identified as a homologue of Human Chk2. It is meaning that a relationship between DNA damage checkpoint and circadian clock exists. But data about the role of prd-4 toward both DNA damage checkpoint and DNA repair was not so much. So, we investigated three genes, that is one human Chk1 homologue and two Chk2 homologues. In general, checkpoint-deficient mutants show higher sensitivity against many mutagens than the wild type. In the S.cerevisiae RAD53 (Chk2 homologue ) mutant is sensitive to some mutagens containing camptothecin (CPT), which causes replication fork collapse, and hydroxy urea (HU), that induces stall of replication fork. The Neurospora Chk1 (ncchk1 ) disruptant indicated sensitivity to both CPT and HU. And two Neurospora Chk2 (nccds1 and prd-4 ) disruptants indicated sensitivity to CPT but not to HU. Moreover, we tried to make the relationship between these genes and other checkpoint genes clear. Surprisingly, CPT sensitivity of mus- 9 (Neurospora ATR ) was suppressed by nccds1 mutation. In yeast and human, CHK2 activation is dependent on ATR or ATM kinase activity, but such a genetic relationship observed in Neurospora had not be reported. These results indicate that Neurospora crassa has a unique mechanism in regulation of the DNA damage checkpoint.


44. A single amino acid determines GAP-specificity of duplicated Rho-GTPases in Ashbya gossypii. Michael Köhli1, Sabrina Buck2 and Hans-Peter Schmitz1,2. 1Applied Microbiology, Biozentrum Universität Basel, Klingelbergstr. 50-70, 4056 Basel, Switzerland 2Department of Genetics, University of Osnabrück, Barbarastr. 11, 49076 Osnabrück, Germany, hans- peter.schmitz@biologie.uni-osnabrueck.de, Tel.: +49 541 969 2289, Fax: +49 541 969 2293


While it is a known fact that gene duplication is a driving force of evolution, the resulting functional diversification is often unknown. We investigated the changes in function of the tandem duplicated homologs AgRho1a and AgRho1b of the Saccharomyces cerevisiae RHO1 gene in the filamentous fungus Ashbya gossypii. We could show that the main differences between the two duplicated copies are due to a single amino acid mutation in the switch I region of one of the two AgRHO1 genes. This exchange alters the specificity for the two Rho1-GAPs AgSac7 and AgLrg1 as shown by mutant analysis, interaction studies and GAP-assays with purified proteins. Our results explicitly show an example how gene duplication and single amino acid changes can rewire signal transduction networks during evolution.


45. Role of Microtubule Organizing Centers for the Distribution, Oscillation and Division of Nuclei in the Filamentous Fungus Ashbya gossypii. Claudia Birrer, Sandrine Grava, Tineke van den Hoorn, Dominic Hoepfner and Peter Philippsen, Molecular Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland, Claudia.lang@stud.unibas.ch


Nuclear positioning is important for normal growth and development of all eukaryotes including filamentous fungi. Among those, Ashbya gossypii is a particularly attractive organism to study nuclear distribution in multi-nucleated hyphae. Its 9 MB genome is completely sequenced and the annotation revealed a high degree of synteny to Saccharomyces cerevisiae. Using GFP-labled histone H4 nuclear dynamics including oscillation, bypassing and division have already been studied earlier (Alberti-Segui et al. 2001, Gladfelter et al. 2006). These processes are most likely co-regulated by the activities of microtubule organizing centers (MTOCs) embedded in the nuclear membrane and the dynamics of an elaborated microtubule (MT) network. We monitored MTOCs in A. gossypii hyphae carrying YFP-labled Tub4, the A. gossypii homolog of gamma-tubulin. Furthermore we studied the locations of re-emerging MTs after nocodazole treatment. Our data exclude hyphal tips or septa as locations for MTOCs and confirm A. gossypii nuclear-localized MTOCs as places of nucleating MTs. By in vivo labeling of alpha-tubulin with GFP and immuno-staining we could show that MTOCs nucleate two kinds of MTs: long ones interconnecting nuclei and short ones contacting the cell cortex. We also analyzed the function of AgSpc72, another component of A. gossypii MTOCs. Its homolog in S. cerevisiae is part of the outer, cytoplasmic layer of MTOCs. Yeast cells lacking this component only generate very short MTs. We show, that AgSPC72-deleted hyphae still form long cytoplasmic MTs, short MTs were not observed. Nuclear migration is substantially disturbed: nuclei are clustered distally from the tip, and nuclear oscillations are abolished. Moreover, the fraction of mitotic nuclei is twofold increased compared to wild type. To gain further insight into the controlled distribution and movement of nuclei we have started to investigate the role of other components on nuclear dynamics in A. gossypii.


46. Mitochondrial transport in Neurospora crassa by the motor proteins NKIN2 and NKIN3. Daniela Justa1, Jolante Reth2, Guenther Woehlke2 and Stephan Seiler1. 1 Institute of Microbiology and Genetics, Goettingen, Germany 2 Institute for Cell Biology, Munich, Germany


The most widely used mechanism for intracellular transport involves molecular motor proteins that carry cargo directionally along cytoskeletal tracks (myosins along actin filaments and kinesins and dyneins along microtubules). Several proteins that interact with motor proteins have been identified, but our knowledge about how the motor-cargo interaction takes place and how it is regulated is still limited. Recently, it has been shown that two kinesins of the kin3 family (named KIN2 and KIN3) are associated with mitochondria in vitro. To further characterize this interaction, we have generated mutants in both kinesins. Phenotypic analysis suggests that KIN2 is the main motor responsible for mitochondrial motility in vegetative hyphae, but also that its loss can be compensated by KIN3. The inability of kin-3 deletion strains to produce spores implicates an important function of this motor during sexual development. Currently, we are generating a kin-2;kin-3 double mutant to analyze the partially overlapping functions of the two motors in more detail. Life imaging of GFP-tagged versions of KIN2 and KIN3 is being developed to study the dynamic localization of the motor proteins. To further dissect the interacting domains of the motors and the responsible mitochondrial receptors, we are generating myc-tagged constructs of both kinesins to purify associated components.


47. Polar tip extension is coordinated by POD6 and COT1 in a motor protein-dependent manner in Neurospora crassa. Sabine Maerz1, Nico Vogt1, Carmit Ziv2, Oded Yarden2 and Stephan Seiler1. 1University of Goettingen, Germany 2The Hebrew University of Jerusalem, Israel


Members of the Ste20 and NDR protein kinase families are important for cell differentiation and morphogenesis in various organisms. We characterized POD6 (NCU02537.2), a member of the GCK family of Ste20 kinases that is essential for hyphal tip extension and coordinated branch formation in N. crassa. pod-6 and the NDR kinase mutant cot-1 exhibit indistinguishable growth defects, characterized by cessation of cell elongation, hyperbranching, and altered cell-wall composition. We suggest that POD6 and COT1 act in the same genetic pathway, based on the fact that both pod-6 and cot-1 can be suppressed by 1) environmental stresses, 2) altering PKA activity, and 3) common extragenic suppressors. Unlinked noncomplementation of cot-1/pod-6 alleles indicates a potential physical interaction between the two kinases, which is supported by coimmunoprecipitation, yeast two hybrid studies, partial colocalization of both proteins in wild-type cells, and their common mislocalization in dynein/kinesin mutants. We conclude that POD6 acts together with COT1 and is essential for polar cell extension in a motor protein-dependent manner in N. crassa. We are currently developing enzyme assays for both kinases and are in the process of establishing an interaction network on the basis of yeast two hybrid studies and co-affinity purifications.


48. Spitzenkörper localization and intracellular traffic of GFP-labeled class I and class VI chitin synthases in living hyphae of Neurospora crassa. M. Riquelme1, S. Bartnicki-Garcia1, J.M. González-Prieto2, E. Sánchez- León1, J. A. Verdín-Ramos1, A. Beltrán-Aguilar1, and M. Freitag3. 1Department of Microbiology. Center for Scientific Research and Higher Education of Ensenada (CICESE). Km 107 Ctra. Tijuana-Ensenada. 22860 Ensenada, Baja California, México; 2Center for Genomic Biotechnology. Blvd. del Maestro s/n. 88710 Cd. Reynosa, Tamaulipas, México; 3Department of Biochemistry and Biophysics ALS 201. Oregon State University, Corvallis, OR 97331-7305, USA.


Chitin synthases (CHS) are essential enzymes involved in cell wall synthesis in fungi and arthropods. Little is known about their intracellular movement in growing fungal hyphae. Here, we present data on the localization of two selected CHS, CHS-3 and CHS-6, from Neurospora crassa. Both CHS-3 and CHS-6 were labeled at their carboxyl terminus with green fluorescent protein (GFP). We used high-resolution laser scanning confocal microscopy (LSCM) to analyze the localization and trafficking of CHS-3 and CHS-6 in growing hyphae. CHS-3-GFP and CHS-6-GFP showed similar distribution patterns along hyphae. In distal regions (beyond 45 µm from the tip), CHS-GFP was found mainly in a highly stained network of large endomembranous compartments and in nearby developing septa. At the subapex, the fluorescence was observed in numerous vesicles or groups of vesicles that moved predominantly forward until reaching the most proximal subapical region (15-20 µm from the tip). At the tip, the fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). Co-labeling of the Spk with the fluorescent marker FM4-64, showed CHS-GFP localized in the inner core of the Spk, the same region in which microvesicles were previously detected by transmission electron microscopy. Analysis of fluorescence recovery after photobleaching (FRAP) suggested that fluorescence at the Spk stems from the immediately surroundings vesicles. There was no co-localization of CHS-GFP fluorescence with stained secretory compartments fluorescence suggesting that CHS-3 and CHS-6 are not transported via the classical ER to Golgi to cell surface secretory pathway.


49. The Phycomyces blakesleeanus madB gene is a member of the White Collar 2 family. Catalina Sanz1 and Arturo P. Eslava1. 1Ãrea de Genetica, Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca, Spain catsof@usal.es, eslava@usal.es


Phycomyces blakesleeanus is a filamentous fungus that has been used as a model in several studies of environmental signals as light, chemicals, wind, gravity and adjacent objects. The P. blakesleeanus genome sequence has led to the identification of three putative homologous genes belonging to the White Collar 1 family of blue- light photoreceptors and four putative homologous genes (wctA, wctB, wctC and wctD) from the White Collar 2 family. In a recent work, we have found that mutations in one of those White Collar 1 homologous genes are responsible for the blind phenotype observed in the madA strains, and as such, this gene has been named madA (Idnurm et al., 2006). In the present work we have analyzed the White Collar 2 family (wct genes) in several P. blakesleeanus strains that exhibit an abnormal sensitivity to light (from madA to madJ) in order to detect possible mutations. All the analyzed madB strains were affected in the wctA gene and present the same G+906A mutation, so it seems likely that all the strains are from the same origin. No other mutations in the White Collar 2 gene family were found among the rest of the mad strains. The G to A change presumably prevents the splicing of the first intron of the gene. We have amplified the cDNA copy of the wctA gene in the madB mutants by PCR and have observed that these cDNAs are partially unspliced. The cDNAs from the madB strains C111, C112 and C109 have been cloned and sequenced to ensure that the mutation does indeed prevent the splicing of the first intron, and to show that there are no secondary splicing events. We have also analyzed some genetic crosses of the wild type strain with some madB strains and have looked for recombinants to confirm whether the wild type recombinants do have the wild type copy of the gene, while the madB recombinants have the G to A change. As all the madB strains show impaired light-sensing this phenotype seems to be link to the mutation found, therefore we have decided to name the wctA gene madB.

 

50. The role of the exocyst in growth and morphogenesis of Neurospora crassa hyphae. A., Beltrán-Aguilar1, S., Seiler2, and M., Riquelme1. 1Center for Scientific Research and higher Education of Ensenada (CICESE), Department of Microbiology. Km. 107 Ctra. Tijuana-Ensenada, 22860, Baja California, México. 2 Institut für Mikrobiologie und Genetik Abt. Molekulare Mikrobiologie und GenetikGeorg-August- Universität Göttingen Grisebachstr. 8D-37077 Göttingen, Germany.


Exocytosis is the last step of secretion and it consists on vesicle incorporation to the plasma membrane. A high fidelity protein-protein interaction (mediated by Sec proteins, SNAREs, GTPases and the exocyst) helps to provide specific recognition at the exocytosis sites. The secretory machinery involved in this process has not been fully described in filamentous fungi, but most of its components are evolutionary conserved among eukaryotes from yeast to mammals’ neurons. The exocyst complex plays an important role in the vesicle-plasma membrane interactions previous to SNAREs assembly and it is compossed of eight proteins (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84). Using a Neurospora crassa sec5 mutant strain we have analyzed the importance of this component for Spitzenkörper formation, polarized growth and hyphal morphology. At low magnification N. crassa sec5 showed a button like colony morphology, with hyperbranched and distorted hyphae. The mutant growth rate was 2.03% that of the wild type strain. At higher magnification the intracellular organization was severely affected. One of the key differences between the wild type and the mutant was the lack of a Spitzenkörper in the latter under high-resolution phase-contrast video- microscopy. A vector containing a sec5::sgfp the fusion was introduced into a N. crassa wild type strain and the localization and traffic of SEC5-GFP was analyzed by laser scanning confocal microscopy. Fluorescence did not accumulate at the tip, but was observed in a punctuate pattern all along the hyphae. This study establishes the basis for future analyses directed to elucidate the role of the exocyst in the apical growth of filamentous fungi.


51. Putative mannose transporters complement a branching/septation defect in Aspergillus nidulans. Loretta Jackson-Hayes, Lauren Fay, Terry W. Hill and Darlene M. Loprete. Departments of Biology and Chemistry, Rhodes College, Memphis, TN 38112. jacksonhayesl@rhodes.edu


In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans, which show hypersensitivity to the chitin synthase inhibitor Calcofluor White (CFW). The phenotype of one of these strains (R205) also shows morphological abnormalities related to branching and septation. We have cloned two DNA fragments from an A. nidulans genomic DNA library which improve resistance to CFW and restore a more normal phenotype. One fragment is gene AN8848.3, "MT1", which shows homology to GDP-mannose transporters. The second fragment is gene AN9298.3, "MT2", which is a similar but distinct gene also homologous to GDP-mannose transporters. When separately cloned, the putative GDP-mannose transporters restore normal phenotype including full restoration of subapical hyphal compartment length and branch density in the mutant. Sequencing reveals a genetic lesion in Exon 5 of MT1 which causes an alanine to proline substitution and no mutation in MT2 in mutant strain R205. Cloned R205 MT1 containing the Exon 5 mutation does not complement the R205 phenotype. Attempts to produce null mutants of MT1 did not produce viable transformants, suggesting that AN8848.3 is an essential gene. MT2 null mutants grow normally under normal growth conditions and show wild type CFW resistance.


52. Constitutive PKA activity prevents growth arrest of Aspergillus fumigatus conidia. Kevin Fuller, Wei Zhao, Lauren Fox, Judith Rhodes. University of Cincinnati, Cincinnati, OH


We have previously generated a mutant of A. fumigatus deficient in the regulatory subunit of PKA and described that conidia are hypersensitive to various forms of oxidative stress. We also noted that the mutant conidia are larger than those of wt in distilled water. Both swelling and increased sensitivity to oxidative stress accompany metabolic activation after exposure of resting conidia to a germinant. Accordingly, we wanted to determine if the increase in conidial size of the pkaR deletion mutant was due to an unexpected initiation of germination in water. To determine the metabolic state of the conidia, we evaluated mitochondrial activity with Mitotracker Orange. Whereas it required 4 hours in a rich medium for the wt and complemented strains to stain positively, the delta pkaR conidia were positive at earlier time points, even in the absence of a germinant. Furthermore, the mutant conidia were killed more readily by hydrogen peroxide when compared to the resting conidia of the other isogenic strains. However, after the 4 hour time point, all members of the isogenic set showed equivalent killing by the treatment. We, therefore, conclude that the hypersensitivity to hydrogen peroxide seen with the conidia of the pkaR deletion mutant is due to failure of resting conidia to maintain growth arrest.


53. Ras signaling in the human pathogen Cryptococcus neoformans. Connie B. Nichols and J. Andrew Alspaugh. Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA. connie.nichols@duke.edu


Cryptococcus neoformans is an opportunistic human fungal pathogen that is the causative agent of cryptococcosis, a life-threatening infection. The ability to grow at high temperature is an important requirement of C. neoformans to establish disease. Previously we found that Ras1 was required for growth at high temperature and for sexual differentiation. We have now identified the downstream components of Ras1 that mediate the response to high temperature, including Cdc24, Cdc42, and Ste20. In the fission yeast Schizosaccharomyces pombe, Ras mediates morphology and sexual differentiation using different upstream activators and downstream effectors. To understand how Ras1 mediates both high temperature growth and sexual differentiation in C. neoformans we have generated a series of ras1 mutant strains including dominant negative, dominant active, farnesylation-defective, and palmitoylation-defective ras1 mutant strains. In addition we have used genetic epistasis experiments to characterize the role of the Cdc24-Cdc42-Ste20 branch of Ras1 signaling in sexual differentiation in C. neoformans.


54. Two-component signaling system for osmotic regulation and fungicide sensitivity in Cochliobolus heterostrophus. Chihiro Tanaka, Kosuke Izumitsu, and Akira Yoshimi* Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, JAPAN. *Present address: New Industry Creation Hatchery Center (NICHe), Tohoku University, Aoba, Sendai 980-8579, JAPAN. E-mail: chihiro@remach.kais.kyoto-u.ac.jp


We have studied the mode of action of dicarboximide/phenylpyrrole fungicides, using resistant mutants of a maize pathogen Cochliobolus heterostrophus. In this fungus, the resistant mutants always showed hyperosmotic sensitivity, as reported in N. crassa. And dic1 encoding a histidine kinase (HK) was characterized as a responsible gene for fungicide resistance and osmotic adaptation. We also elucidated that exposure to the fungicides led to Dic1p mediated improper activation of Hog1-MAPK (BmHog1p). HK and MAPK are core components in two- component signaling and MAPK signaling systems, respectively. These results suggested that both signaling systems were involved in the mode of the action of the fungicides and high-osmorality adaptation in this fungus. To investigate further, we isolated mutants of ChSln1 (HK, whose orthologue is a sole HK in yeast and involved in its osmo-adaptation), ChSsk1 (response regulator: RR) and ChSkn7 (another RR) and compared their phenotypes with a wild- type, dic1 and Bmhog1. ChSln1 disruptants showed no apparent phenotypic changes with respect to osmo-sensitivity. However, both of Chssk1 and Chskn7 mutants showed moderate resistance to the fungicides, and some sensitivity to high osmorality. The phenotype comparisons and phosphorylation analyses of Hog1- MAPK in these mutants revealed that C. heterostrophus has two streams of two-component signaling pathways for the osmotic adaptation and fungicide sensitivity: one is Dic1p-ChSsk1p driving Hog1-MAPK cascade, another is Dic1p-ChSkn7p, and both streams were controlled by Dic1p.


55. Live cell imaging of microtubule dynamics in Neurospora crassa using total internal reflection fluorescence microscopy. Maho Uchida1, Rosa R. Mouriño-Pérez2, Michael Freitag3 and Robert W. Roberson1 1School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA 2Departamento de Microbiología. Centro de Investigación Científica y Educación Superior de Ensenada. Ensenada, B. C. Mexico 3Department of Biochemistry and Biophysics, Oregon State University, Eugene, OR 97331-7305, USA

Total internal reflection fluorescence (TIRF) microscopy is an optical technique that uses evanescent waves to excite fluorophores within 200 nm of the specimen and coverslip interface. This effectively eliminates background fluorescence from other regions of the cell and improves spatial resolution by increasing the signal-to-noise ratio. Furthermore, living cells can be viewed over extended time periods due to low phototoxic effects. Here, we used TIRF microscopy to study cortical microtubule (MT) dynamics in living primary hyphae of Neurospora crassa expressing β-tubulin- GFP. Detection of plus-end MT dynamics was much improved with this approach compared to confocal and widefield fluorescence microscopy methods. Surprisingly, MTs in N. crassa polymerize twice as fast as those reported in Aspergillus nidulans, while MT depolymerization rates in both species were similar. During this study, we observed events of MT fragmentation and fragment motility. These behaviors were compared to those observed in MT-motor mutant strains in order to address the role(s) played by dynein/dynactin and kinesin in MT behavior.


56. Micro and nano morphology of Aspergillus species in response to carbon starvation. Mark R. Marten, Judith Kadarusman, Liming Zhao, Youghyun Kim, Bill Moss Zebulon Jones and David Schaefer2. UMBC, Chemical & Biochemical Engineering, Baltimore, MD 21784, 2Towson Univ., Physics Dept, Towson, MD 21252


We are using Aspergillus species as models to characterize fungal morphological response to carbon starvation. To study micro-morphology we use a parallel-plate flow chamber, with a narrow (50 micron) gap, forcing two dimensional fungal growth. This chamber is perfused with oxygenated growth medium, and mounted on a microscope stage, allowing us to follow morphological development of individual mycelial as a function of time and growth environment. In response to carbon deprivation (i.e., step change, glucose+ to glucose-), A. oryaze enters a lag phase where extention ceases, vacuolation increases, and duration depends on the initial size of the mycelium. When complete, regrowth begins at a relatively constant rate independent of mycelial size. When this behavior is compared to that during rapamycin-induced autophagy we find quantitative differences implying autophagy may comprise only a portion of a more complicated starvation stress response. To study nano-morphology we use an atomic force microscope (AFM). We have developed the ability to use the AFM for testing material properties of fungal cell walls as a function of axial position. We will discuss development of this technique and how we are using it to study changes in A. nidulans morphology during carbon deprivation.


57. Withdrawn


58. A functional analysis of the DopA protein of A. nidulans and A. fumigatus. Holger Hannemann and Bruce Miller. University of Idaho, MMBB. Moscow, Idaho


Aspergillus nidulans (An) is a widely used model organism whereas A. fumigatus (Af) is an airborne pathogen, causing mostly fatal invasive mycoses in immunocompromised patients. Polarized growth is one of the key factors of fungal pathogenesis. DopA, whose homologs are found in fungi, worms and chordata, is a protein involved in polarization of cells. An An strain expressing a temperature sensitive DopA protein (DopAts) displays aberrant cellular morphology of all cell types, asynchronous cell pattern formation and loss of both asexual and sexual reproductive cycles at the restrictive temperature. Successive shortening of the An and Af dopA open reading frames demonstrated that the leucine zippers, located at the C-terminal end of the proteins, serve different functions in these two organisms. DopA-GFP fusions in both An and Af revealed that the proteins are localized to the Golgi and the endosomal membrane networks in both species. Genetic interactions between An dopA1ts and An rasA suggest that DopA modulates signaling through a RasA-mediated signal transduction pathway that controls cell polarity and morphogenesis. Additionally, the N-terminal dopey domain of An DopA interacts physically with BemA, the S. cerevisiae (Sc) Bem3 homolog. Therefore, the interactions between An RasA and An DopA, An BemA and An DopA and signaling interactions between Sc Ras and the Sc Cdc42 regulatory module suggests that the DopA/Dop1 protein plays a central role in integrating signal transduction and mechanisms regulating cell polarity in the fungi.


59. The Aspergillus nidulans snoA inhibitor of cell division associates with the BRDF checkpoint domain of nimODbf4. Steve James, James Barra, Megan Campbell, and Matthew Denholtz. Biology Department, Gettysburg College, Gettysburg, PA. sjames@gettysburg.edu


In Aspergillus nidulans, nimODbf4 and cdc7 encode regulatory and catalytic subunits of the conserved DBF4-dependent kinase (DDK). DDK initiates DNA synthesis by phosphorylating the replicative DNA helicase to trigger DNA unwinding at origins of replication. In addition, DBF4 plays an important role in the DNA damage response. This role is mediated by an N-terminal BRDF motif (BRCT and DBF4 similarity domain), as revealed by mutations in yeast homologs that confer enhanced sensitivity to DNA damage agents and failure to restrain DNA synthesis during genotoxic stress. In Saccharomyces cerevisiae, RAD53/CHK2 kinase is the only checkpoint mediator known to associate directly with the DBF4 BRDF motif. We identified a novel inhibitor of nimODbf4 called snoA (suppressor-of-nimO). Loss of snoA rescues nimO18 ts-lethality and hypomorphic nimO+ expression. Conversely, snoA overexpression confers a dose-dependent, lethal interphase cell cycle arrest in nimO18 cells. Here we report a novel interaction between the nimO BRDF motif and snoA. Using yeast two-hybrid analysis, we demonstrate that a short (~100 amino acid) serine- and proline-rich region in the snoA C-terminus can associate with the nimO BRDF. This novel discovery suggests that snoA may act to regulate normal DNA synthesis or to exert S phase checkpoint control by direct association with the A. nidulans DBF4-dependent kinase. (Supported by NSF-RUI #01-14446 to SJ)


60. Where is calcium in the cell and how does it get there? Barry Bowman, Stephen Abreu, Marija Draskovic and Emma Jean Bowman. Department of MCD Biology, University of California, Santa Cruz, CA 95060 email:bowman@biology.ucsc.edu


Calcium gradients are hypothesized to have a central role in polar growth. However, little is known about the distribution of calcium within fungal cells or the proteins that transport it. By fractionating cells we have found that mitochondria, vacuoles, and unidentified light organelles contain roughly equal amounts of calcium. The Neurospora crassa genome contains at least four P-type ATPases that appear to pump calcium - nca-1, nca-2, nca-3, and pmr-1. In addition seven genes encode transport proteins that may exchange calcium for H+ or Na+, cax being the best characterized. We have characterized nine mutant strains that lack a calcium transporter and have fused several transporters to GFP. The types of transporters and the distribution of calcium are more complex in N. crassa than in S. cerevisiae, the best studied system. Disruption of the cax gene causes the complete loss of calcium from the vacuoles, but does not affect other compartments in N. crassa. Yeasts lack a homolog of the endoplasmic reticulum Ca-ATPase of animal cells (the SERCA ATPase), but N. crassa has a homolog, nca-1. Interestingly, nca-1::GFP localizes largely to the nuclear envelope, suggesting the nuclear envelope forms a major part of the endoplasmic reticulum in fungi. Two of the mutant strains, pmr-1 and one of the homologs of cax, are significantly altered in hyphal morphology. Do they help to generate calcium gradients at the hyphal tip?


61. Withdrawn


62. Withdrawn


63. Two-component response regulators, ChSsk1p and ChSkn7p, additively regulate high-osmolarity adaptation and fungicide sensitivity in Cochliobolus heterostrophus. Kosuke Izumitsu, Akira Yoshimi* and Chihiro Tanaka. Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, JAPAN. *Present adress: New Industry Creation Hatchery Center (NICHe), Tohoku University, Aoba, Sendai 980-8579, JAPAN.


Filamentous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two- component signaling systems. We have characterized the response regulator genes ChSsk1 and ChSkn7 in the Southern corn leaf bright fungus Cochliobolus heterostrophus. Both Chssk1- and Chskn7- disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The ChSsk1/ChSkn7 double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1-type and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase.


64. The role of cAMP-receptor-like genes in Neurospora crassa development. Svetlana Krystofova and Katherine Borkovich, Plant Pathology, University of California Riverside, Riverside, CA 92521, svetlana@ucr.edu


G-protein coupled receptors (GPCRs) are 7-transmembrane proteins that sense a variety of extracellular stimuli, such as light, odor, chemoattractants, peptides, neurotrasmitters, hormones and lipids. Putative GPCRs have been identified in the Neurospora crassa genome sequence. Based on phylogenetic analysis, three of these genes, gpr-1, gpr-2 and gpr-3, belong to a cAMP-receptor-like gene family that includes Arabidopsis GCR1, Dictyostelium crlA-crlC, Cryptococcus neoformans gpr4 and Aspergillus nidulans GprH. Deletion of gpr-1 leads to very specific defects during sexual development (lack of ostioles), while asexual development is not affected. The absence of ostioles in mature perithecia negatively contributes to the ability to release ascospores by an ejection mechanism. Moreover, perithecia are frequently ruptured leading to the release of inner material. The functions of gpr-2 and gpr-3 remain unknown. The expression profiles indicate that these genes are expressed in early developmental stages. Deletion mutants have been isolated and phenotypically analyzed. The possibility of redundancy between gpr-1, gpr-2 and gpr-3 has been investigated. Both genetic and biochemical approaches have been used to identify possible downstream effectors of N. crassa cAMP-receptor-like proteins.


65. Role of cytoplasmic bulk flow and microtubule-related motor proteins in nuclear displacement in Neurospora crassa mature hyphae. Silvia L. Ramos-Garcia1, Rosa R. Mouriño-Pérez1, Michael Freitag2, Salomon Bartnicki-Garcia1. 1Departamento de Microbiologia. Centro de Investigación Científica y Educación Superior de Ensenada. Ensenada B. C. Mexico. rmourino@cicese.mx. 2 Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing. Oregon State University. Corvallis, USA.


Nuclear movement and localization are multifactorial events that have been intensely studied over the last two decades. Here we report the results of a study to evaluate nuclear dynamics in the subapical region of Neurospora hyphae. Nucleus movement may be controlled by many factors, for example, cytoplasmic bulk flow, the cytoskeleton (microtubules and actin microfilaments) and motor proteins (dynein/dynactin complex and conventional kinesin). By confocal microscopy, the nuclear displacement of five strains of Neurospora crassa that express histone H 1 tagged with GFP was compared. We included a control strain, ropy-1, ropy-3, nkin and a ropy-1; nkin double mutant. Additionally, we treated the H1-GFP control strain with benomyl to inhibit microtubule formation and with cytochalasin A to inhibit actin microfilaments. Anterograde and retrograde movement of nuclei were observed in all strains, except the double mutant. The displacement rate of nuclei was highly correlated to hyphal elongation rate. The exception was the nkin mutant and cells treated with cytochalasin A. The shape of nuclei in growing hyphae was different: In the control, almost all nuclei were elongated, while in all other strains varying numbers of spherical nuclei were observed. The distance between the hyphal apex and the first nucleus varied between all strains and treatments: The ropy-1 mutant showed the largest exclusion region and the cytochalasin A-treated cells completely lacked this nucleus exclusion zone. In conclusion, the movement and distribution of nuclei in mature hyphae appear determined by a combination of forces, with cytoplasmic bulk flow being a major determinant. Nevertheless, motor proteins bind nuclei to microtubules and actin microfilaments appear to act as a scaffolding to move all components in unison with the cytoplasm.


66. Characterization of signaling genes ncSCD2,ncSHK1 in Neurospora crassa. Masashi Kawamura, Makoto Kataoka, Makoto Fujimura, and Akihiko Ichiishi. Faculty of Life Sciences, Toyo University, Gunma, Japan.

Schizosaccharomyces pombe has the SCD1 signaling pathway regulated by RAS1. This pathway is necessary for proper morphogenesis such as polarized growth or actin cytoskeleton, and pheromone response. In this pathway, SCD1 that is GDP/GTP exchange factor (GEF) for CDC42 (small GTPase) interacts with SCD2 (scaffolding protein). Then, GTP-bound CDC42 and SHK1 (p-21 activated kinase) also interact with SCD2. This complex transfers phosphate signals from up stream to down stream. Therefore, SCD2 and SHK1 function in the same pathway, both proteins are important factors mediate this pathway. Neurospora crassa also has the homologous of fission yeast SCD2 and SHK1, so we each named these genes ncSCD2 and ncSHK1, respectively. Then, we constructed strains defective in ncSCD2 and ncSHK1 to characterize and analyze these genes function. Both null mutants grew more slowly than wild type strain, and displayed morphological defects that appear to have abnormal branching pattern and frequently septations. Moreover, both null mutants couldn't fertilize. But there were some differences in phenotype between ncSCD2 mutant and ncSHK1 mutant. SHK1 mutant resisted osmotic stress more strongly than ncSCD2 mutant. This data suggests that N.crassa also has the SCD1 signaling pathway, and that ncSCD2 and ncSHK1 function resemble to fission yeast scd2 or shk1. In addition, the fact that SHK1 mutant had resisted high osmotic stress implied possibility that the SCD1 signaling pathway link to the HOG (High-osmolarity glycerol) pathway in N.crassa.


67. Spatially segregated SNARE protein interactions in filamentous fungus. Mari Valkonen1, Markku Saloheimo1, Merja Penttilä1, Nick D. Read2, and Rory R. Duncan3, 1VTT Biotechnology, P.O. Box 1000, FIN-02044 VTT, Finland. 2Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3JH, UK. 3Membrane Biology Group, University of Edinburgh, George Square, EH8 9XD, UK


The machinery for trafficking proteins through the secretory pathway is well conserved in eukaryotes, but remains poorly characterized in filamentous fungi. We describe the isolation of the snc1 and sso1 genes encoding exocytic SNARE proteins from Trichoderma reesei. The encoded SNCI protein can complement Snc protein depletion in S. cerevisiae whilst the T. reesei SSOI protein was unable to complement depletion of its yeast homologues. The localization and interactions of the T. reesei SNARE proteins were studied with advanced fluorescence imaging methods using fluorescent fusions of the SNARE proteins. The SSOI and SNCI proteins co-localized in sterol-independent clusters on the plasma membrane in sub-apical but not apical hyphal regions. The v-SNARE SNCI localized to the apical vesicle cluster within the Spitzenkörper of the growing hyphal tips when expressed under the T. reesei cbh1 promoter. Using fluorescence lifetime imaging microscopy (FLIM) and fluorescence energy transfer (FRET) analysis, we quantified the interactions between these proteins with high spatial resolution in living cells. Our data showed that the site of SNARE complex formation between these proteins is on the plasma membrane of non- growing hyphae in old sub-peripheral regions of the colony, but that there is no interaction between the proteins in growing hyphal tips at the colony margin.


68. A RasGAP protein involved in polarity establishment and maintenance in Aspergillus nidulans. Laura Harispe1,2,3, Lisette Gorfinkiel2, Cecilia Portela2, Miguel A. Peñalva3, and Claudio Scazzocchio1. 1IGM, Univ. Paris-Sud XI. Bat 409 Centre d´Orsay. 91405 Orsay(France). 2F. Ciencias, Univ. de la República. Iguá 4525. 11400 Montevideo(Uruguay) 3CIB, CSIC, Ramiro de Maeztu 9. 28040 Madrid(Spain)


Filamentous fungi represent an extreme example of polarised growth(a). We report on the identification and characterisation of GapA, an A. nidulans RasGAP involved in polarity establishment and maintenance. GapA was identified after serendipitously isolating a partial loss-of-function mutation, designated gapA1, in a genetic screen. Phenotypic characteristics resulting from gapA deletion include compact colony morphology, a marked delay in polarity establishment during conidial germination, impairment of polarised hyphal extension, a conspicuous developmental defect typically manifested by the absence of one layer of sterigmata in the conidiophore and a defect in the otherwise polarised distribution of the actin cystoskeleton. GapA-GFP protein fusion expressed from a gene replacement allel appears localises to hyphal tips and septa. This localisation suggests that a Ras protein(s), whose activity is antagonised by GapA plays a role in the regulation of the actin cytoskeleton at the hyphal tip and that this abnormal regulation underlies the polarity phenotypes associated with gapA loss-of-function. (a)Momany M. (2002),Curr. Opinion in Microbiology, 5:580–585.


69. Optical tweezer micromanipulation of living fungal cells. Graham Wright1, Jochen Arlt2, Wilson Poon2 & Nick Read1. 1Institute of Cell Biology, University of Edinburgh, Edinburgh, UK. 2School of Physics, University of Edinburgh, Edinburgh, UK graham@fungalcell.org


Optical tweezers facilitate the non-invasive micromanipulation of both inert and biological microscopic particles solely by using light. We are using optical tweezers in a range of cell biological applications. They have been used in various experimental live-cell studies to manipulate intracellular organelles, hyphal growth and branching, and whole cells. Optically trapped microbeads have been used for the localized delivery of chemicals and to mechanically stimulate cells. Finally, the effects of optical trapping on fungal cell viability have been assessed.


70. Characterization of radC, the Aspergillus nidulans homolog of RAD52, a key gene for DNA repair by homologous recombination. Michael Lynge Nielsen, Gaëlle Lettier, Jakob Blæsbjerg Nielsen and Uffe Hasbro Mortensen. Technical University of Denmark, BioCentrum-DTU


Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome integrity and failure to repair even a single DNA DSB is lethal as it causes loss of part of a chromosome during cell division. In the yeast, Saccharomyces cerevisiae, Rad52 plays a fundamental role in the repair of DSBs by homologous recombination (HR) and among genes involved in DNA DSB repair and HR deletion of RAD52 produces the most dramatic phenotype. Hence, S. cerevisiae cells lacking Rad52 are impaired in DSB repair and all types of HR including targeted integration of transforming DNA. Orthologs of RAD52 have been identified in other organisms, including Schizosaccharomyces pombe, Aspergillus nidulans, chicken, mouse, and human. However, in contrast to S. cerevisiae, the absence of RAD52 in higher eukaryotes does not result in a severe phenotype, due to the evolution of additional repair pathways and the role of Rad52 in DNA DSB repair and HR remains unclear. To address the role of Rad52 in higher eukaryotes, we have recently constructed a radC (homolog of RAD52) deletion mutant in A. nidulans and performed an initial characterization of the mutant strain. Unlike in S. cerevisiae, where Rad52 is essential for the repair of all types of DNA lesions that require HR, we show that in A. nidulans it is only required for the repair of a subset of these lesions. Hence, in an evolutionary perspective, HR repair in A. nidulans may represent an intermediate state between S. cerevisiae and human and A. nidulans may therefore be a useful model to further the understanding of HR in higher eukaryotes.


71. A new cell type produced by conidia that is involved in sexual reproduction in Neurospora crassa. Hsiao- Che Kuo, Gabriela Roca, Chris Jeffree and Nick D. Read. Institute of Cell Biology, University of Edinburgh, UK


We describe a new cell type, the conidial sex tube (CST), which is produced by macroconidia and arthroconidia of Neurospora crassa. It is morphologically and physiologically distinct from germ tubes and conidial anastomosis tubes (CATs), and under separate genetic control. CSTs are characterized by being long, thin (thinner than germ tubes), straight and unbranched. They contain few nuclei and do not avoid or grow towards each other. The CST surface texture and chemistry is different to that of germ tubes and CATs. CSTs require the presence of protoperithecia of opposite mating type to be induced (germ tube and CAT formation are inhibited under these coniditions). Trichogynes show strong chemotropisms towards CSTs but CSTs do not grow towards trichogynes. On making contact, trichogynes coil around CSTs. Trichogynes are not attracted to or coil around CSTs in mutants not producing sex pheromones when male only and trichogynes are not attracted to CSTs in mutants lacking sex pheromone receptor when female only. Nuclear division is inhibited in the presence of CSTs. CST formation is weakly conidium density dependent in contrast to CAT formation which is strongly conidium density dependent and germ tube formation which is conidium density independent.


72. Organelle organization and behavior during CAT fusion and heterokaryon formation in Neurospora crassa. Gabriela Roca1, Michael B. Freitag2 & Nick D. Read1. 1Inst. of Cell Biology, University of Edinburgh, UK. 2Dept. of Biochemistry and Biophysics, Oregon State University, USA


Conidia of Neurospora crassa form conidial anastomosis tubes (CATs) which are specialized hyphae that are distinct from germ tubes. CAT induction, homing and fusion were analyzed using strains expressing different GFP- or mRFP-fusion proteins, and compatible heterokaryons resulting from the fusion of these strains. Various organelle- selective dyes were also used. Cytoplasm passed through fused CATs prior to microtubules, and were followed by mitochondria and then nuclei. When heterokaryons were generated in which one of the parent homokaryons expressed either H1::GFP or H1::mRFP, it took > 3 h for nuclei in the unlabelled homokaryon to become labeled. Mitosis occured more slowly in ungerminated macroconidia (~ 1.5 h) than in germ tubes (~ 15 min). Mitosis was arrested during CAT homing and nuclei did not enter CATs. We also analysed heterokaryons resulting from CAT fusion between vegetatively incompatible strains differing in mating type. Nuclear immobilization and cell vacuolarization started 6 h after fusion but was not immediately followed by cell death. Heterokaryon incompatibility following CAT fusion is therefore delayed in contrast to the rapid cell death that follows incompatible fusions in mature colonies. This suggests that N. crassa possesses a mechanism to escape from vegetative incompatibility during the early stage of colony establishment.


73. Intracelular Analysis of Neurospora crassa Germlings by Confocal Microscopy. Castro-Longoria E., Araujo-Palomares C. and Riquelme M. ecastro@cicese.mx. Department of Microbiology, CICESE, Km. 107 C. Tij-Ensenada, 22860 Ensenada, B. C., México.


By laser scanning confocal microscopy and using the amphiphilic styril dye FM4-64 and Neurospora crassa wild type strain whose microtubules are labeled with GFP we have analyzed the intracellular changes prior to the appearance of a mature Spk. Observations began at the early stages of spore germination and were carried out until a conspicuous Spk could be observed. Before the Spk appearance, germ tubes ( <150 µm) displayed a uniform distribution of organelles along the length of the cells. Once the germlings reached approximately 66.4 + 4.5 µm in length, a small accumulation of vesicles stained with FM4-64, but not detected by phase-contrast microscopy could be observed at the apex of the cell. When germlings reached more than 150 µm in length visible organelles such as mitochondria and nuclei experienced a displacement towards the subapical region of the cell and a small exclusion zone free of organelles formed at the apex. The position of this exclusion zone within the apex seemed to determine the germling growth direction, which was highly erratic. Few minutes after it first appeared, upon growth of the germling, the exclusion zone started to become occupied by an accumulation of material that gradually concentrated into a light gray body that we describe as an immature Spk, the accumulation of FM4-64 colocalized with the position the immature Spk. Our preliminary observations on microtubule organization during germ tube elongation showed that during the early stages of germination of N. crassa, cytoplasmic microtubules were less abundant, shorter and differently organized when compared to those previously reported in mature vegetative hyphae. At later stages, when a mature Spk was observed, cytoplasmic microtubules were more abundant, longer, and were distributed along the main longitudinal axis of growth of the germ tube. The formation of a mature Spk and a longitudinal and abundant distribution of microtubules coincided with the stabilization of the growth direction of the germling, therefore suggesting that it is at this stage when the transition from germling to vegetative hypha occurs.


74. Reversion Analysis of the Cytoplasmic Dynein Motor: a Trip Around the Ring. Michael Plamann, David Madole, Robert Schnittker, and Elizabeth Wulff. School of Biological Sciences, University of Missouri-Kansas City


Cytoplasmic dynein is a large, microtubule-associated motor complex that facilitates minus-end-directed transport of various cargoes. Dynein heavy chain (DHC) is >4000 residues in length, with the last two-thirds of the heavy chain forming the motor head. Six domains within the dynein motor exhibit varying degrees of homology to the AAA+ superfamily of ATPases. These domains are followed by a distinct C-terminal domain and together form a ring-like structure from which a microtubule-binding domain (MTBD) protrudes. Using a genetic assay, we have isolated over 50 DHC mutants of Neurospora that produce full-length proteins that are defective in function. We have identified DHC point mutations in nearly all domains within the dynein motor head. We have now isolated revertants for a subset of these DHC mutants and have identified the respective intragenic suppressor mutations in >100 revertants. Interestingly, we have found that two mutations within the MTBD are suppressed by mutations in various domains around the ring. These results suggest that the conformational states of the MTBD and motor head are tightly coupled. Most DHC mutations examined to date revert exclusively by intragenic suppression. However, 30 to 90% of the AAA#6 and C- terminal domain DHC revertants contain extragenic suppressors. These results suggest that AAA#6 and C-terminal domain mutations are readily bypassed by mutations in other genes.


75. Endocytosis and polarized growth in Aspergillus nidulans. Araújo-Bazán L., Peñalva M.A & Espeso E.A. CIB, C.S.I.C., Ramiro de Maeztu, 9 28040 Madrid (Spain).


In Aspergillus nidulans polarity maintenance during hyphal growth involves the polarised deposition of secretory vesicles at the extending tip. Cortical actin patches are present along the hyphal tube but strongly predominate in the apical region One third of the endocytic proteins directly regulate actin assembly and/or bind to actin. The unique Abp1 and SlaB orthologue in Aspergillus were found using bioinformatics and being designated as abpA and slaB, respectively. After cDNA sequencing, we determined that AbpA shows strong similarity with Abp1p having one conserved cofilin domain and two, instead of one, SH3 domains. AbpA localises to highly motile and transient peripheral punctuate structures overlapping with actin patches. SlaB also localises to peripheral patches, which are markedly more abundant and cortical than those of AbpA but their motility is notably restricted as compared to AbpA patches. Similar subcellular localisation has been determined for actin and the amphiphysin orthologue AnRvs167. AbpA patches show random distribution during the isotropic growth phase preceding polarity establishment, but polarise as soon as a germtube primordium emerges from the swelled conidiospore. Thus, while endocytosis can occur along hyphae, the apical predominance and spatial organization of these endocytic machinery proteins as a slightly subapical ring strongly suggest that tight spatial coupling of secretion and compensatory endocytosis underlies hyphal growth.



Population and Evolutionary Biology


76. Recombination hotspots flank the Cryptococcus mating-type locus: implications for the evolution of a fungal sex chromosome. Yen-Ping Hsueh, Alexander Idnurm, and Joseph Heitman Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina


Recombination increases dramatically during meiosis to promote genetic exchange and generate recombinant progeny. Interestingly, meiotic recombination is unevenly distributed throughout genomes and as a consequence genetic and physical map distances do not have a simple linear relationship. Recombination hotspots and coldspots have been described in many organisms and often reflect global features of chromosome structure. In particular, recombination frequencies are often distorted within or outside sex determining regions of the genome. Here we report that recombination is elevated adjacent to the mating-type locus (MAT) in the pathogenic basidiomycete Cryptococcus neoformans. Among fungi, C. neoformans has an unusually large MAT locus, and recombination is suppressed between the two >100 kb mating-type specific alleles. When genetic markers were introduced at defined physical distances from MAT, we found the meiotic recombination frequency to be ~20% between MAT and a flanking marker at 5, 10, 50 or 100 kb from the right border. As a result, the physical/genetic map ratio in the regions adjacent to MAT is distorted ~10- to 50- fold compared to the genome-wide average. Moreover, recombination frequently occurred on both sides of MAT and negative interference between crossovers was observed. Sequence analysis revealed a correlation between high G + C content and these hotspot regions. We hypothesize that the presence of recombinational activators may have driven several key events during the assembly and reshaping of the MAT locus, and may have played similar roles in the origins of both metabolic and biosynthetic gene clusters. Our findings suggest that during meiosis the MAT locus may be exchanged onto different genetic backgrounds and therefore have broad evolutionary implications with respect to mating-type switching in both model and and pathogenic yeasts.


77. Survey of mating-type (MAT) gene distribution in the Penicillia. Carly Eagle1, Rob A. Samson2, Paul S. Dyer1 1School of Biology, University Park, University of Nottingham, Nottingham, NG7 2RD 2Fungal Biodiversity Centre, CBS, P.O. Box 85167, 3508 Utrecht, The Netherlands sbxcee@nottingham.ac.uk


The genus Penicillium contains various species that are important in medicine, industry and food spoilage. This genus is polyphyletic, containing four subgenera; Biverticillium, Furcatum, Aspergilloides and Penicillium. Studies are underway to investigate the genetic basis of reproductive mode in the Penicillia, initially involving a screen of mating-type (MAT) genes in asexual and sexual taxa. Penicillium marneffei has previously been shown to contain either a MAT1-1 or MAT1-2 locus. This species is classified within the subgenus Biverticillium which is closely related to the sexual genus Talaromyces. The focus of current work is the subgenus Penicillium which contains the asexual species Penicillium chrysogenum (produces penicillin) and P. griseofulvum (produces griseofulvin), and is closely related to the sexual genus Eupencillium. Using a degenerate PCR approach, MAT1-1 and MAT1-2 loci have been amplified and sequenced from a number of species belonging to this subgenera.


78. Reproductive strategy and genetic polymorphism in Pleurotus pulmonarius populations in Russia. Alla Shnyreva. Department of Mycology and Algology, Moscow State University, Moscow, Russia, e-mail: shnyreva@herba.msu.ru


The genetic structure of populations of P. pulmonarius was inferred from variation among 157 individuals at 14 polymorphic allozyme loci and RAPD-profiles to determine the extent of geographic differentiation in this widespread mushroom species in Central Russia. Population genetic analysis was carried out by employing homokaryotic and dikaryotic strains (parents – offspring). To define interbreeding fungal populations, crosses between homokaryons were performed. Genetic exchange was shown to be not only limited to the sexual cycle and to occur in heterokaryon- homokaryon mating (he-ho-mating). The majority of the genetic variation was contained within populations; however, considerable genetic differentiation was observed among populations from three distant regions (FST = 0.750). The low level of inbreeding (FIS = 0.018) suggested that the populations were panmictic. The most common type of mating events was confirmed to be a spore-mediated outcrossing reflected in spatial distribution of mating type factors. However, gene flow between geographic localities was restricted. Clustering analysis demonstrated that genetic distance was correlated with geographic distance and that a large component of the genetic variation was due to allele frequency differences among populations. Population structure of P.pulmonarius will be discussed with a special reference to sexual reproduction, clonality, and outcrossing events. The research was supported by RFBR grant 06- 04-08161.


79. Watching crystal balls: Asexual life-cycles and morphology of cultured mesomycetozoans. Wyth Marshall1, Gail Celio2, Mahajabeen Padamsee2, David McLaughlin2, Mary Berbee1. 1Department of Botany, University of British Columbia, Vancouver, B.C. 2Department of Plant Science, University of Minnesota, St. Paul, MN. WythMarshall@Yahoo.ca

Several clades of unicellular organisms diverged near the animal/fungal divide. Of these, the Mesomycetozoans or Ichthyosporids are perhaps the most diverse and are comprised of approximately 70 spp. of animal commensuals and parasites. Mesomycetozoans have been isolated from animals ranging from arthropods and molluscs to vertebrates but very few have been propagated in culture. We have successfully cultured five new species isolated from the gut contents of various marine invertebrates. We characterized the species using ribosomal DNA sequences and have studied their life- cycles and ultrastructure using microscopy. We documented and photographed four mechanisms of dispersal and the asexual life-cycles of three species. One species, which we plan to name ‘Creolimax fragrantissima,’ disperses via numerous slug-shaped amoebae and produces multicellular clusters. Further details of amoeboid behaviour, phylogenetic placement and ultrastructure of this new species will be presented. Understanding of the biology of these closely related fungus-like protists is integral to our understanding of how fungus-like forms evolved and why the true fungi became such a successful multicellular kingdom.


80. Evolutionary Aspects of the Alkaloid Biosynthesis in Claviceps sp. Nicole Lorenz1, Caroline Machado2, Ella Konnova2, Chris Schardl2, Paul Tudzynski1. 1Institute of Botany, WWU Muenster, Germany. 2Department of Plant Pathology, University of Kentucky, USA


The genus Claviceps consists of many phytopathogenic ascomycets producing ergot alkaloids during a specific stage of their lifecycle. Ergot alkaloids show high homology to some neurotransmitters and bind to the same receptors in the CNS leading to an application in a variety of clinical conditions. The genes encoding for the enzymes needed for alkaloid production are organized in a cluster. To study the evolution of the alkaloid biosynthesis the work focused on a comparison of the cluster sequences of three Claviceps species producing different kinds of alkaloids as endproducts of their pathways. While C. purpurea is able to synthesize ergopeptides the other species fulfill only a truncated pathway leading to an accumulation of species-specific intermediates. C. fusiformis shows a corresponding cluster including the genes for the early steps within the pathway. Two additional genes for further synthesis are present but complementation of the corresponding knock-out mutants of C. purpurea failed (the genes seem to be not functional due to a rearrangement event) whereas the implementation of a functional gene of C. purpurea results in a modified alkaloid spectrum of C. fusiformis. C. hirtella synthesizes ergometrine, a simple ergopeptide and should only lack the large NRPS genes. First sequence analysis confirmed these assumptions.


81. Rapid evolution by genetic decay of mating type (mat) genes in homothallic Neurospora. Lotta Wik, Magnus Karlsson, Hanna Johannesson Dept of Evolutionary Biology, Uppsala University Hanna.Johannesson@ebc.uu.se


In filamentous ascomycetes, three basic sexual reproductive modes are present: heterothallism, homothallism and pseudohomothallism. The mat locus is the sole determinant of the mating type in Neurospora, and is the master-regulator of sexual reproduction in this genus. We sequenced the four mat-genes from Neurospora taxa with different reproductive systems, and used likelihood-based methods to reveal that two mat-genes (mat a-1 and mat A-2) evolve under positive selection in heterothallic taxa, while all four mat-genes in homothallic taxa are subjected to an evolutionary decay. In all four genes a lower ratio of non-synonymous to synonymous substitutions (dN/dS) was found in heterothallic than in homothallic taxa. This is due to a higher proportion of conserved sites in the heterothallic dataset and a higher proportion of neutrally evolving sites among the homothallic species. While the heterothallic taxa show a codon or domain specific selective pressure, the codons with elevated dN/dS were more evenly distributed along the genes in the homothallic dataset. This indication of mat-gene degeneration is further supported by the observations of a disrupted ORF of mat a-1, mat A-2 and mat A-3 in homothallic taxa, and the finding of mat A-2 being silenced in N. africana and N. galapagosensis, thus becoming a pseudogene in these two taxa. Our result indicate that mat-genes evolve in an adaptive manner in heterothallic taxa, while in homothallic taxa they are not needed to be functionally conserved.


82. Many Globally Isolated AD Hybrid Strains of Cryptococcus neoformans Originated in Africa. Anastasia P. Litvintseva, Irka Templeton, Joseph Heitman, and Thomas G. Mitchell. Duke University Medical Center, Durham, USA


AD strains of C. neoformans are hybrids between C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D). While most isolates of serotype A and D are haploid, AD strains are diploid or aneuploid, contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type; however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele. Previously we described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by AFLP, and discovered that AD hybrid strains possessing the rare serotype A MATa allele are clonally related and cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrids are more resistant to UV radiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in Botswana from a cross between strains of serotype A and D; (ii) This fusion produced strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to propagate globally.


83. Horizontal gene transfer as the source of extra genes in Aspergillus oryzae. Nora Khaldi*, Kenneth H. Wolfe. khaldin@tcd.ie Smurfit Institute of Genetics. University of Dublin Trinity College Dublin 2. Ireland.

Although horizontal gene transfer (HGT) is a major aspect of prokaryotic evolution and is becoming increasingly documented between prokaryotes and eukaryotes, very few cases of HGT of nuclear genes from eukaryote to eukaryote have been documented so far. Several obstacles impede the unequivocal demonstration of HGT. The detection of foreign genes is facilitated if the recipient and donor genomes are from two different kingdoms, but if they are relatively closely related then observations suggestive of HGT may be equally consistent with alternative scenarios of gene duplication and gene loss. Aspergillus oryzae is a filamentous fungus that has a large gene complement compared to its annotated relatives. Here we test three alternative explanations for the existence of these extra genes in Aspergillus oryzae: recent gene duplication, whole genome duplication, and HGT. We show using gene location, evolutionary rate, and phylogeny based methods that HGT took place from the close sister subphylum Sordariomycetes into an ancestor of Aspergillus oryzae. Finally, we investigate the role of the transferred genes and show that transfer played a role in enriching Aspergillus oryzae's gene repertoire, specifically by expanding its hydrolytic enzyme gene complement.


84. The influence of CAT fusion on colony establishment in Neurospora crassa. M. Gabriela Roca 1*, Natalia Angarita-Jaimes2, Catherine Towers2, David Towers2 and Nick Read1 1 Institute of Cell Biology, University of Edinburgh, UK 2 School of Mechanical Engineering, University of Leeds, UK * Gabriela.Roca@ed.ac.uk


Hyphal network formation is a key feature of filamentous fungi. We are using Neurospora crassa as a model to study hyphal networks which are formed during colony establishment by means of conidial anastomosis tube (CAT) fusion. The role of CAT fusion in colony establishment is presently unclear. We have addressed this issue by recording germling growth and nuclear dynamics during the first 10 h following conidial germination. Nuclei were labeled with H1::GFP and imaged by confocal microscopy. Advanced image processing techniques were used to track nuclear motion and determine germ tube growth rates. Nuclei were identified by a particle mask correlation method and then tracked with a particle tracking velocimetry (PTV) algorithm. Germling growth was monitored by means of a level set algorithm for centerline extraction. Germ tubes within microcolonies derived from single conidia grew significantly more slowly ( < 0.03 ìm/sec) than colonies formed from conidia that had fused via CATs ( < 0.085 ìm/sec). Nuclei in microcolonies derived from fused conidia achieved higher velocities ( < 1.2 ìm/sec) than those formed from single unfused conidia (< 0.8 ìm/sec). Our initial results provide evidence for cooperative behavior between fused conidial germlings; CAT fusion seems to stimulate germ tube growth and actively promote nuclear mixing.


85. Molecular characterization of the Fusarium graminearum species complex in Japan. H. Suga1, G.W. Karugia1, T. Ward2, L.R. Gale3, K. Tomimura4, T. Nakajima4, A. Miyasaka4, S. Koizumi4, K. Kageyama1, and M. Hyakumachi1. 1Gifu University, Gifu, Japan. 2Agricultural Research Service, USDA, Peoria, USA. 3University of Minnesota, St. Paul, USA. 4National Agricultural Research Center, Tsukuba Japan.


We collected 298 strains of the the Fusarium graminearum species complex from wheat or barley from 2001 to 2004 in Japan and investigated species and trichothecene chemotype compositions. Species-diagnostic PCR-RFLP revealed the presence and differential distribution of Fusarium graminearum s. str. and Fusarium asiaticum in Japan. F. graminearum s. str. is predominant in the north, while F. asiaticum is predominant in southern regions. In the Tohoku area, distinct co-occurrence of these species was observed. Chemotyping by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by PCR-RFLP markers, as interspecies hybridization of F. graminearum s. str. and F. asiaticum was previously achieved in vitro. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.


86. Withdrawn


87. Phytophthora database: An integrated resource for detecting, monitoring, and managing Phytophthora diseases. Seogchan Kang1, Jaime Blair1, Dave Geiser1, Izabela Makalowska1, Sook-Young Park1, Bongsoo Park1, Mike Coffey2, Kelly Ivors3, Yong-Hwan Lee4, Jong-Seon Park4, and Frank Martin5. 1Penn State, University Park, PA. 2UC, Riverside, CA. 3NCSU, Raleigh, NC. 4Seoul National Univ., Seoul, Korea. 5USDA-ARS, Salinas, CA.


High virulence of Phytophthora species and their ability to spread rapidly establishes Phytophthora as one of the most important groups of plant pathogens. The ability to accurately and rapidly identify the causal agent of a disease is crucial for developing effective regulatory and management strategies against Phytophthora. The Phytophthora database project ( Link here ) was initiated to enhance our capability of rapid detection and diagnosis of Phytophthora spp. by archiving known genotypic and phenotypic diversity in a highly integrative database. To compliment the species morphological descriptions and serve as a molecular reference for isolate identification, ~2000 accessions representing 71 morphological species maintained in the WPC Genetic Resource Collection have been genetically characterized. A seven-locus phylogeny of the whole genus supports the division of Phytophthora into approximately eight major groups. In addition to these nuclear genes, four mitochondrially encoded genes are being sequenced from the same isolates to construct a mitochondrially based phylogenetic framework. This project is on-going; sequence and species data are continually being deposited, and new data analysis and visualization tools are being developed to increase utility and breadth of this database. An overview of database functions will be presented.


88. A novel kind of cooperation facilitates conidial germination in Neurospora crassa. Franck Richard 1, N. Louise Glass 2, and Anne Pringle 1. 1 Harvard University, Organismic and Evolutionary Biology, 16 Divinity Avenue, Cambridge MA 02138. 2 University of California Berkeley, Plant and Microbial Biology, 111 Koshland Hall, Berkeley CA 94720 pringle@oeb.harvard.edu

The cooperative behaviors of filamentous fungi have long fascinated mycologists. In these experiments we use micromanipulations of individual conidia to demonstrate that time to germination is significantly reduced when two or more conidia are grouped. Single conidia take longer to germinate and both the length of the germ tube, as well as the perimeter of a colony, are significantly smaller as compared to germ tubes and colony perimeters of two or more conidia. A similar result is found with two wildtype strains as well as the soft mutant (which lacks the ability to fuse), proving that the pattern is a general one and is likely to be controlled by early signals working independently of the fusion process. Experiments with isolates from geographically distant locations suggest that this kind of cooperation functions even when isolates are genetically distinct, but whether one isolate is co-opting the mechanism for its own benefit remains to be tested.


89. Interspecific transfer of host-specific toxin genes in Stagonospora nodorum. Richard P Oliver1, Peter S. Solomon1, James Hane1, Eva H. Stukenbrock2, Bruce A McDonald2, Zhaohiu Liu3, Justin D Faris3, Timothy L Friesen3. 1ACNFP, Murdoch University, Australia 2ETH, Zurich, Switzerland 3USDA and NDSU, ND


The host-specific toxin ToxA, produced by P. tritici-repentis, confers virulence on wheat genotypes carrying Tsn1. The Stagonospora nodorum gene, SnToxA was identified from a reannotation of the genome sequence and is nearly identical to PtrToxA. Disruption of SnToxA reduced disease on wheat lines carrying Tsn1. ToxA genes from a large isolate collection from P. tritici-repentis were identical but those from S. nodorum were highly variable. P. tritici-repentis was first identified in 1902 but it was not until 1941 that typical tan spot symptoms were first described. Nowadays, P. tritici- repentis is a regular and abundant pathogen. Our evidence strongly suggests that ToxA was horizontally transferred to P. tritici-repentis some time prior to 1941. These new forms appear to have rapidly spread around the world, probably in grain shipments (Nature Genetics 38 953-956). Our work indicates that S. nodorum produces several other toxins. The presence of multiple host-specific toxins and their transfer between species raises many interesting evolutionary questions; 1. How does toxin possession and expression affect the fecundity of fungal strains carrying the toxins? 2. How can we explain the expression of toxin receptor genes? 3.What are the mechanisms of sequence diversification in toxin genes?


90. Explosively launched ascospores are shaped to minimize drag. Marcus Roper, Michael P. Brenner and Anne Pringle, Harvard University Cambridge MA 02138, pringle@oeb.harvard.edu


Many species of ascomycete fungi have a sexual phase in which ascospores are forcibly ejected clear of the fruiting body. Reproductive success is dependent upon the spores germinating away from the parent fungus, and thus the spores must be ejected at very large launch speeds in order to pass through the layer of still air surrounding the fruiting body, into the vigorous air currents beyond. To do this these fungi have evolved mechanisms for applying initial accelerations that are unmatched in the plant or animal kingdoms. Spores are tiny, fast moving objects, so the distance that they can travel in still air is severely limited by fluid drag. We present evidence that drag minimisation is a primary determinant of spore shape. This results leads to several predictions, including upper and lower bounds upon spore size (spores are predicted to be no smaller than 1.8 micrometers and no larger than 30 micrometers) and a constant launch speed of 2 m/s across the phylum.


91. Sexual mating of B. cinerea (fuckeliana) illustrates PRP8 intein HEG activity. Annika A.M. Bokor and Russell T.M. Poulter, University of Otago, Dunedin, New Zealand


Inteins are proteins that are removed post-translationally from a host protein. This is an autocatalytic splicing process necessary for the maturation of the host protein. Inteins typically contain not only the necessary splicing domains but also a homing endonuclease (HEG). The HEG enables the intein encoding sequence to spread through the gene pool of a species and potentially to also undergo horizontal transmission to other species. In a diploid, heterozygous for the presence/absence of the intein gene, the HEG recognises a 30-50bp target sequence at the ‘empty’ allelic site. It introduces a double-stranded DNA break at this target. This double-strand break triggers the host’s recombinational repair system, which in the repair process copies the full-length intein into the target site. Our group has discovered several nuclear full-length inteins (located in the critical PRP8 gene) in species including the human fungal pathogens Aspergillus fumigatus, Histoplasma capsulatum and the plant pathogen Botrytis cinerea. To demonstrate intein replication a species must have a sexual cycle and there must be polymorphism for presence/absence of the intein in the gene pool of the species. We have demonstrated that B. cinerea is polymorphic for presence/absence of the PRP8 intein. Characterisation of HEG activity in B. cinerea sexual cycle will be presented. This is the first mycelial system in which the intein replication cycle has been demonstrated.


92. Global molecular epidemiology of Cryptococcus gattii VGII isolates traces the origine of the Vancouver Island outbreak to Latin American. Wieland Meyer 1, Sirada Kaocharoen 1, Luciana Trills 2, Alexandro Jover-Botella 1, Patricia Escandón 3, Elizabeth Castañeda 3, Clement Tsui 1, Ferry Hagen 4, and Teun Boekhout 4. 1 Molecular Mycology Research Laboratory, CIDM at Westmead Hospital, Westmead Millennium Institute, The University of Sydney Western Clinical School, Westmead, NSW, Australia. 2 Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil. 3 Grupo de Microbiología, Instituto Nacional de Salud, Bogotá, Colombia. 4 Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

The basidiomycetous yeast Cryptococcus gattii causes life-threatening disease mainly in immunocompetent patients. Four molecular types have been identified: VGI/AFLP4-VGIV/AFL7. Two subtypes of molecular type VGII/AFLP6 have emerged in 2000 as a primary pathogen on Vancouver Island, Canada, causing a large scale cryptococcosis outbreak in humans and animals: VGIIa/AFLP6A, the major and more virulent and VGIIb/AFLP6B, the minor and less virulent genotype. A global molecular epidemiological study of more than 160 VGII strains recovered since 1986 was conducted using PCR fingerprinting, AFLP and MLST analysis (8 polymorphic loci: ACT1, CAP59, IDE, IGS, LAC1, PLB1, SXI1alpha, URA5). VGIIa is also present in Colombia, Brazil, Venezuela, Argentina, USA, Thailand and Greece. Australian VGII isolates clustered with VGIIb. Brazilian VGIIa isolates are identical with the Vancouver Island outbreak strains. Colombian VGIIa isolates are closely related but not identical to the Vancouver Island outbreak strains and are avirulent. The vast majority of Colombian isolates are MATa compared with the Vancouver Island outbreak isolates (all MATalpha). In vitro mating experiments showed that the avirulent Colombian MATa strains mate with the virulent VGII MATalpha strains from Brazil and Vancouver. The fact that there are VGIIa and VGIIb isolates recovered as early as in 1986 in South America indicates that these genotypes may have been present for a long time in the Americas rather than being a result of a recent recombination event between a less virulent genotype introduced to North America from Australia and an unknown mating partner as suggested by Farser et al. (Nature 437,2005).


93. Evolution and phylogeny of Candida species inferred from multigene analyses. Clement Tsui 1, Heide-Marie Daniel 3, Wieland Meyer1,2. 1Molecular Mycology Research Laboratory, Center for Infectious Diseases and Microbiology, Westmead Hospital, Westmead, Australia; email: clementsui@gmail.com 2Western Clinical School, Department of Medicine, The University of Sydney, Australia; 3BCCM/MUCL culture collection, Université catholique de Louvain, Unite de Microbiologié, Louvain-la- Neuve, Belgium.


Candida species are ubiquitous human pathogens, plant endophytes and insect symbionts. Phylogenies based on rDNA and actin sequences suggested that the genus is not monophyletic, and did not clearly resolved the relationships among related genera and species. Protein coding genes are useful to resolve taxonomic positions among a broad range of fungi. We selected over 70 taxa from Candida and allied genera, and investigated their phylogenetic relationships using nuclear sequences of the largest subunit (RPB1) and second largest subunit (RPB2) of RNA polymerase II gene, actin, mitochondrial cytochrome oxidase subunit 2 (COX2) gene, and LSU rDNA. The DNA sequences were subjected to maximum parsimony and Bayesian analyses. Four major phylogenetic groups were recognized according to Bayesian probabilities. Group A contained six pathogenic species of Candida, seemed to derive from non-pathogenic species, while Group B contained species of Clavispora, Metschinikowia, and Pichia guilliermondii. Species of Debaryomyces were related to groups A and B but formed an independent clade. Group C consisted species in the Saccharomycetaceae, in sibling relationship with a clade containing Pichia jadinii. Pichia fermentans and other environmental species concentrated in Group D. The phylogenetic relationships of species and the evolution of codon usage would be discussed.


94. Phylogeny of clusters of genes with predicted roles in biosynthesis of epipolythiodioxopiperazine toxins in ascomycetes. Nicola Patron1, Anton Cozijnsen1, Bill Nierman2, Ross Waller1 and Barbara Howlett1. 1School of Botany, the University of Melbourne, Australia; 2 The Institute for Genomic Research, USA


Epipolythiodioxopiperazines (ETPs) are toxins made by phylogenetically diverse filamentous fungi. Ten clustered genes are responsible for biosynthesis of the core ETP moiety. Clusters with most or all the core genes are present in Magnaporthe grisea, Chaetomium globosum, Fusarium graminaerum, Trichoderma virens, T.reesei, Leptosphaeria maculans, Penicillium lilacinoechinulatum, Aspergillus terreus, A.clavatus, A.flavus, A.oryzae, A.fumigatus, Neosartorya fischeri. Each of six cluster genes examined is closely related phylogenetically to non-cluster paralogues within filamentous fungi. Similar relationships were inferred for all six genes, each one sharing a common ancestry to the exclusion of non-cluster paralogues, excepting cluster genes from C.globosum and F.graminarium. This suggests that most ETP gene clusters have a common origin and that the cluster was inherited relatively intact, rather than assembling independently in the different ascomycete lineages. Internal topologies of clusters do not reflect phylogeny of ascomycete lineages. Gene clusters fall into three phylogenetic classes, which do not necessarily reflect the structures of toxins produced. The disjunct heredity of these clusters may arise from multiple independent losses of cluster class or lateral gene transfer of clusters between lineages.


95. Horizontal Gene Transfer and Recombination in Histone 3 Gene Evolution of Alternaria. Soon Gyu Hong, and Barry M. Pryor. Korea Polar Research Institute, Incheon, Korea.


Evolution of H3 gene was analyzed by comparing H3 gene phylogeny with reference phylogeny constructed by alt a 1, gpd, and ITS sequences in Alternaria and related species. Several H3 gene types defined by intron presence and absence were recovered and distribution of each gene type on the phylogeny was very complex. We postulated four alternative hypotheses to explain the findings: 1) maintenance of different types of H3 gene in a strain with one of them as a major copy and the others as minor copies; 2) repeated insertion and deletion of introns during evolution of H3 genes; 3) maintenance of multiple copy of the gene in the ancestral species and independent loss of different type of genes in each descendent; 4) exchange of genetic materials among strains leading to horizontal transfer of the gene and recombination between different copies of the gene to achieve concerted evolution. Among them, the fourth hypothesis of horizontal gene transfer and recombination was chosen as the most plausible explanation for the evolution of H3 gene in Alternaria and related species. The first hypothesis of maintaining multiple types of H3 gene could explain the multiple PCR bands of radicina species-group and Stemphylium group.


96. Extent of Genetic Diversity in a Single Fruiting Body of Parmelia laevior. Jin Sung Lee and Soon Gyu Hong. Korea Polar Research Institute, Incheon Korea.

There have been debates in defining species boundary in fungi with emphasis on phenotypic or genotypic characteristics. To test the extent of genetic diversity in lichen-forming fungus, Parmelia laevior, sequence variations of three genes (ITS, RPBII and mtSSU rDNA) were analyzed from 20 spore-germinated cultures from an identical fruiting body. All of the isolates contained identical mtSSU rDNA sequences, whereas two types of ITS and RPBII sequences were observed. Two types of ITS sequences showed 7 nucleotide difference among 639 bases (98.9% similarity). Two types of RPBII sequences showed 16 nucleotide difference among 1192 bases (98.83% similarity). To discriminate mixed cultures from homogeneous single spore cultures, two types of single-copy RPBII gene was specifically detected by oligonucleotide ligation assay (OLA) based upon the single nucleotide polymorphism. As a result, 13 isolates turned out as mixed cultures by showing both types of RPBII sequences. To detect incomplete concerted evolution of ITS sequences, OLA was applied for ITS sequences and it was found that all of the single-spore isolates had only one type of ITS sequences. RPBII and ITS alleles were segregated together in all of the single-spore isolates, implying that they are closely linked on the chromosome. From the identical sequences of mtSSU rDNA and high variation of ITS and RPBII sequences, it is concluded that boundary of species in Parmalia laevior is broader than have been expected from previous researches.


97. The evolution of mating type idiomorphs in the genus Mycosphaerella. Mahdi Arzanlou1,2, Lute-Harm Zwiers1, and Pedro W. Crous1,2. 1Evolutionary Phytopathology Group, CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. 2Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands. arzanlou@cbs.knaw.nl


The genus Mycosphaerella is one of the largest genera of ascomycetes, comprising several thousand species. Despite the diverse ecological habitats inhabited by species of Mycosphaerella, many species are specialized plant pathogens, occurring on a wide range of hosts ranging from monocots, dicots to even gymnosperms. The fact that different Mycosphaerella species can co-occur on a single host and even within a single lesion, as well as their ability to jump between hosts, makes Mycosphaerella a good model to study the (co) evolution of closely related species. The mating strategy for the majority of Mycosphaerella species is yet unknown; for a few species a heterothallic or homothallic mating system has been observed. However, for the majority of the Mycosphaerella species the understanding of sex remains a puzzle. A large number of species in this genus are known only from their teleomorphs, while for many other species no Mycosphaerella state has been discovered, and only the anamorphic stage is known. In order to examine the importance of sexual reproduction in evolution and speciation of Mycosphaerella species occurring on banana, we analyzed the structure of the mating type idiomorphs of several additional Mycosphaerella species. Analysis of the structure of the mating type genes from homothallic and heterothallic members of this genus showed unique features in the mating type loci. In all mating type idiomorphs additional open reading frames (“ORFs”) can be distinguished which are seemingly unique to the Mycosphaerellaceae. Furthermore, the obtained data suggest that heterothallism is in most cases the ancestral state, and evidence exists that that some heterothallic species have evolved from a homothallic ancestor.


98. Molecular Analysis and Evolution of Gibberellin Biosynthetic Gene Clusters in Fungi. Christiane Bömke and Bettina Tudzynski. WWU Münster, Institut für Botanik, Schlossgarten 3, Germany


Although gibberellins (GAs) are ubiquitous diterpenoid plant hormones, they were first identified as secondary metabolites of Fusarium fujikuroi. Today it is known that they are also produced by several bacteria and some other fungi. Since a horizontal gene transfer of the GA genes from plants to fungi can be excluded due to fundamental differences at the chemical (pathway), biochemical (enzymes) and genetic level, we are interested in the origin of the fungal GA cluster genes. To elucidate this question, we focus on the distantly related GA producing fungus Sphaceloma manihoticola. The spectrum of the GAs present suggests that the GA biosynthesis in Sphaceloma follows the pathway known for Fusarium. However, in contrast to F. fujikuroi, the biosynthesis does not go to the end product GA3 but stops two steps earlier with GA4. The assumption that the responsible genes (P450-3 and des) are missing was confirmed after cloning of the S. manihoticola GA gene cluster: homologues of P450-3 and des which flank the gene cluster in Fusarium are missing in this cluster. The order of the other five genes is similar but not identical to F. fujikuroi due to gene inversions. The functional analysis of the genes was done by a combination of knock-outs and complementation of the corresponding F. fujikuroi-mutants. A heterologous gene expression of Ff-P450-3 and Ff-des will show if the complete pathway can be restored.


99. An Unprecedented Shift in the Composition of Fusarium Head Blight Pathogens in North America due to the Rapid Spread of a Highly Toxigenic Population of Fusarium graminearum. Todd J. Ward1, Randall M. Clear2, Alejandro P. Rooney1, Kerry O’Donnell1, Don Gaba2, Jeannie Gilbert3, Susan Patrick2, and David E. Starkey4. 1Microbial Genomics and Bioprocessing Research Unit, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois, USA. 2Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada. 3Agriculture and Agri-Food Canada, Winnipeg, Manitoba, Canada. 4University of Central Arkansas, Conway, Arkansas, USA.


To date, a fraction of the combined species and chemotype diversity of Fusarium head blight (FHB) pathogens is present in North America. However, our active surveillance with a unique multilocus genotyping assay revealed for the first time that 3ADON-producing isolates of F. graminearum are prevalent in North America. In addition, we identified a dramatic East-West (3ADON-15ADON) chemotype cline in Canada, and documented a recent and unprecedented shift in FHB pathogen composition in North America by demonstrating that the 3ADON chemotype frequency in Canadian prairie provinces increased 14-fold between 1998 and 2004. Analyses of population structure based on nine VNTR markers demonstrated that 3ADON and 15ADON isolates from western Canada represent distinct populations (FST = 0.4, P < 0.001) with low levels of gene flow evidenced by the identification of a small number of admixed isolates. On average, isolates from the 3ADON population produce significantly (P < 0.001) higher levels of trichothecene toxins, grow faster, and produce more spores in vitro than isolates from the 15ADON population. Combined with the rapid and significant shift in chemotype frequency that we have documented, these data suggest that a recently introduced or emergent population of F. graminearum with a 3ADON chemotype has a selective advantage and is rapidly changing the FHB landscape in North America. In addition, the demonstration that highly toxigenic FHB pathogens with a novel trichothecene profile have rapidly displaced less toxigenic isolates has significant implications for food safety and disease control efforts.


100. Profiling Fungal Diversity in Green Lake. Heather Michael, Michael L. McCormick, Jinnie M. Garrett. Department of Biology, Hamilton College, Clinton, NY, USA. jgarrett@hamilton.edu

Exhibiting permanent geochemical stratification, Green Lake near Fayetteville New York, is an example of a meromictic lake. The lake’s physical environment is structured like a layer-cake: two chemically distinct ecozones— an upper oxic mixolimnion and lower anoxic monimolimnion— remain separated by a stable redox transition zone, or “chemocline”. Previous diversity studies have been conducted at other meromictic lakes. While related research has focused on bacterial and archaeal assemblages; few, if any, attempts have been made to characterize fungal diversity in meromictic environments. The goal of the present study is to determine how fungal diversity in Green Lake varies with depth and the associated changes in water chemistry. Our geochemical analysis revealed distinct stratification, with dramatic shifts in pH, temperature, oxidation-reduction potential, and specific conductance occurring within the chemocline. We expect that niche differentiation and community structure will exhibit vertical stratification in association these changes in water chemistry. In June of 2005, Microbes were collected from Green Lake via sterile syringes deployed at 23 depths spanning the chemocline. Sampling intervals varied from 5 m in chemically uniform regions of the mixolimnion and monimolimnion to 25 cm across the chemocline. A 500bp region of fungal 18s rDNA was selectively amplified using taxon-specific primers. We are currently in the process of using Denaturing Gradient Gel Electrophroesis (DGGE) to profile fungal diversity from water samples collected at various depths. Following DGGE, unique 18s rDNA amplicons will be sequenced to determine community composition. Preliminary DGGE of a chemocline community indicates a richness of 8 to 12 fungal species. Depth-related trends in fungal diversity will be presented.


101. Structural and Functional Evolution of Non-Ribosomal Peptide Synthetases. Kathryn E. Bushley1 and B. Gillian Turgeon1. 1Department of Plant Pathology, Cornell University.


Non-ribosomal peptide synthetases (NRPSs) are multimodular enzymes that make non-ribosomal peptides (NRPs) through a thiotemplate mechanism independent of ribosomes. These structurally diverse molecules have important roles in basal and niche-specific developmental success of filamentous fungi. In addition to roles they play in their producers’ lives, NRPs have diverse biological effects on other organisms including antibiotic, immunosuppressant, antitumor and virulence-promoting activities. Previous studies suggested that NPS genes have a highly discontinuous distribution in filamentous ascomycetes (Lee et al., Eukaryotic Cell, 2005). Comparative analyses of a larger dataset suggest that NPSs show varying levels of functional and structural conservation. Genes with functions critical to the fungal cell (stress resistance, involvement in sexual and asexual reproduction, virulence, morphology, etc) are highly conserved. For example, Cochliobolus heterostrophus NPS6, responsible for extracellular siderophore biosynthesis (Oide at al., Plant Cell, 2006) is important for virulence and resistance to oxidative stress and is conserved both structurally and functionally across all fungi examined to date. Homologs of ChNPS2, responsible for biosynthesis of an intracellular siderophore, are also found in most fungi but show a history of duplication into two clades, NPS2 and NPS1/SidC/Sid2 (represented by Fusarium graminearum NPS1/Aspergillus nidulans SidC/Ustilago maydis Sid2), with corresponding differences in function. At the other end of the spectrum are taxon-specific genes such as ChNPS1 and ChNPS3 which are intact in only a few closely related Cochliobolus species. We are using a variety of phylogenetic tools to address the roles of recombination, natural selection, and duplication/loss, in generating diversity of NPSs and their corresponding peptide products.


102. Evolution of the Cryptococcus Mating-type Locus: a Comparative Genomics Approach. Keisha Findley1, James Fraser2, and Joseph Heitman1.Department of Molecular Genetics and Microbiology1, Duke University, Durham, North Carolina, School of Molecular and Microbial Sciences2, University of Queensland, Brisbane, Australia. kmf13@duke.edu


Many basidiomycetous fungi have tetrapolar mating-type systems in which mating is regulated via two unlinked loci that control and establish cell identity and both must differ for sexual reproduction. One MAT locus encodes pheromones and pheromone receptors, while the other MAT locus encodes homeodomain transcription factors. In contrast, the fungus Cryptococcus exhibits a bipolar mating system with a single locus that encodes the pheromones, receptors, and transcription factors that establish mating-type. The ability of this fungus to reproduce sexually has been studied extensively and underlies development and is linked to pathogenicity. To characterize the evolution of the mating-type locus of this pathogenic fungal species complex, a comparative genomics approach is being applied with distantly and closely related species. This analysis involves cloning and sequencing the MAT locus from the closely related species Tsuchiyaea wingfieldii, Filobasidiella depauperata, Bullera dendrophila, Cryptococcus amylolentus, Cryptococccus heveanensis and the more distantly related species Cryptococcus laurentii and Cryptococcus albidus. To identify genes associated with MAT in these species, degenerate primers are applied for two genes flanking MAT (FAO1 and NOG2) and two highly conserved genes within MAT (RPO41 and LPD1). High-density filters of genomic fosmid libraries are produced and probed with these genes to identify MAT-specific fosmids. Positive clones are confirmed by Southern analysis and pooled to generate sequencing libraries. Thus far, preliminary assemblies have been generated for T. wingfieldii and C. heveanensis. Our results reveal that homologs of the homeodomain proteins Sxi1α and Sxi1a are both present and divergently transcribed similar, to bE and bW in Ustilago maydis. A second unlinked gene cluster encodes pheromone and pheromone receptor homologs and many other MAT associated genes. This analysis reveals that the translocation event that collapsed a tetrapolar to a bipolar system, and loss of one or the other homeodomain genes, occurred recently, perhaps concomitant with the emergence of this pathogenic species complex. Further MAT sequencing and studies on sexual reproduction potential are in progress.


103. Cryptic Sexuality in Aspergillus parasiticus and A. flavus. Jorge H. Ramirez-Prado1, Geromy G. Moore1, Bruce W. Horn2, Ignazio Carbone1. 1Center for Integrated Fungal Research, Department of Plant Pathology, North Carolina State University, Raleigh, NC. 2National Peanut Research Laboratory, USDA, ARS, Dawson, GA.


Ascomycetous fungi of the genus Aspergillus comprise a wide variety of species of biotechnological importance (e.g. A. sojae, A. oryzae, A. niger) as well as pathogens and toxin producers (e.g. A. flavus, A. parasiticus, A. fumigatus, A. nidulans). With the exception of A. nidulans, which is a homothallic fungus, these species were previously thought to be strictly asexual. Recent studies report A. fumigatus to be heterothallic and possibly undergoing sexual reproduction. Our population genetic analysis of the aflatoxin gene cluster in a Georgia population of A. parasiticus showed evidence of four distinct recombination blocks each showing a pattern of significant linkage disequilibrium and shared evolutionary history, thus providing indirect evidence of the possibility of sexual recombination in nature. We therefore investigated whether compatible mating type “MAT” genes (MAT1-1 alpha box gene and MAT1-2 HMG gene) were present. We found that each distinct A. parasiticus chemotype lineage (OMST and G1 dominant) was heterothallic and possessed a single MAT locus, containing either a MAT1-1 (G1 dominant) or a MAT1-2 (OMST) idiomorph. We observed a similar heterothallic organization of the MAT locus in A. flavus sampled from the same Georgia field but there was no association of mating type idiomorph with a specific chemotype lineage. The existence of both mating type idiomorphs in equal proportions in A. parasiticus and A. flavus populations indicates the potential for a cryptic sexual state in these agriculturally important species.


104. What makes a pathogenic fungus? A comparative genomic analysis of Coccidiodies reveals the evolutionary rise of pathogenicity. Thomas J. Sharpton, Jason E. Stajich and John W. Taylor. Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA email: sharpton@berkeley.edu


Knowledge regarding the evolutionary origins of pathogenicity is currently limited, but ultimately necessary to tackle disease. We employed evolutionary comparative genomic techniques to evaluate the origins of the pathogenic phenotype of Coccidioides, a Eurotiomycete capable of causing disease in healthy human adults. Comparisons of the pathogenic genomes of C. immitis, C. posadasii and the close, non-pathogenic relative Uncinocarpus reesei revealed genes unique to the pathogens and genes rapidly evolving under selective pressure, perhaps to escape the host immune system. This analysis reveals putative antigens and pathogenicity genes that will be investigated further in vivo. Additionally, surveys of gene family size across the fungal kingdom reveal unique characteristic expansions and contractions of certain families in the Onygenales, which contains several dimorphic pathogens including Coccidoides. Strikingly, families that experience significant reduction in the dimorphic pathogens appear to play a role in plant pathogenicity. Additionally, Coccidioides appears to have undergone expansions in gene families known to contribute to pathogenicity in Coccidioides as well as non-Onygenalen pathogens. This analysis should serve to identify possible drug and vaccine targets for Coccidioides and ultimately clarify the origins of the genetic architecture that enables a pathogenic phenotype in the Onygenales.


105. Evidence of sexual reproduction in the Septoria speckled leaf blotch pathogen, Septoria passerinii, in the upper Great Plains. Seonghee Lee and Stephen M. Neate. Department of Plant Pathology, North Dakota State University, Fargo 58105- 5012, USA. Email: sul22@psu.edu


Fifty seven polymorphic AFLP markers and mating type idiomorphs of Septoria passerinii were used to examine the sexual reproduction of the barley pathogen under field conditions. A total of 496 Septoria passerinii isolates were collected from 19 widely distributed barley fields in North Dakota and western Minnesota. The level of genetic and genotypic diversity was low within a lesion, but the higher level was found within a leaf and field. Population differentiation was low among field populations, and a significant correlation between geographical distance and genetic distance was not found. In addition, two mating types of Septoria passerinii, MAT-1 and MAT-2, were frequently found in isolates taken from individual leaves. The chi-square test revealed that equal frequencies of both mating types were observed in a leaf and field population. Moreover, when the data for all 19 sites was pooled, the overall ratios for mating types did not statistically differ from 1:1 ratio. The equal frequencies of both mating types are consistent with a sexually reproducing fungal population. Thus, we conclude that sexual recombination is occurring in S. passerinii under field conditions in the upper Great Plains.


106. Comparative functional genomics: Reconstructing the evolution of central carbon metabolism in 13 fungal species. Dawn Thompson1 and Aviv Regev1,2. 1Broad Institute of MIT and Harvard and 2Department of Biology, MIT, Cambridge, MA USA


Molecular networks are the information processing devices of cells and organisms, transforming extra- and intra-cellular signals into coherent cellular responses. Networks are also remarkably flexible and can re-configure in an adaptive response to perturbation. We seek to understand the mechanisms by which molecular networks accommodate changes by analyzing genomics data through the unifying abstraction of the functional module. Specifically, we focus on evolutionary changes that occur in the organization and regulation of central carbon metabolism in Ascomycota fungi. This is the cornerstone metabolic module of yeast cells. In free-living Ascomycete yeast species the constantly changing environment dictates that nutrient availability is the major factor controlling growth and development. To survive in a highly competitive ecosystem yeasts have evolved to take up and metabolize a variety of carbohydrates, the most important of which is glucose. In the model organism Saccharomyces cerevisiae the central carbon metabolic network has been clarified in great detail, but the regulatory network is less well characterized. Although central carbon metabolism follows the same general theme in all yeasts, important biochemical, genetic and regulatory variations exist. For example, physiologically yeasts can be classified into respiro-fermentative and respiratory during aerobic growth on glucose. The former is characterized by high glucose uptake and ethanol secretion rates, and low biomass yield (Saccharomyces cerevisiae and close relatives) and the latter by low glucose uptakes and high biomass yield (e.g. Kluyveromyces). In addition, for species other than Saccharomyces cerevisiae, many of which have important industrial uses and some are human pathogens, basic knowledge of this and other fundamental pathways is limited. We are setting up a genomics platform to study a set of 13 fully sequenced fungal species spanning over 300 million years of evolution. We grow each organism under a variety of steady state conditions (e.g. rich glucose, alternative carbon sources) and along transitions from one condition to another. We will then measure a variety of phenotypes, most notably growth characteristics and genome wide expression profiles (followed by metabolomics and proteomics). We will use our novel computational algorithms for orthology mapping (SYNERGY) and regulatory network reconstruction (CISPROF), along with novel algorithmic development, to reconstruct the regulatory modules in each extant species, and the modules that existed in ancestral (extinct) ones. We will then mine those reconstructions for general patterns of how module content and regulation evolves and how changes occur at different molecular levels


107. The course of evolution of Magnaporthe oryzae Eleusine pathotype inferred from phylogenetic trees and structures of the flanking region of avirulence gene PWL1. Masaki Tanaka, Hitoshi Nakayashiki, Yukio Tosa, Shigeyuki Mayama. Faculty of Agriculture, Kobe University, Hyogo, Japan.

The blast fungus Magnaporthe oryzae is composed of host-specific subgroups. We examined the population structure of a subgroup, Eleusine isolates, using DNA makers (MGR586, rDNA-ITS, and grasshopper). The population of Eleusine isolates was divided into two groups, A and B. Interestingly, this grouping corresponded to the distribution of avirulence gene PWL1; all the B group isolates carried PWL1 whereas the A-group isolates did not. Furthermore, the presence/absence of PWL1 in Eleusine isolates was correlated with their pathogenicity toward weeping lovegrass (Eragrostis curvula); the A group, non-carrier of PWL1, was pathogenic on Eragrostis species. We assumed that the A group may be derived from an Eragrostis pathogen. To test this hypothesis, we constructed phylogenetic trees of M. oryzae isolates using rDNA-ITS, NUT1, Mdim2, beta-tubulin, Calmodulin, and EF-1alpha genes. In a combined tree the A group was clustered with Eragrostis isolates, not with the B group., as expected. Then, we cloned flanking regions of PWL1 in the A group, the B group, and Eragrostis isolates, and compared their structures. The A group and Eragorstis isolates carried a gene for a hypothetical protein (Acc. XM_369238) in a region corresponding to PWL1. In the B group isolates, however, most of the DNA fragment for the hypothetical protein was substituted with PWL1. These results suggest that the B group may have acquired PWL1 by horizontal transfer or recombination. The two-groups in the Eleusine isolates seem to have evolved through different courses.


108. Homing in on sexual recombination and spore production in the Cryptococcus neoformans species complex. Nathan Saul*^, Tien Bui†, Mark Krockenberger^, Richard Malik^ and Dee Carter†. *Molecular & Microbial Biosciences, and ^Faculty of Vet. Science, University of Sydney, NSW, Australia. d.carter@mmb.usyd.edu.au


Cryptococcosis, caused by Cryptococcus neoformans and C. gattii initiates with the inhalation of an infectious propagule. This is likely to be a basidiospore as yeast cells are encapsulated and unable reach lung alveoli. Spores can be produced sexually via the union of MATalpha and MATa cells, however most populations are overwhelmingly MATalpha. Recent studies have found recombinant spores can also be formed following MATalpha-MATalpha unions. Finally, some MATalpha strains can produce asexual spores, but there is little evidence of this in the genotypes responsible for most cryptococcal infections. A population genetics approach was used to investigate whether sexual recombination, and by implication the production of recombinant spores, had occurred in populations consisting of MATa and MATalpha cells, and in those that were MATalpha only. C. gattii isolates were obtained from a single Eucalyptus camaldulensis tree hollow that contained both mating types, and from a hollow in an area where only MATalpha cells occur. C. neoformans var. grubii isolates obtained from infected animals known to live exclusively in Sydney where MATa cells have never been reported, were also investigated. Sexual recombination was evident in all populations. Key to this finding were 1) selection of populations restricted in time and geographic space; and 2) preliminary phylogenetic analysis to identify recombining subpopulations within larger populations that initially appeared clonal.


109. Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of Leptosphaeria maculans. L. Gout1,3, M.L. Kuhn1, L. Vincenot1, L. Cattolico2, S. Bernard-Samain2, A. D’Armaillé1, M.-H. Balesdent1, and T. Rouxel1. 1INRA, PMDV, Route de St Cyr, 78016 Versailles, France. 2Genoscope-centre national de séquençage, 2 rue Crémieux, 91057 Evry, France. 3Protection des Plantes, INA-PG, 78850 Thiverval-Grignon, France


The Dothideomycete Leptosphaeria maculans is the most damaging disease of oilseed rape (Brassica napus) worldwide. In France, disease control relies mainly on the use of disease-resistant cultivars. The Rlm genes effectively control the disease as long as the corresponding avirulent allele (AvrLm) dominates in the pathogen population. However, L. maculans has the ability to rapidly adapt to the selection pressure exerted by a novel resistance gene as exemplified by the 3-year evolution towards virulence at the AvrLm1 locus in French field conditions. AvrLm1 was recently cloned and shown to be a solo gene within a 269-kb non-coding, heterochromatin-like region consisting of mosaics of degenerated repeats. We fully or partly sequenced the AvrLm1 genomic region in one avirulent and two virulent isolates. The gain of virulence was linked in both cases with a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar for both the virulent isolates. Among 343 field isolates analyzed, a similar large deletion leading to chromosome length polymorphism was evidenced by multilocus haplotype analysis in > 90% of the virulent isolates. Deletion breakpoints were strongly conserved in most of the virulent isolates leading to the hypothesis that a unique event of large deletion leading to the avrLm1 virulence has diffused in populations or is strongly constrained by the genome environment. Surprisingly, even though a large chromosomal fragment was lost to gain virulence, no effect on fungal and pathogenic fitness could be evidenced, either in vitro or under natural field infection conditions.


110. Intron evolution in basidiomycete laccase genes. Sreedhar Kilaru 1, Pierre-Emmanuel Courty 2, Patrik J. Hoegger 3, Timothy Y. James 4, Francis Martin 2 and Ursula Kües 3. 1 School of Biological Sciences, University of Bristol, Bristol, UK 2 Centre INRA de Nancy, Champenoux, France 3 Molecular Wood Biotechnology, Georg-August-University, Göttingen, Germany* 4 Department of Biology, Duke University, Durham, NC, USA


Most but not all homobasidiomycetes possess laccase genes. Coprinopsis cinerea was shown to contain 17 different laccase genes that could be divided into two gene subfamilies by intron positions. Here, we analyze a total of 835 introns in 74 different homobasidiomycete laccase genes related to the C. cinerea subfamily 1 laccase genes. These introns distribute over 75 different positions over the length of the genes. An intron phase-analysis shows a 2:1:1 distribution for phase-zero, phase-one and phase-two insertion within codons and thus a bias towards phase-zero. Two ancient introns occur in phase-zero position of histidine codons, regardless of the taxonomical order. Other ancient introns were lost from individual orders whilst new ones were apparently acquired at different time points during laccase gene evolution. Some of these other intron positions distinguish genes from Agaricales, Aphyllophorales and Ceratobasidiales. Within an order, there are also species- or genus-specific intron positions. The data suggest that particularly in the Agaricales, laccase genes duplicated several-fold after division into families, genera or species. A phylogenetic tree constructed by intron position conservation grouped laccase genes primarily according to taxonomy. In contrast, in a phylogenetic tree based on protein similarity, laccases clustered partially by the fungal life-style (white rot versus litter-decomposing species). *Work in our laboratory is supported by the DBU (Deutsche Bundesstiftung Umwelt).


111. Missing links in Mycosphaerella graminicola. Sarrah Ben M’Barek, Alexander H.J. Wittenberg, Theo A.J. van der Lee, Sarah B. Ware, Henk J. Schouten and Gert H.J. Kema Plant Research International B.V., P.O. Box 16, 6700 AA Wageningen, The Netherlands

Mycosphaerella graminicola (Fückel) J. Schröt. in Cohn is a haploid ascomycete that causes septoria tritici blotch in durum ( Triticum turgidum L.) and in bread wheat (T. aesitivum L.). Two high-density genetic linkage maps with over 2000 Diversity Arrays Technology (DArT), AFLP and SSR markers were generated from two mapping populations derived from crosses between the sequenced isolate IPO323 and either isolate IPO94269 or IPO95052. Graphical genotyping revealed that some of the progenies lacked one or more linkage groups that were present in both parental isolates. PCR’s with primers derived from the SSR and DArT markers positioned on these linkage groups were used to verify the absence of these linkage groups in off spring isolates. We confirmed the absence of six linkage groups in the IPO323 x IPO94269 and IPO323 x IPO95052 progenies. We could not conclusively confirm the absence of two additional linkage groups, maybe due to the presence of translocations. We studied the origin and effect of this phenomenon. Our results demonstrate that the chromosomes were lost during meiosis possibly by non disjunction. The loss of chromosomes did not seem to affect viability, pathogenicity or fertility as back crosses between these progeny isolates and the parents produced significant numbers of off-spring isolates. The finished genome sequence of isolate IPO323 and the aligned DArT markers from the high-density map enabled us to identify the missing chromosomes containing apparently redundant genes. Interestingly, we also identified isolates in the IPO323 x IPO94269 progeny that were diploid for one linkage group. We hope our data will contribute to a better understanding of the genetics in Mycosphaerella and redundancy of its genomic content.


112. Simple sequence repeat (SSR) markers for Pythium aphanidermatum, P. irregulare, and P. cryptoirregulare. Seonghee Lee and Gary W. Moorman. Department of Plant Pathology, The Pennsylvania State University, University Park, 16802, PA. USA. Email: sul22@psu.edu


Six microsatellite-enriched genome libraries from three Pythium species, P. aphanidermatum, P. irregulare, and P. cryptoirregulare were constructed to develop simple sequence repeat (SSR) markers. Four synthetic di-, (AG)12, (AC)12, (GT)12, and (CT)12, and three trinucleotides repeats, (GGT)6, (AAG)8, and (AAC)6, were used to screen microsatellite loci in the three Pythium species. Approximately 600 positive recombinant clones for each Pythium species were selected and sequenced. About 35 % in P. aphanidermatum, 17 % in P. irregulare, and 25 % clone sequences in P. cryptoirregulare contained the unique simple sequence repeats (> 4 repeats). Total 110 SSR primer pairs for P. aphanidermatum, 73 SSRs for P. cryptoirregulare, and 82 SSRs for P. irregulare were developed and tested for amplifications and polymorphisms on four isolates of each Pythium species. Twenty three in P. aphanidermatum, 39 in P. irregulare, and 41 polymorphic SSRs in P. cryptoirregulare were found. After screening the polymorphic SSR markers to 8 isolates of each species, the most polymorphic and reproductive SSR markers were developed for each Pythium species; 9 in P. aphanidermatum, 18 in P. irregulare, and 23 in P.cryptoirregulare. These newly developed SSR markers can be useful for monitoring the introduced isolates and population genetic studies.


113. Development of Phlebiopsis gigantea as a biocontrol agent for annosum root disease in the southeastern United States. Brian Higgins and Sarah F. Covert. Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA


The white-rot basidiomycete Heterobasidion annosum causes root and butt rot in conifers across temperate climate zones worldwide. In freshly thinned pine stands in the southeastern U.S., the ability of H. annosum to spread from cut stumps to live trees often results in significant volume loss. Phlebiopsis gigantea, another white- rot basidiomycete, has been used successfully across Europe to control H. annosum, but the U.S. Environmental Protection Agency (EPA) has not approved the use of P. gigantea in the U.S for this purpose. In order to develop a P. gigantea biocontrol product that will meet EPA regulatory approval, we have collected 64 isolates of P. gigantea from 14 sites in the southeastern U.S. Our early results suggest that P. gigantea isolates from the southeastern U.S. make sufficient spores in culture for commercial application of a spore suspension to cut stumps, and they are at least as effective in controlling H. annosum infection in southern pine wood as two commercially available P. gigantea isolates from Europe, known as RotStop (Finland) and PG1 (U.K.). We have developed 8 microsatellite markers to measure P. gigantea genetic diversity. These data will indicate how commercial application of selected P. gigantea strains is likely to impact local P. gigantea population structure. Current data on P. gigantea genetic diversity also be presented.


114. Relationships between Alleles at Lineage Diagnostic Loci in Fusarium graminearum. L.L. Anderson1, Y-.W. Lee2, R.L. Bowden3, and J.F. Leslie1. 1Department of Plant Pathology, Kansas State University, Manhattan, KS; 2School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea; 3USDA-ARS Plant Science and Entomology Research Unit, Manhattan, KS.


DNA sequences of three nuclear genes (MAT-1-1-3, TRI101 and RED) were examined from over 500 isolates of Fusarium graminearum collected from South American and Korean wheat, maize and sorghum. Genetic networks were developed for each gene that illustrate the relationships between the DNA sequences of isolates in this study based on the minimum number of base pair changes that separate the isolates. Some lineage diagnostic single nucleotide polymorphisms (SNPs) are not conserved in this strain set. The lack of dichotomous branching results in polytomies, which suggests that the lineages did not evolve in a stepwise fashion. This is further supported by the inconsistent location of lineages within the networks. For example, in the MAT network, Lineage 3 is in close proximity to the center of the network, whereas in TRI101 it is one of the most distal. Additionally, several of the genetic networks cannot be resolved without cycles, which is consistent with recent gene flow between the lineages.


115. Genetic dissection of Neurospora crassa circadian clock using two independent QTL mapping analyses. Tae Sung Kim1, Benjamin Logsdon2, Jason Mezey2, Kwangwon Lee1. 1Dept of Plant Pathology, 2Dept of Biological Statistics and Computational Biology, Cornell University, Ithaca NY 14853


Neurospora crassa has been a successful model organism in the circadian clock study for the past four decades. In an attempt to identify novel genetic components that are contributing the clock phenotypes, we relied on the quantitative genetics approach, Quantitative Trait loci (QTL) analysis. We constructed three independent mapping populations, whose parents are from geologically isolated locations. Two circadian clock phenotypes, period and phase, were evaluated in the 188 F1 progenies of each mapping population for the QTL analyses. Simple sequence-repeat- markers-based linkage maps that cover the whole genome with an average 13 cM interval were developed for the current study. To identify the clock QTLs, we performed two independent QTL mapping analyses, composite interval mapping (CIM) and Bayesian QTL mapping (BQM). For the two clock phenotypes, the CIM analysis identified 17 genetic loci from the three populations. BQM analysis confirmed all the 17 QTLs from CIM and furthermore detected additional 23 QTLs, suggesting that BQM has more power in identifying QTLs than CIM. 15 QTLs were co-localized with the previously identified clock genes, however, 25 of the identified QTLs were not co-localized with any previously identified clock genes. Our findings demonstrate that QTL analysis is a powerful tool identifying novel genetic elements on complex phenotypes.


116. Mites act in distribution of fungal spores in Coprinopsis cinerea. Monica Navarro-Gonzalez 1, Wassana Chaisaena 1, Olivia Sanchez-Hernandez1, Stefan Schütz2 and Ursula Kües 1. 1Institute of Forest Botany, and 2Institute of Forest Zoology and Forest Conservation, Georg- August-University, Göttingen, Germany


Mites are attracted to fungal cultures of Coprinopsis cinerea in order to graze on the mycelium. Both monokaryotic and dikaryotic mycelium are eaten by the mites including asexual spores (oidia). In vegetative cultures, only the melanized sclerotia are left, supporting a function in nature for duration: on fresh media, these sclerotia germinate. Upon fruiting body induction, primordia are also refused by the mites. When fruiting bodies matures, one can observe mites to climb up the stipe to feed on the autolysing cap tissues with the basidiospores. Stipes are refused as the sclerotia. Basidiospores are ingested but not digested. Faecal pellets are formed with around 400 basidiospores. These spores germinate and can easily form a dikaryon since the high number of eaten spores ensures that all four possible mating types arising from meiosis are represented. The fast moving mites thus contribute to the distribution of basidiospores in the environment and possibly to mixing populations in the nature. Mites lay eggs in close vicinity to spore pellets. This raises the idea that there might be a form of symbiotic interaction between mites and the fungus since germinated fungal colonies will serve as food for the hatching larvae. MNG, WC and OSH were funded by CONACYT (Mexico), the Rajamangala University of Technology and the DAAD.


117. Adaptive evolution of the blast pathogen Magnaporthe oryzae populations on cereal hosts in Africa. S. Sreenivasaprasad1, J. P. Takan1, S. Muthumeenakshi1, J. Chipili1, N. J. Talbot2, E. O. Manyasa3 and Y. Sere4. 1Warwick HRI, University of Warwick, Warwickshire, CV35 9EF, UK. 2University of Exeter, UK. 3ICRISAT, Kenya. 4ARC-WARDA, Benin.


Blast caused by Magnaporthe oryzae is a major production constraint to key cereals such as rice and finger millet in sub-Saharan Africa. We have used molecular, biological and pathological assays to characterise the pathogen populations on these hosts. In West Africa, where intensive rice cultivation is relatively recent (less than 500 years), blast pathogen populations showed typical lineage based structure established by MGR586 fingerprinting. Skewed distribution of the two mating types MAT1-1 and MAT1-2, high female sterility and low fertility were observed. Pathogen isolates revealed clear differences in compatibility on rice differentials exhibiting R gene-type interactions. In contrast, on finger millet, domesticated in East Africa a few thousand years ago, more than 190 blast pathogen haplotypes were identified. The pathogen populations revealed continuous variation (lack of clonality) reflecting sexual recombination. These indigenous blast populations were genetically distinct to those in Asia, and showed a near equal distribution of the mating types and high fertility. All 70 isolates tested showed basic compatibility to a range of finger millet varieties suggesting polygenic quantitative interactions. Thus the M. oryzae populations on rice and finger millet reveal distinctive adaptive evolutionary patterns related to crop domestication and agricultural intensification in sub-Saharan Africa.


118. Evolution of Mitogen-activated Protein (MAP) Kinase Cascade Components in Filamentous Fungi. C. Greenwald and H. H. Wilkinson. Texas A&M University, Plant Pathology and Microbiology, College Station, TX


All filamentous fungi contain three paralogous MAP kinase signaling cascades within their genomes. Each cascade consists of three phosphorylating enzymes (MAPKKK, MAPKK, and MAPK), which act sequentially to transduce and amplify an environmental signal from a receptor protein to downstream genes that are directly involved in phenotypic responses. Mutations in fungal cascade components are well known for dramatic phenotypic consequences including effects on pathogenicity, mating, asexual development, abiotic stress response, etc. We predicted that since MAP kinase cascades are central to fungal development and life history there would be evidence for strong conservation of these proteins. We compared the gene family trees for MAP kinase pathway components from eight filamentous fungal genomes representing a diversity of filamentous fungi (Neurospora crassa, Aspergillus nidulans, Fusarium graminearum, Magnaporthe grisea, Botrytis cinerea, Chaetomium globosum, Sclerotinia sclerotiorum, Aspergillus flavus). The analysis has revealed a higher rate of synonymous vs. non-synonymous mutations in the cascade component genes, from which we inferred they have undergone a history of purifying selection. As one might expect, evolution of genes upstream (e.g. environmental sensors) and downstream (e.g. transcription factors) from these cascades do not mirror this degree of conservation, in fact, orthologous genes are seldom maintained in all eight species.


119. Mating type in Coccidioides species. M. Alejandra Mandel1,3, Bridget M. Barker1,3, Scott Kroken1, John N. Galgiani2,3,4, Steve. D. Rounsley1,2 and Marc J. Orbach1,2,3 1Department of Plant Sciences, 2Bio5 Institute, 3Valley Fever Center for Excellence, University of Arizona and 4Southern Arizona Veterans Administration Health Care System, Tucson, AZ

Coccidioides immitis and C. posadasii are the causative agents of coccidioidomycosis, also known as Valley fever, a respiratory disease endemic to the desert southwestern US, along with parts of Mexico, and Central and South America. It is caused by inhalation of asexual spores that undergo differentiation to initiate the parasitic phase in host lungs and, if not controlled, disseminates to other parts of the body. Coccidioides is only known to propagate asexually by segmentation of hyphae to produce arthroconidia, although details of its growth in the environment are limited. However, each species of Coccidioides has a recombined population structure, suggesting sexual reproduction. The presence of mating-type genes is necessary, although not sufficient, for fungal sexual reproduction. In order to investigate the potential for sexual reproduction in Coccidioides we analyzed genome sequences for potential mating-type loci and have identified idiomorphs in Coccidioides indicative of a typical heterothallic ascomycete. Data will be presented that define the MAT1-1 and MAT1-2 idiomorphs of both C. immitis and C. posadasii, and show that they are highly conserved between the two species. The idiomorphs in Coccidioides are the largest reported for an ascomycete; MAT1-1 is 8 kb and MAT1-2 is 9 kb in length. The MAT1-1 idiomorph contains four putative ORFs, and the MAT1-2 idiomorph has five putative ORFs. Analyses of ~400 isolates of C. posadasii and C. immitis revealed that they contain either a MAT1-1 or a MAT1-2 locus, and that they are distributed in a 1:1 ratio. A detailed analysis of the putative ORFs, their expression and a comparison of the MAT loci to those of related fungi will be presented.


120. Characterizing the mating-type locus of the graminicolous Colletotrichum: Patterns of sex, selection and host specialization. Jo Anne Crouch1, Michael R. Thon2, Michele Groenner-Penna3, Bruce B. Clarke1, Adlane Vilas-Boas3, Lisa J. Vaillancourt4 and Bradley I. Hillman1. 1Rutgers University, New Brunswick, NJ; 2 Texas A&M University, College Station, TX; 3Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 4 University of Kentucky, Lexington, KY.


While most filamentous ascomycetes have a one locus, two allele (idiomorph) mating system, extensive sampling of Colletotrichum species (teleomorph=Glomerella, order Phyllacorales) has identified only a single idiomorph (MAT1-2, with HMG box), regardless of reproductive lifestyle, i.e., heterothallic (self sterile) or homothallic (self fertile). Genetic analysis of C. graminicola has suggested the Colletotrichum mating system is governed by at least two unlinked loci. In order to explore its unique mating strategy, we have cloned and characterized the entire ~17-kb MAT locus from representatives of the falcate-spored, grass-inhabiting Colletotrichum group. First, using multi-locus phylogenetics (3,400-bp, 4 genes), we identified 11 unique species in a highly resolved tree topology that was almost entirely congruent with host plant association. Next, using a combination of transposon-mediated cosmid sequencing, cosmid chromosome walking and PCR amplification, we identified 6 genes at the MAT locus, including the MAT1-2, DNA lyase, and putative anaphase-promoting complex. Locus organization was identical in all 11 species, irrespective of homothallism (e.g. C. falcatum) or heterothallism (e.g. C. graminicola), with no evidence for a MAT1-1 idiomorph. Comparison of the MAT locus with the distantly related species C. gloeosporioides, a cosmopolitan pathogen of dicots, showed that this region has been broadly conserved across evolutionary time, with all genes arranged in the same chromosomal orientation, although several differences in gene length and the number of introns/exons were detected. An examination of polymorphism, divergence and selection pressures on the locus are currently in progress and will be discussed.


121. Evolution of an avirulence homolog (Avh) gene subfamily in Phytophthora ramorum, causal agent of sudden oak death. Erica M. Goss, Caroline M. Press, and Niklaus J. Grunwald Horticultural Crops Research Laboratory, USDA ARS, Corvallis, OR gosse@science.oregonstate.edu


Pathogen effectors can serve a virulence function on behalf of the pathogen or trigger a rapid defense response in resistant hosts. Sequencing of the Phytophthora ramorum genome and subsequent analysis identified a diverse superfamily of approximately 350 genes that share two protein motifs (RXLR and dEER) with the four known effectors in plant pathogenic oomycetes. These have been termed Avh (avirulence homolog) genes. While as a whole the genes in this superfamily share modest sequence similarity, small groups of closely related genes can be identified. We have investigated the molecular evolution of one such group of seven Avh genes. Microarray data suggests that four of these genes are expressed in isolate Pr-102. We sequenced the full coding region (approximately 400 bp) and flanking noncoding regions of each gene in the three P. ramorum lineages. The number of polymorphic sites within P. ramorum genes ranges from 1 to 12, suggesting different evolutionary pressures among genes. Analysis indicates that these genes contain both amino acids under purifying selection (e.g. in the signal peptide and RXLR and dEER motifs) and under positive selection. We have also been able to obtain the sequence of homologous Avh genes in the sister taxa P. hibernalis and P. lateralis, allowing for examination of the evolution of these genes across species.


122. Withdrawn


123. Evaluating genetic diversity of Fusarium proliferatum from orchids in Hawaii. Cassandra Swett1, Laurel Anderson2, John F. Leslie2, and Janice Y. Uchida1. 1Department of Plant and Environmental Protection Sciences, University of Hawaii, Manoa, HI, USA. 2Department of Plant Pathology, Kansas State University, KS, USA.


Fusarium proliferatum is a recently introduced pathogen to the Hawaiian Islands, where it causes disease on orchids in commercial nurseries. The pathogen origin is unknown, although south-east Asia has been suggested as a source. In this study, we evaluated the genetic diversity of F. proliferatum haplotype populations from diseased orchids in three nurseries, one on the Island of Oahu, and two on Hawaii. Amplified fragment length polymorphisms (AFLP’s) were generated for between 29 and 49 single spore isolates per nursery, using three primer sets. Total genetic diversity of F. proliferatum is described for the state of Hawaii, and haplotype population diversity compared within and among nurseries. This work can be used to monitor the F. proliferatum strain populations, to evaluate if introduction is still occurring, and for future comparisons to strains populations from potential sources, to establish pathogen origins.


124. Implications of repeat-induced point mutated transposons during the evolution of Colletotrichum cereale. Jo Anne Crouch, Bruce B. Clarke and Bradley I. Hillman. Rutgers University, New Brunswick, NJ

The evolution of eukaryotic genomes can be greatly influenced by the activities of transposable elements; conversely, transposons can serve as valuable tools that may increase our understanding of genome evolution and diversification. Here, we report the discovery of five transposon species from the two major lineages (clades A and B) of Colletotrichum cereale: the DNA transposons Collect1 and Collect2 (pogo and Tc1/mariner families, respectively), the long terminal repeat (LTR) retrotransposons Ccret1 and Ccret2 (Pseudoviridae and Metaviridae) and the non-LTR retrotransposon Ccret3. Using computational tools, our data show that 21 of 35 unique transposon loci display the signature pattern of recurring rounds of repeat-induced point mutation (RIP), a genome defense mechanism unique to filamentous fungi that mutates duplicated sequences by inducing GC-to-AT transitions. Analysis of over 100-kb of sequence data showed the RIPped transposons possess a skewed A+T base composition (genome=51%; RIPped=68%), increased incidence of TA, CT and AG dinucleotides, and decreased representation of the dinucleotide pairs TG, CA, CG, GA and TC. RIP index values were also consistent with those observed for RIPped transposons from Neurospora, Fusarium, Podospora and Aspergillus species, with CA, CG and TG the most common context for GC-to-AT transitions. Although our data supported the identification of RIP mutation acting only on transposons in the C. cereale clade B genome, none of the clade A transposon sequences displayed any evidence of RIP activity. Nevertheless, identification of a RIPped Ccret2 element in the closely related species C. falcatum suggests that RIP mutation predated cladogenesis between these taxa.


125. Patterns of recombination and population differentiation among Colletotrichum cereale isolates from 27 host plant genera. Jo Anne Crouch, Bruce B. Clarke and Bradley I. Hillman. Rutgers University, New Brunswick, NJ


In populations of the turfgrass anthracnose fungus Colletotrichum cereale, the prevailing mode of reproduction is thought to occur clonally, as inferred primarily through the lack of a teleomorph. Coupled with the visual absence of a sexual state, previous reports of clonal genotypes from RAPD and isozyme analyses contribute to the notion that C. cereale has endured throughout history as an asexual organism. Because a pathogen’s mode of reproduction strongly influences the course of its evolution and the alternative hypothesis has never been adequately tested, we are evaluating patterns of variation in this species. In the present study, we used an extensive nucleotide sequence dataset (4 genes, 3,400 nucleotides) to examine a large, worldwide sampling of pathogenic isolates of C. cereale from turfgrass and their counterparts isolated from cereal crops (wheat, oats, barley) and prairie grasses. This dataset illustrated that C. cereale is subdivided into several lineages, each composed of primarily (but not exclusively) either turfgrass or non- turfgrass derived isolates. Next, RFLP analysis of three transposon species provided evidence for recombination by C. cereale, even in relatively small populations. Furthermore, 21 of 35 unique transposon loci, when evaluated for dinucleotide compositional bias, displayed the signature pattern of repeat-induced point mutation (RIP), a genome defense mechanism that functions only during meiosis. Taken together, our data rejects the presumption of clonality for C. cereale and, given the severe losses sustained in turfgrass systems due to this pathogen, strongly emphasizes the need for additional inquiry into the biology, mating and dispersal mechanisms of C. cereale.


126. Examining diversity in populations of Anisogramma anomala. Sara N. Baxer, Jo Anne Crouch, C. Reed Funk, Thomas J. Molnar, Bradley I. Hillman. Department of Plant Biology, Rutgers University, New Brunswick, NJ


While Anisogramma anomala (Gnomoniaceae) is a relatively harmless inhabitant of the native American hazelnut (Corylus americana), this fungus is a destructive pathogen of the introduced, commercially important European hazelnut (C. avellana). Using systematic greenhouse and field inoculations, we have shown the breakdown of several sources that have provided long-term resistance to A. anomala in the Pacific Northwest. These findings raise questions about the possibility of geographically isolated populations of the fungus and whether variability in the fungus could be responsible for the breakdown of resistance. To address these questions, we have collected and are evaluating 185 isolates of A. anomala from 37 sites across North America. Our initial survey by sequence analysis at 4 gene regions (ITS, EF-1alpha, BT2, CAL; 1865 nucleotides) demonstrated an extremely low level of genetic variation between the isolates, despite the fact that A. anomala is only known to reproduce sexually. Using a hybrid-capture protocol, we are currently assembling a genomic DNA library enriched for microsatellite marker sequences to examine diversity in A. anomala populations at high levels of resolution.


127. Modern arbuscular mycorrhizal fungi are clonal: evidence based on population structure of Glomus etunicatum. Henk C. den Bakker1, Joseph B. Morton2, Teresa E. Pawlowska1. 1Department of Plant Pathology, 334 Plant Science Building, Cornell University, Ithaca, NY 14853-5904; 2 Division of Plant and Soil Sciences, 1090 Agricultural Sciences Building, West Virginia University, Morgantown, WV 26506-6108


Arbuscular mycorrhizal fungi (phylum Glomeromycota) are generally believed to represent a group of ancient asexual organisms. To test the hypothesis of clonality, we explored the population structure of globally distributed isolates of Glomus etunicatum with a particular focus on two intensely sampled populations from California, USA. We used 9 anonymous markers, two protein coding loci (Beta tubuline and GAPDH) and ribosomal markers to (1) infer the phylogenetic relationship of G. etunicatum and its closest relatives, (2) to detect recombination and (3) to infer global and local haplotype distribution. (1) Phylogenetic analyses showed that G. etunicatum represents a monophyletic group, that is nested within an undescribed species Glomus ‘yellow’. (2) Gene trees inferred from the individual markers exhibited phylogenetic concordance. Based on this phylogenetic concordance and the outcome of various tests of recombination we have to conclude that G. etunicatum reproduces in a clonal fashion. Our population genetic study revealed 11 distinct haplotypes among the globally distributed isolates of G. etunicatum. Eight of these haplotypes were found in tropical and subtropical regions, one haplotype was only found in the Californian populations, while two haplotypes were found to occur worldwide, without any distinct geographic pattern. We hypothesize that these haplotypes may represent two clonal lineages that have dispersed rapidly all over the world due to human agricultural activities.


128. Microsatellite analysis of environmental isolates of the human pathogenic fungus Coccidioides. Bridget M. Barker1,2, Scott Kroken2, Marc J. Orbach2. 1Graduate Interdisciplinary Program in Genetics, University of Arizona. 2Department of Plant Sciences, Division of Plant Pathology and Microbiology, University of Arizona, Tucson, Arizona

Coccidioides consists of two species: C. immitis, proposed to be restricted to California, and C. posadasii in Arizona, Texas, Mexico, and Latin America. Their geographical separation is putative, as some Californian patients were infected with C. posadasii and some non-Californian patients were infected with C. immitis. Geographical separation has also been observed at the population level within each species. Atypical infections may be due to patients traveling to and being exposed in other regions than where they were diagnosed, or due to long distance wind dispersal of infective arthroconidia. Alternatively, the ranges of the two species and their constituent populations may overlap. To test these alternative hypotheses, we determined the genotypes of 65 isolates obtained from soil samples in Tucson, Arizona, and genotypes of isolates from domesticated and wild animals from Tucson. These new genotypes were added to the database of all previously characterized isolates, and submitted to phylogenetic and population genetic analyses. Without exception, soil and animal isolates from Tucson are C. posadasii. Additionally, we have not yet observed any obvious sub-structuring among isolates that infect humans, domesticated and wild animals, and those found in the soil. These Tucson isolates encompass a significant amount of the genetic diversity of C. posadasii, suggesting that gene flow is sufficient to maintain high diversity within the Tucson subpopulation. There also appears to be greater gene flow than previously observed between Arizona and adjacent Mexico, suggesting that the strongest population structuring of C. posadasii may occur between the Sonoran and Chihuahuan Deserts.


129. Evolutionary genomics of the ectomycorrhizal fungus Paxillus involutus. Anders Tunlid, Tomas Johansson, Antoine LeQuere, Kasper Astrup Eriksen, Andres Schutzendubeln, Balaji Rajashekar, Björn Canbäck, Jenny Hedh, Peter Samson, Susanne Erland Department of Microbial Ecology, Lund University, SE 223 62 Lund, Sweden (anders.tunlid@mbioekol.lu.se)


It is well known that ectomycorrhizal (ECM) fungi can differ markedly in their ability to form mycorrhizae and to promote the growth of the host plant. Generally such phenotypic differences could be the result of variations in gene content, quantitative differences in gene expression, and structural differences in gene products. We have uced cDNA microarrays to compare the trancriptome and genomes of strains of Paxillus involutus . The analyses included Nau, that is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. The array contained reporters for 1075 putative unique genes in P. involutus , derived from a collection of expressed sequence tags (ESTs). On genomic level, Nau and Maj were very similar. Only 16 out of 1,075 genes analyzed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1,075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes showed that two of them have evolved at an enhanced rate in Nau due to relaxed or positive selection.


130. Phylogenetic markers for the Genus Aspergillus developed from complete genome sequences. S.M. Witiak1, R.A. Samson2, J. Varga2, A. Rokas3 and D.M. Geiser1. 1Department of Plant Pathology, Penn State University, University Park, PA 16802 USA, 2Centraalbureau voor Schimmelcutures, 3508 AD Utrecht, NETHERLANDS, 3 Broad Institute, 7 Cambridge Center, Cambridge MA 02142 USA. email: dgeiser@psu.edu


The genus Aspergillus comprises tremendous biological and ecological diversity, and comparative genomic research in the genus would be aided greatly by a detailed multilocus phylogeny. Complete genome sequences of eight Aspergillus species were used as a guide to identify gene regions suitable for multilocus phylogenetic analysis of the genus. Amino acid sequences identified as conserved among animal and fungal species were used to design PCR primers, based on DNA sequences aligned among the Aspergillus genomes. Primers were then screened among seventeen or more Aspergillus species representative of the known phylogenetic breadth of the genus, as well as Penicillium outgroups, to determine the breadth of their utility. To date, ten new loci have been amplified and/or sequenced successfully. These loci will be used to infer phylogenetic relationships at the genus level, as well as among closely related species within sections and subgenera. Their utility as markers for multilocus sequence typing (MLST) and for inference of species boundaries will also be assessed.



Genomics and Proteomics


131. Comparative genomics of Candida glabrata clinical strains and closely related species. Cécile Fairhead1*, Christiane Bouchier2, Christophe Hennequin3, Laurence Ma2, Héloïse Muller1 and Bernard Dujon1 1 and 2: Génétique Moléculaire des Levures and Plate-Forme Génomique, Institut Pasteur, 25 rue du Dr Roux 75015 Paris, France 3: Parasitologie-Mycologie, Faculté de Médecine St-Antoine, 27 rue Chaligny 75012 Paris, France *cfair@pasteur.fr


The hemiascomycete Candida glabrata is currently the second agent of candidiasis in humans after C. albicans. The genome of C. glabrata strain CBS138 was released in 2004. This species is in fact closely related to Saccharomyces cerevisiae and they share the same ancestor which has undergone a whole genome duplication. Although it has apparently retained the genes of the mating pathway; C. glabrata is only isolated as a haploid and no mating has been observed in this species. We have studied several isolates of C. glabrata by microsatellite analysis, and also by obtaining and examining Random Sequence Tags from three of them. The same strains were also examined for several features, such as electrophoretic karyotype and mating type. We will present evidence for the mainly clonal reproduction of this species, and discuss the mechanisms that lead to chromosome length polymorphism. We have also produced and examined Random Sequence Tags from three species that are described as the closest to C. glabrata: K. delphensis, C. castellii and K. bacillisporus. We will report on the divergence between these species and evolution of their gene functions and genomes.


132. Genome evolution in hemiascomycetous yeasts. Claude Gaillardin1 and The Génolevures Consortium2. 1Microbiologie et Génétique Moléculaire, INA-PG INRA CNRS, Thiverval-Grignon, 78500, FRANCE. 2Génoscope (Evry), Institut Pasteur (Paris), U. Strasbourg, U. Orsay, CNRS (Bordeaux), CEA (Saclay), IBMC (Strasbourg), Laboratoire de Chimie Bactérienne (Marseille), U. Louvain (Louvain-la-Neuve), INA-PG (Thiverval-Grignon). claude.gaillardin@grignon.inra.fr

Within the Génolevures project, we have initiated a large scale genomic survey of the entire phylum of Hemiascomycetes. Genetic maps were found to be poorly conserved. Rates and types of genome rarrangements appeared conserved between lineages but highly variable between lineages. This suggested that distinct evolutionary mechanisms operated in each phylogenetic branch within this otherwise homogeneous phylum. To confirm these findings, we choose to populate clades that were currently less scrutinized than the Saccharomyces or the Candida albicans clades. We focussed on the Kluyveromyces clade, which contains several species of applied interest. We compared the genomes of K. thermotolerans and Zygosaccharomyces rouxii to those of K. lactis, K. waltii and Eremothecium gossyppii. The proximity of these genomes, the absence of recent whole genome duplication and the overall conservation of large syntenic blocks facilitated whole genome alignments thus permitting robust annotation. Phylogenetic relationships between these species were revised using conserved othologues. Clusters of orthologous proteins were individually examined for possible cases of accelerated evolution, showing that species with high or low gene evolution rates coexist in the same clade.


133. Analysis of the fumitremorgin gene cluster in a human pathogen, Aspergillus fumigatus. Shubha Maiya1, Alexander Grundmann2, Kosalec I 3, Shu-Ming Li2 and Geoffrey Turner1. 1Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 3QX, UK; 2Eberhard- Karls-Universität Tübingen, Pharmazeutische Biologie, Auf der Morgenstelle 8, 72076 Tübingen, Germany. 3 Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb g.turner@shef.ac.uk.


Aspergillus fumigatus strains have been reported to produce a variety of secondary metabolites, including toxic prenylated alkaloids such as fumitremorgin C,and many putative secondary metabolic gene clusters have been identified in the genome reference strain Af293 following the completion of the genome sequence.A gene ftmA encoding a dimodular non-ribosomal peptide syntetase (NRPS) was found within one of the putative secondary metabolic gene clusters. ftmA was overexpressed in strain Af293 by insertion of multiple copies,and in the naïve host Aspergillus nidulans, which lacks the equivalent gene cluster,under the control of the alcA promoter.Though neither fumitremorgins nor the dipeptide intermediate brevianamide F,cyclo-L-Trp- L-Pro,could be detected in wild-type strains,brevianamide F accumulated in liquid cultures of both species following increased expression of the NRPS gene.The cyclic dipeptide brevianamide F is the precursor of a variety of prenylated alkaloids,including fumitremorgins A,B,C,tryprostatin B and verruculogen.RT-PCR indicated that some of the genes in this cluster are poorly transcribed in Af293.We have also observed that overexpressing both ftmA and ftmB,a prenyl transferase,under a regulatable promoter alcA in A. fumigatus and in A.nidulans led to the accumulation of tryprostatin B which is the next intermediate in the fumitremorgin pathway.In order to determine the function of other genes in the cluster and to understand why it is poorly expressed in Af293,we have begun analysis of a fumitremorgin producing strain Af6460.


134. Transposons in biotechnologically relevant strains of Aspergillus niger and Penicillium chrysogenum. Ilka Braumann1, Marco van den Berg2 & Frank Kempken1. 1Abteilung für Botanische Genetik und Molekularbiologie, Botanisches Institut und Botanischer Garten, Christian-Albrechts-Universität zu Kiel, Olshausenstraße 40, 24098 Kiel, Germany 2DSM Anti-Infectives, Alexander Fleminglaan 1, 2613 AX, Delft, The Netherlands


Despite of the relatively late discovery of fungal transposons and despite of the small fraction of repetitive DNA in fungal genomes (1.4%, 2.9%, and 3% in the genomes of Aspergillus oryzae, A. fumigatus and A. nidulans (Galagan et al., 2005)), today virtually all types of eukaryotic transposons have been identified in fungi (reviewed in Pöggeler and Kempken 2004). However, the impact of transposons on their fungal hosts’ genomic development is still a matter of debate. Transposition might play a role in enhancing recombination in asexual fungi (Rep et al., 2006). However, it often failed to identify actively transposing mobile elements in the genomes of fungal laboratory strains, possibly because of ongoing selection for phenotypic stability (Daboussi and Capy, 2003) . It is particularly interesting that relatively little is known about the role transposons have in industrial producing strains. We set out to investigate the transposon content in the genomes of biotechnologically important strains of the two fungi Aspergillus niger and Penicillium chrysogenum using the annotated genome sequences of strains CBS 513.88 and ATCC 28089. A compilation of transposon-like sequences identified in the two genomes is given. Single sequence sets have been analysed in more detail: For example the P. chrysogenum class II element PeTraII was identified, to our knowledge the first transposon known in this fungus. We further compared the distribution of selected elements in a set of different fungal strains, all emanating from each other, to investigate transposon mobility in classical strain improvement programs. As an additional approach to test for transposon activity we performed transposon trap analysis for both fungi. Moreover we are currently developing a system which allows us to test the activity of putative transposable elements identified during database analysis. References: Daboussi MJ, Capy P (2003) Annu Rev Microbiol 57, 275-99; Galagan JE, Calvo SE, Cuomo C et al. (2005), Nature 438, 1105-15; Pöggeler S, Kempken F (2004) In: The Mycota II, Genetics and Biotechnology, 2nd edition (ed. Kück U). Springer Verlag, Heidelberg, New York, Tokyo; Rep M, van der Does HC, Cornelissen BJC (2005) Fungal Genet Biol 42, 546-553


135. Horizontal gene transfer as the source of extra genes in Aspergillus oryzae. Nora Khaldi*, Kenneth H. Wolfe. Smurfit Institute of Genetics. University of Dublin Trinity College Dublin 2. Ireland.


Although horizontal gene transfer (HGT) is a major aspect of prokaryotic evolution and is becoming increasingly documented between prokaryotes and eukaryotes, very few cases of the HGT of nuclear genes from eukaryote to eukaryote have been documented so far. Several obstacles impede the unequivocal demonstration of HGT. The detection of foreign genes is facilitated if the recipient and donor genomes are from two different kingdoms, but if they are relatively closely related then observations suggestive of HGT may be equally consistent with alternative scenarios of gene duplication and gene loss. Aspergillus oryzae is a filamentous fungus that has a large gene complement compared to its annotated relatives. Here we test three alternative explanations for the existence of these extra genes in Aspergillus oryzae: recent gene duplication, whole genome duplication, and HGT. We show using gene location, evolutionary rate, and phylogeny based methods that HGT took place from the close sister subphylum Sordariomycetes into an ancestor of Aspergillus oryzae. Finally, we investigate the role of the transferred genes and show that transfer played a role in enriching Aspergillus oryzae’s gene repertoire, specifically by expanding its hydrolytic enzyme gene complement.

 

136. Characterisation of the Trichoderma reesei proteasome. L. Kautto1,2, J. Grinyer1, P. L. Bergquist1,2,3 , V. S. J. Te’o1,2, and K.M. H. Nevalainen1,2 1Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney NSW 2109, Australia. 2Macquarie University Biotechnology Research Institute, Macquarie University, Sydney NSW 2109, Australia. 3Department of Molecular Medicine and Pathology, University of Auckland Medical School, Auckland, New Zealand


The filamentous fungus Trichoderma reesei is one of the most efficient eukaryotic cell factories available. Considering its extraordinary secretion capacity, between 40 – 100 grams L-1 of protein, this species can be characterised as “a professional” protein secretor. High-level secretion proposes a challenge for the cellular machinery responsible for protein quality control, which is mainly carried out by UPR and ERAD. Ubiquitin-proteasome pathway plays a key role in the post –translational regulation in eukaryotic cells. Proteins that are not folded correctly or not fully assembled are recognised in the early secretory pathway featuring a large (approximately 2.4 MDa) multicatalytic protease, the proteasome. We are especially interested in exploring the function of the proteasome in ERADication of misfolded proteins from the secretory pathway by using mutant forms of an endogenous efficiently secreted cellulase enzyme as experimental molecules. We have produced a 2D master map for the T. reesei 20S proteasome. From this reference map, 13 of the 14 20S proteasome subunits and many interactive proteins that were co-purified with the 20S proteasome have been identified. We have prepared a series of mutant forms of the main cellobiohydrolase enzyme CBHI to trace their secretion and presumed degradation in the proteasome. We are currently in a process of characterising T. reesei transformants expressing the mutant proteins.


137. Systems Biology of the Biological Clock. Dong, Wubei Xiaojia Tang, Yihai Yu, Roger Nilsen, James Griffith, Jonathan Arnold, and H.-Bernd Schuttler. University of Georgia, Athens, GA 30602. arnold@uga.edu.


A model-driven discovery process called computing life is used to guide a series of 3 microarray experiments. As much as 25% of the N. crassa transcriptome is found to be circadian. A clock mechanism involving the genes wc-1, wc-2, and frq appears to have many different kinds of outputs including genes whose products are in DNA metabolism, RNA metabolism, cell cycle, protein metabolism, transport, carbon metabolism, development, isoprenoid (i.e., including carotenoid) biosynthesis, development, and varied signaling processes. The known clock mechanism involving the FRQ oscillator appears to explain only about 43% of the genes, which are circadian and light-responsive. Only about 58% of the White-Collar Complex (WCC)- controlled genes appear to be circadian or light-responsive, implying other control functions for WCC. In each of three cycles of microarray experiments through the computing life paradigm microarray data support that wc-1 and wc-2 should be autoregulated by WCC. Among 11,000 genes in the N. crassa genome a total of 295 genes, including a large fraction of phosphatases/kinases, appear to be under the immediate control of the FRQ oscillator as validated by 3 independent microarray experiments.


138. Withdrawn


139. Comparative genome hybridization to examine genome variability in Cryptococcus species. GG Hu, I Liu, A Sham, JW Kronstad. Michael Smith Laboratories, The University of British Columbia, Vancouver, BC, Canada


The basidiomyceteous fungal pathogen Cryptococcus neoformans includes four serotypes, A, B, C, and D, based on antigenic differences in the polysaccharide capsule that is the major virulence factor. The A and D serotypes mainly infect immunocompromised individuals, while B and C are now classified as a separate species, C. gattii, which infects both immunocompromised and immunocompetent patients. Strains with hybrid serotypes (AD and AB) have also been identified from clinical and environmental sources. There are also two mating types (MAT alpha and MATa) in each serotype, although strains of the alpha mating type are more frequently isolated and only genomes for MAT alpha strains have been sequenced. Genome sequence information is now available for five Cryptococcus strains (serotype A strain H99, serotype D strains JEC21 and B3501, and serotype B strains R265 and WM276). In the present study, we used the complete genome sequences of H99, JEC21 and WM276 to obtain high-density oligonucleotide genomic microarrays for comparative genome hybridization (CGH) experiments. We first examined the genomes of the MATa strain NIH433 and the MATalpha strain NIH12 that were the original parents of a cross that generated the strain JEC21. The hybridization of these genomes to a JEC21 array allowed the mapping of putative recombination sites and a comparison of the MAT regions. We then examined the genomic differences between MATalpha and MATa strains in serotype A strains (H99, 125.91, BT63), and serotype B strains (WM276 and E566). The CGH data for the MATalpha and MATa regions allowed us to establish the relationship between the CGH signal log ratio and sequence identity, and to interpret additional differences in genome content between the strains. The differences for a set of these regions were confirmed by PCR and sequencing. We further examined genome content of the AD hybrid strains KW5, CDC228 and CDC304 using both H99 (serotype A) and JEC21 (serotype D) genomic arrays. Interestingly, the CGH data indicated that chromosome 1 of all three AD hybrid strains and chromosomes 6 and 7 of KW5 most likely originated only from a serotype A genome. In addition, chromosome 5 of CDC304 and chromosome 8 of KW5 appear to originate from a serotype D genome. These conclusions are supported by PCR-RFLP analysis of selected sites on these chromosomes. Taken together, the CGH data provide the first insight into the differences in whole genome content among MATalpha and MATa strains and AD hybrids.


140. Transcriptome analysis of Lentinula edodes mature fruiting bodies by novel pyrosequencing. W.W.Y. Chum, I.S.W. Kwok, T.C.H. Au, H.S. Kwan. The Chinese University of Hong Kong. Shatin, HKSAR


Developmental processes of basidiomycetes from vegetative mycelium to mature fruiting bodies for sporulation are dramatic morphological changes. We attempted to understand the biological mechanisms through transcriptome analysis of fruiting bodies of Lentinula edodes using high-throughput Genome Sequencer 20 System (454 Life Science). Combined cDNA from mature fruiting bodies before and during sporulation of L. edodes were sequenced to isolate a total of 7481 cDNA contigs. Each contig was generated from a number of reads of cDNA that represent the number of cDNA fragments aligned for the contigs. Numbers of reads/length may be proportional to gene expression levels of the contigs revealed by comparison with LongSAGE results of L. edodes. All contigs were homology searched by BLASTX. However, there is no genome data available for L. edodes studies, we developed a program to match the contigs from other databases followed by categorization with Gene Ontology (GO). Among a total 1202 annotated contigs, 909 contigs matched to sequences in database and their biological processes were classified by GO. Top 100 contigs in numbers of reads/length were analyzed and some of them may be related to mature fruiting body development or spore formation. About 50 contigs were novel. To our knowledge, this is the first study in sequencing fungi cDNA by using the GS20 for transcriptome analysis. The results greatly advance our knowledge in biological mechanisms of mature fruiting body and sporulation of mushroom.

 

141. Transcriptome analysis reveals differentially expressed genes involved in increased nuclear kinetics and polar growth in the Aspergillus nidulans ATM (atmA) null mutant. Iran Malavazi and Gustavo H. Goldman, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil


Ataxia telangiectasia (AT) is an inherited disorder characterized by progressive loss of motor function and susceptibility to cancer. Numerous studies have emphasized the role of the affected gene product, ATM, in the regulation of the DNA damage response. Recently, in addition to its expected role in the DNA damage response, we found that the Aspergillus nidulans homologue, AtmA, is also required for nuclear proliferation and polarized hyphal growth. For a comprehensive evaluation of genes that have their expression modulated by ATM, here by using a microarray hybridization approach, we performed two sets of comparisons of the mutant with the wild type strain: (i) growth in complete medium for 60, 90, and 120 minutes, and (ii) HU block-release experiments for polar growth (60, 90, and 120 minutes release after 5 hours HU-growth). The first set of experiments revealed that several genes involved in DNA replication and in the pentose phosphate pathway (ppp) are more expressed in the delta atmA mutant. We constructed double mutants for delta atmA and two mutations impaired in the ppp, pppA (probably encoding a transaldolase) and pppB. The double mutants have a decreased nuclear proliferation rate when compared to the delta atmA mutant, suggesting that the increased expression of the ppp genes could be important for increased nuclear proliferation in the delta atmA mutant. The second set of experiments with global expression associated to polar growth revealed a decreased mRNA expression for the following group of genes in the delta atmA mutant: (i) genes involved in the formation of a polarized hyphae (e.g., cdc42) and control of polar growth; (ii) genes involved in the synthesis of phosphatidic acid (1,2-diacyl-sn-glycerol 3-phosphate); and (iii) genes involved in the ergosterol biosynthesis (plasma membrane microdomains, lipid rafts). We are currently investigating some of these genes by constructing double mutants with them and delta atmA. Financial support: FAPESP and CNPq, Brazil


142. Transcriptional profiling of cross pathway control in Neurospora crassa: Comparative analysis of the Gcn4p and CPC1 regulogs. Chaoguang Tian, Takao Kasuga and N. Louise Glass. Plant and Microbial Biology Department, University of California, Berkeley, CA 94720


Identifying and characterizing transcriptional regulatory networks is an important task in elucidating gene function and how these regulatory networks evolve. In Saccharomyces cerevisiae one of the most intensely studied transcription factors is the bZIP transcription factor, Gcn4p. Gnc4p is essential for transcriptional response when S. cerevisiae experiences amino acid starvation. In the filamentous ascomycete fungus, Neurospora crassa, the ortholog of GCN4, called cross pathway control (cpc-1) is required for the ability of N. crassa to induce a number of amino acid biosynthetic genes upon response to amino acid starvation. In this study, we decipher the CPC1 regulog by transcriptional profiling wild-type and a cpc-1 mutant using full genome N. crassa 70-mer oligonucleotide microarrays. We show that 443 genes are either direct or indirect CPC1 targets, including 67 amino acid biosynthetic genes, 16 tRNA synthetase genes and 13 vitamin-related genes. Comparison between the N. crassa CPC1 and S. cerevisiae Gcn4 regulogs showed that regulation of only 18 genes is conserved between these two species, and primarily include amino acid biosynthetic genes. Exploration of regulatory networks in N. crassa and S. cerevisiae sheds light on how gene interaction networks evolve.


143. Development and evaluation of an Affymetrix array for Aspergillus flavus. Andrea Dolezal1, David Ryan Georgianna1, Greg OBrian1, Charles Woloshuk2, Nancy Keller3, Jiujiang Yu4, Dahlia Nielsen1, Gary Payne1. 1 North Carolina State University, Raleigh, NC. 2 Purdue University, West Lafayette, IN. 3 University of Wisconsin, Madison, WI. 4USDA/ARS/SRRC, New Orleans, LA.


A multi-species Affymetrix GeneChip array was developed to study development, metabolism, and pathogenicity of A. flavus. This chip, based on the whole genome sequence of A. flavus, contains 13,000 A. flavus genes, 8,000 maize genes, and 25 human and mouse innate immune response genes as well as the fumonisin and trichothecene clusters from Fusarium. These arrays were used to monitor gene expression of A. flavus during aflatoxin biosynthesis in defined media and during infection of developing maize seeds. A parallel study comparing this array with a 5002 element cDNA array showed the same expression profile for the aflatoxin biosynthetic genes when A. flavus was grown in culture on defined media at conducive and non conducive temperatures for aflatoxin production. Gene expression was also monitored in A. flavus during the infection of field grown maize seeds. The profile of aflatoxin gene expression by A. flavus in infected maize kernels was similar to that observed for A. flavus grown in the lab under conductive temperatures for aflatoxin production. In addition, several genes encoding enzymes for the metabolism of complex carbohydrates and for transporters were also elevated during infection of maize seeds. Nonspecific hybridization across species has not been observed in any of our experiments using the Affymetrix GeneChip. These initial observations show that these multi-species arrays will be a powerful tool for studying the complex ecology and metabolism of A. flavus. This research was funded by USDA/NRI/CGP 2006-35604-16666.


144. Gene expression analysis of the basidiomycete wood decay fungus (Heterobasidion parviporum) during adaptive growth on wood and toxic cell wall phenolic (ferulic acid). Fred O. Asiegbu1,*, Guosheng Li1 and Jason Osborne2. 1Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, 750 07, Uppsala, Sweden 2Department of Statistics, North Carolina State University, Raleigh, USA. Email: Fred.Asiegbu@mykopat.slu.se


The success of many wood rotting fungi lies in their ability to overcome and survive in a phenol-phytoalexin rich toxic environment within litter, wood debris and tissues. Although so much has been learned about the ecology, taxonomy and physiology of several wood decaying basidiomycete fungi, the molecular basis for their survival in a diverse range of substrates and ecological habitats has been very little studied. Using the necrotrophic wood decay fungus (Heterobasidion sp.) as an experimental model, we investigated its transcriptome response during growth on a woody substrate and further compared how the gene expression pattern is affected by exposure to toxic cell wall phenolics (ferulic acid), simple sugars (glucose) and oxalic acid (a component of hyphal exudates). We documented elevated transcription of significantly more genes in glucose starved media (such as in ferulic acid) which probably indicates that the phenolic compound may constitute an important alternative energy source during periods of glucose starvation. The result also revealed similarity in expression profiling pattern of oxalate and glucose media or ferulic acid and wood which suggest that the fungus might use the same genetic programs for responding to growth on certain substrates. The results also indicate that some genes in fungi that determine the outcome of growth on a diverse range of substrates may share common regulatory pattern. The importance of the results in the identification of genes controlling important traits and their regulation during exposure to different substrates as well as how it applies to other related wood rotting fungi is discussed.


145. Comparative Verticillium Genomics. Steven J. Klosterman1 Krishna V. Subbarao2, Katherine F. Dobinson3, Paola Veronese4, Seogchan Kang5, Scott E. Gold6 and Li-Jun Ma7. 1USDA, Agricultural Research Service, Salinas, CA 2University of California, Davis, CA 3Agriculture and Agri-Food, London, ON Canada 4North Carolina State University, Raleigh, NC 5Penn State University, University Park, PA 6University of Georgia, Athens, GA 7The Broad Institute of MIT and Harvard, Cambridge, MA

The genus Verticillium contains several phytopathogenic species, the most important being V. dahliae, V. albo-atrum, and V. longisporum. The soil-borne habitat of these species, and their capacity to infect a variety of crops, make them chronic economic problems in crop production. Verticillium wilt causes billions of dollars in annual losses worldwide. A comparative genomic approach has been initiated to study the evolutionary processes and genetic mechanisms that underpin the pathogenic development of soilborne fungi among Verticillium spp. For this purpose, we will generate a 7X genome assembly of V. dahliae and a 4X genome assembly of V. albo- atrum, two verticillium species that have distinct phenotypic variation and host ranges. Both of these assemblies, coupled with gene annotation, will provide the tools for comparative analyses to improve annotation, identify SNPs and chromosome structure changes, and create sets of conserved and species-specific genes among these two soilborne pathogens. In addition, 0.5X Fosmid sequence reads of V. longisporum will enable the assessment of whether the diploidization of V. longisporum arose as a result of inter- or intraspecific hybridization events. The synergistic power of comparative genomics will speed Verticillium research toward a greater understanding of pathogenicity. This knowledge will enable new methods for improving disease detection and management.


146. Comparative genomic analysis of Aspergillus fumigatus and sequenced close relatives. William C. Nierman1, Vinita Joardar1, Rama Maiti1, Paolo Amedeo1, David Denning2 Jennifer Wortman1, and Natalie Fedorova1. 1The Institute for Genomic Research, Rockville, MD, USA. 2School of Medicine and Faculty of Life Sciences, The University of Manchester, Manchester, UK


Comparative genomic analyses of pathogenic Aspergillus fumigatus, and closely related species revealed a large pool of variable genes, which may ensure the rapid adaptation to a range of environments. These variable genes are structurally distinct from the rest of the genome and tend to function in secondary metabolite biosynthesis, transport, and detoxification of xenobiotics. Many are up-expressed in a mammalian host suggesting that they may be essential for survival in this environment. In contrast, all previously identified virulence-associated genes are well conserved in all sequenced aspergilli and seem to be evolving under strong purifying selection. The variable genes tend to cluster together in subtelomeric and intrasyntenic chromosomal blocks called accessory zones that share several characteristics with prokaryotic genomic islands. The origin of accessory genes in aspergilli does not appear to involve lateral gene transfer. This analysis also shows that the aspergilli have utilized several mechanisms to ensure a quick diversification and turnover of secondary metabolism gene clusters in the course of evolution.


147. Transcriptome analysis of shiitake mushroom Lentinula edodes development using multiple approaches. W.W.Y. Chum, I.S.W. Kwok, X.L. Bian, K.T.P. Ng, R.S.M. Shih, G.S.W. Leung, T.C.H. Au, and H.S. Kwan. The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR


Fruiting body development is an important area in mushroom biology and have been analyzed at the molecular level. We aim to analyze the transcriptome during development of Shiitake mushroom Lentinula edodes. First, we used RNA fingerprinting with arbitrarily primed polymerase chain reaction (RAP or Differential Display) to isolate genes differentially expressed. Over 100 genes were isolated and sequenced. Fifteen were studied further. Second, cDNA clones were randomly sequenced to generate about 1000 Expressed Sequence Tags (ESTs). Differential expression of the ESTs were analyzed by dot-blot hybridization and cDNA microarray analysis. Third, Serial Analysis of Gene Expression (SAGE) and LongSAGE were used to determine the proportion of each mRNA among total transcripts in various growth stages. About 30,000 transcripts were counted from seven developmental stages. Expression profiles of the tags were annotated and clustered. About 800 tags matched to our cDNA sequence collection. Forth, over 13000 cDNA sequence contigs were obtained by high-throughput sequence-by-synthesis technology. These cDNA were annotated using programs we developed. Fifth, differential expression of selected genes were confirmed using Northern analysis and real-time RT-PCR. A few genes were localized in various tissues by in situ RNA-RNA hybridization. Gene expression profiles revealed by different approaches were compared and were generally consistent.


148. Isolation and analysis of genes expressed in lignocellulose-grown Lentinula edodes using novel sequencing-by- synthesis approach. Iris S.W. Kwok, Winnie W.Y. Chum, Tommy C.H. Au and H.S. Kwan. Department of Biology, The Chinese University of Hong Kong, Shatin, HKSAR, China


The edible shiitake mushroom Lentinula edodes degrades lignin and cellulose efficiently and completely, so it has been widely used in bioconversion and bioremediation. A cDNA pool prepared from mycelium grown on lignocellulose was sequenced using pico-titered plate based genome sequencer (Roche). 5894 cDNA contigs were obtained. Their sizes ranged from 100bp to 2 kb. More than 2600 contigs could be annotated by BLAST homology search, one third of the matches were highly significant (E-value >10-5). Using Gene Ontology (GO), these contigs were categorized according to their biological functions and processes involved. We found genes encoding ligninolytic enzymes and cellulolytic enzymes, such as chloroperoxidase, laccase, a and b-glucosidases, and endo-1,3(4)-b-glucanase. We have also obtained genes encoding antioxidative thioredoxins, glutaredoxin, superoxide dismutase (SOD) and catalase. The expression levels of SOD and catalases in mycelia grown in lignocellulose and non-lignocellulose media were compared with quantitative RT-PCR. Surprisingly, their expressions are higher in non-lignocellulose medium. This indicates that growing in lignin, L. edodes is not encountering strong oxidative stress despite that free radicals are produced to degrade lignin. These findings provide new insights on the lignocellulytic system and also the antioxidative activity during lignin degradation of L. edodes.


149. The Neurospora crassa e-Compendium: integrating genetic and genomic data. A. Radford, Biological Sciences, The University of Leeds.

The e-Compendium is the logical development of earlier printed Compendia (Barratt et al (1954) Adv Gen 6: 1-93; Perkins et al (1982) Microbiol Rev 46: 426-570; Perkins et al, The Neurospora Compendium, Academic Press, 2001), integrating the genetic data accumulated over seven decades with the genome sequence and annotations of the last few years. It presents on line, in searchable form, data on over 2,100 genes structured into fields for gene symbol, name, linkage, product, phenotype, contig, sequence, reference, etc., with web links to sequence at Broad, bibliography at Pubmed, enzyme at Expasy, structure at PDB, pictures of morphological mutants at various locations, etc.. The search function permits keyword searches singly and in combination in different database fields. The e-Compendium also includes compiled chromosome maps based on both genetic and genomic data, and including over 1,700 sequenced genes. These include the products of the author's recent studies of novel genes for tRNA, rRNA, proteases, glycosyl hydrolases etc.. Monitoring software shows that the e-Compendium has been used by over 1,300 individual users in the past 12 months, over half of whom have used the site repeatedly.


150. Searching for the Rosetta stones in the multifunctional proteins of the Phytophthora genomes. Tom Wittenschlaeger 1, Ryan Austin2, Nicholas Provart2, Paul F Morris 1. 1 Department of Biological Sciences, Bowling Green State University, Bowling Green OH 43403, 2Cell and Systems Biology University of Toronto, Toronto ON M5S 3G5.


Eukaryotic genomes have in common a large number of multifunctional proteins. A global survey of the oomycete Phytophthora sojae genome, identified 274 novel multifunctional proteins using strict criteria that excluded multi- exonic gene models without EST support. These P. sojae proteins have significant BLAST hits to two or more different proteins. Such proteins have been posited as Rosetta stones, since their association in one genome has been used to infer the association of orthologous proteins in other genomes. In our analysis, we adopted the reciprocal smallest distance algorithm (Wall et al 2003) to identify potential orthologs in 34 sequenced genomes. Surprisingly, this approach identified only seven potential Rosetta stones, where each domain of a multifunctional protein had an ortholog in the same organism. A separate phylogenetic analysis has identified several examples where each half of a multifunctional protein, clusters in a node with homologs from separate kingdoms. The evolutionary history of oomycetes involved the endosymbiotic acquisition of a red algae, and subsequent transfer of nuclear and plastid genes to the host nucleus. We postulate that this endosymbiotic event (genome acquisition and recombination) has enabled the ancestral genome to develop metabolic and regulatory pathways that are distinct from those of the animal, fungal and plant genomes. Oomycete pathways that include genes from plant and animal ancestral genomes may have metabolic and regulatory efficiencies that are not present in other organisms. Our observations suggest that the evolutionary strategy of genome acquisition and recombination should also be assessed in other members of the Chromalveolates.


151. Full-Length cDNA Sequences for Genome Annotation in Phytophthora infestans. William Morgan1, Joe Win1, Liliana Cano1, Michael C. Zody2, Chad Nusbaum2, Sophien Kamoun1. 1Department of Plant Pathology. The Ohio State University, Ohio Agriculture Research and Development Center, Wooster, OH 44691, USA. 2The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.


Phytophthora infestans is a devastating phytopathogenic oomycete that causes late blight on tomato and potato. Recent genome sequencing of P. infestans and other Phytophthora species resulted in vast amounts of sequence data providing opportunities to unlock the complex nature of pathogenesis. However, accurate annotation of Phytophthora genomes is a significant challenge. Full-length cDNA (FLcDNA) sequences are essential for the correct annotation of genomic sequences and for downstream functional analyses. We initiated three separate approaches to generate novel FLcDNA resources for P. infestans. First, bioinformatics analysis of 31592 P. infestans 5’ ESTs identified 4943 putative FLcDNAs predicted to contain a complete open reading frame. Of these, we sequenced the inserts of 480 clones corresponding to unique sequences. Second, we constructed and sequenced normalized FLcDNA libraries using mRNA isolated from various developmental stages of P. infestans strain T30-4 to acquire additional FLcDNA sequences. Finally, we constructed new cDNA libraries that are adapted for high throughput sequencing of 5’ mRNA ends using 454 sequencing. The accurate picture of P. infestans gene structure that emerges will help us develop more precise gene predictions algorithms. In addition, the FLcDNA sequences will allow us to gauge the degree of sequence conservation between P. infestans genes and those of other oomycetes, and identify patterns of gene conservation between P. infestans and various eukaryotes.


152. Proteomic analyses of developmental stage specific proteins in Aspergillus nidulans. Yun-Hee Park1, Eun Hye Kang1, Sujin Kim1, Ja-Young Yun1, Kwang- Yeop Jang2, Keon-Sang Chae2, Dong-Min Han3, Suhn-Kee Chae4, Pil Jae Maeng1 and Hee-Moon Park1. 1Dept. Microbiol., SBB, Chungnam National University, Daejeon; 2Div. Biol. Sci., Chonbuk University, Chonju; 3Div. Life Sci., Wonkwang University, Iksan; 4Dept. Life Sci. & Technol., Paichai University, Daejeon, South Korea.


Aspergillus nidulans which has three different cycles, such as asexual, sexual and parasexual cycle, showed various morphological changes during vegetative and development. For a global analysis of developmental stage specific proteins, we investigated the changes in protein expression profiles during developmental stage. We decided the time points for harvesting cultures which revealed specific change of morphology, such as vegetative growth 9 and 14 hr, asexual development 9 and 18 hr, hypoxia condition 12 hr and sexual development 1, 6, and 24 hr. We extracted total proteins from each stage and performed two-dimensional polyacrylamide gel electrophoresis (2-DE). All 2-DEs were independent and repeating three-times for every stages. The PDQuest image analysis of silver stained 2D-gel revealed about 2400 protein spots. Among those 301 spots with meaningful changes along with the developmental stages, were identified by peptide mass fingerprinting (PMF).


153. Transcriptome analysis during sexual development in Aspergillus nidulans. Yeong Man Yu1, Yong Jin Kim1, Dong-Min Han2, Kap-Hoon Han3, Hee-Moon Park1, Suhn-Kee Chae4, Kwang-Yeop Jahng5, Keon-Sang Chae5, Jong-Hwa Kim3, Tae Jeong Oh6, and Pil Jae Maeng1*. 1Chungnam National Univ., Daejeon; 2Wonkwang Univ., Iksan, Chonbuk; 3Woosuk Univ., Wanju, Chonbuk; 4Paichai Univ., Daejeon; 5Chonbuk National Univ., Chonju, Chonbuk, 6GenomicTree Inc., Daejeon, Korea


In Aspergillus nidulans, although its genome has been fully sequenced, microarray analysis is still in its infancy. Here, we describe analysis of microarrays for A. nidulans using a 70-base-oligomer microarray that assays 10,580 representing ORFs from. To estimate the relative gene expression levels and changes in gene expression during sexual development, we analyzed the microarrays throughout a time course during sexual development using a AnURR (A. nidulans Universal Reference RNA) in all hybridization. In the oligomers designed for annotated genes, we obtained 9,698 reliable genes (92%). The array revealed time-dependent expression of distinct genes set. The most significantly regulated genes (P < 0.01) were grouped in six clusters based on their expression profile. Functional group of the genes contained in each cluster were analyzed by using the MIPS classification. We found several groups of genes that are expected to be involved in the regulation of sexual development at transcriptional level.


154. Microarray profiling of gene expression of Aspergillus nidulans in response to hypoxic stress. Yeong Man Yu1, Hye Jun Yun1, Dong-Min Han2, Kap-Hoon Han3, Hee-Moon Park1, Suhn-Kee Chae4, Kwang-Yeop Jahng5, Keon-Sang Chae5, Jong-Hwa Kim3, Tae Jeong Oh6, and Pil Jae Maeng1*. 1Chungnam National Univ., Daejeon; 2Wonkwang Univ., Iksan, Chonbuk; 3Woosuk Univ., Wanju, Chonbuk; 4Paichai Univ., Daejeon; 5Chonbuk National Univ., Chonju, Chonbuk, 6GenomicTree Inc., Daejeon, Korea


In all aerobic organisms, oxygen is the terminal electron acceptor in the respiratory chain and is required for ATP generation via oxidation phosphorylation. Thus hypoxia is expected to affect the expression of many genes involved in cellular bioenergetics, sterol, heme, and fatty acid synthesis. In order to figure out the change of transcriptome under hypoxic conditions, we analyzed the profiles of mycelia exposed to hypoxic stress by using a 70-base-oligomer microarray. The array revealed hypoxia-specific expression of distinct genes set. The most significantly regulated genes (P < 0.01) were grouped in four clusters based on their expression profile. The clusters analyzed by using the MIPS classification. Our transcriptional profiling data seems to correlate well with the change in biochemical and physiological processes caused by the hypoxic stress. Several groups of genes expected to be involved in the regulation of hypoxic genes were found.


155. Gene clusters for secreted proteins in smut fungi: Determinants for the biotrophic lifestyle. Jan Schirawski, Thomas Brefort, Julia Schöning, Kerstin Schipper and Regine Kahmann. Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany


The two related basidiomycetes Ustilago maydis and Sporisorium reilianum cause smut disease in maize. Upon penetration of the maize cuticle both fungi proliferate extensively within the plant apoplast. While U. maydis induces tumors filled with fungal spores on all aerial parts of its host, spore formation in S. reilianum occurs without prior tumor induction exclusively in the inflorescence. To define determinants of compatibility, we focused on the predicted secretome. In U. maydis a surprising number of secreted protein-encoding genes occur clustered. These clusters seem to encode virulence factors, since deletion of individual clusters in U. maydis in many cases affected pathogenicity. To define conservation/divergence between S. reilianum and U. maydis potentially explaining differences in disease progression, we sequenced the genome of S. reilianum and compared the cluster loci. In U. maydis deletion of the largest cluster (19A) containing 23 genes for potentially secreted proteins resulted in loss of tumor induction and spore formation without affecting preceding developmental stages. A detailed microscopic analysis of the cluster 19A deletion mutant in planta suggested recognition by the plant defense machinery at late stages of development. Intriguingly, interspecies comparison of the cluster loci revealed significantly lower conservation between cluster genes than between neighboring genes. These findings suggest that the secreted cluster proteins are under diversifying selection, potentially arising from the different modes of disease development. Currently we determine localization of secreted proteins of U. maydis cluster 19A in planta and analyze the role of the corresponding genes during biotrophic development of S. reilianum.


156. Functional characterization of acetylglutamate synthase and phosphoribosylamine-glycine ligase genes in Gibberella zeae. Jung-Eun, Kim1, Kilseon Myong1, Won-Bo Shim2, Sung-Hwan Yun3, and Yin-Won Lee1. 1School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea 2Department of Plant Pathology and Microbiology, Texas A&M University, USA 3Department of Biological Resources and Technology, Soonchunhyang University, Asan 336-745, Korea


Gibberella zeae (anamorph, Fusarium graminearum) is an important pathogen of cereal crops in many regions of the world. In this study, we characterized two auxotrophs (designated S4B1279 and S4B3008) that were recovered from a collection of insertional mutants of G. zeae created by the restriction enzyme-mediated integration (REMI) procedure. Both mutants showed pleiotropic phenotypes including reduction in mycelial growth and virulence, and no sexual reproduction. Molecular analyses of the REMI mutations revealed that genes responsible for the arginine and adenine auxotrophy in the strains S4B1279 and S4B3008 are ARG2 (encoding a putative acetylglutamate synthase) and ADE5 (encoding a phosphoribosylamine-glycine ligase), respectively. Subsequent gene deletion and complementation analyses confirmed the functions of ARG2 and ADE5 in G. zeae. In addition, complementation of the auxotrophs with intact copy of ARG2 or ADE5 indicated that both ARG2 and ADE5 can be used as new dominant selectable markers for G. zeae


157. Symbiosis Insights from the Genome of the Mycorrhizal Basidiomycete Laccaria bicolor. Francis Martin, Joint Genome Institute and Laccaria Genome Consortium (fmartin@nancy.inra.fr)


Laccaria bicolor is a ubiquitous fungal symbiont of trees in boreal and temperate forest ecosystems. This biotroph has a beneficial impact on plant growth, yet our understanding of the interactions driving this ectomycorrhizal association are poorly known. To elucidate the genetic basis of this ecologically important behavior, we have sequenced the 65 Mb L. bicolor genome The genome assembly contains about 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences (i.e. 21% of the assembled sequence) which contribute to its large size compared to known fungal genomes. Analysis of this gene set yields insights into unexpected aspects of the symbiont biology including the identification of genes potentially associated with saprobic organic matter decay and pathogenesis. This fungus also possesses expanded protein families associated with plant interactions, such as hydrolases, signaling proteins, and small secreted proteins. WD40 repeat- and TPR-containing proteins – key integrators of stress and nutrient availability signals - are very abundant. Comparison of the genomes of pathogenic and saprobic fungi with the L. bicolor genome reveals that the symbiont versatile genome encodes all the features corresponding to the major fungal lifestyles, i.e., saprophytism, pathogenesis and symbiosis. I will close with some thoughts on other strategies, including transcriptomics and proteomics, we are undertaking to fully appreciate the molecular basis of fungal symbiosis.


158. Browsing C. albicans Genomic Information at the Candida Genome Database. Martha B. Arnaud, Maria C. Costanzo, Marek S. Skrzypek, Prachi Shah, Gail Binkley, Stuart R. Miyasato, and Gavin Sherlock. Candida Genome Database, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305- 5120


Candida Genome Database (CGD, freely accessible at http://www.candidagenome.org/ ) contains genomic sequence data and information about the genes and gene products of the opportunistic human fungal pathogen Candida albicans. CGD supports genome viewing and browsing using GBrowse, an open-source tool developed as part of the Generic Model Organism Database project ( http://www.gmod.org/ ). CGD provides access to the two most current versions of the C. albicans genome sequence, Assemblies 19 and 20. The Assembly 19 contigs and the Assembly 20 chromosomes are independently navigable. The tool displays a user- configurable set of Tracks, which each show a class of features, for example ORFs, tRNAs, or contigs. CGD has generated sequence-based mappings of the contigs from Assemblies 4, 6, and 19 and the ORFs from Assemblies 6 and 19, onto the chromosomes from Assembly 20, and mappings of the older assembly information onto the Assembly 19 contigs. These historical tracks are available in GBrowse, where they serve as a graphical representation of the evolution of the genome assembly. GBrowse may also be used to view the results of any BLAST query. To view the genomic context of the sequence match, select the link from CGD BLAST results page. GBrowse also allows downloading of any selected region of DNA or protein sequence, annotation of restriction sites recognized by a configurable list of restriction endonucleases, and export of publication-quality images. CGD curators welcome comments and suggestions by email, at candida- curator@genome.stanford.edu. CGD is supported by grant RO1 DE15873 from the NIDCR at the NIH.


159. Determining mode of antifungal activity of phytochemicals using S. cerevisiae and genetic array technologies. Nadereh Hannah Mir-Rashed1, Imelda J. Galvan2, John T. Arnason3, Myron L. Smith1. 1 Biology Department, Carleton University, Canada.2 Chemistry Department, Universidad Autónoma de Aguascalientes, México. 3 Biology Department, University of Ottawa, Canada.


Host toxicity, a limited spectrum of activity, and propensity to induce or select for resistant fungal pathogens by many current antifungals has created a need for additional agents to treat fungal infections. We have studied how S. cerevisiae responds to sub-lethal exposures of plant-derived compounds, including the alkaloid berberine, cinnamodial, chimaphilin and those in Echinacea extracts. DNA microarray analysis of yeast grown in the presence of berberine revealed significant differential RNA transcript levels for 106 genes which were assigned to seven categories including cell cycle/division, metabolism, cell wall, gene expression, transport/secretion and unknown functions. Based on this transcriptional response, we hypothesize that berberine is an effective antifungal because, at least in part, it is not apparently recognized as a xenobiotic. A yeast Gene Deletion Array (GDA, ~4600 gene deletion mutants) was also used to examine chemical-genetic interactions with yeast exposed to each compound. These experiments provide insight into antifungal mode of action and thus add to the value of these bioactive compounds. For example, a significant pattern to emerge from the GDA analysis was that Echinacea extracts interfere with fungal cell wall functions. Furthermore, the experiments provide a template in medicinal plant research. The future direction of the study is to link the collected data to the antifungal drug discovery and development processes.


160. Telomere instability in Magnaporthe oryzae caused by highly mobile, telomere-targeted tranposons. Mark Farman & John Starnes. Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA


 Magnaporthe oryzae isolates that infect perennial ryegrass have unusually unstable telomeres that undergo continual rearrangements in culture and in planta. Sequencing revealed that the chromosome ends of the ryegrass pathogens are organized very differently to those of a rice-infecting strain with stable telomeres. Specifically, the subtelomeres consist of tandem arrays of two types of repetitive elements, with canonical telomere repeats at the ends of the arrays. The elements are found only in subtelomeric locations, so we have named them M. oryzae telomere- exclusive repeats (MoTERs). MoTER1 is 4.6 kb in length and codes for a reverse transcriptase. It lacks terminal repeats and, therefore, appears to be a non-LTR retrotransposon. MoTER2 is only 1.7 kb long and potentially codes for a 204aa protein of unknown function. We have identified two new insertions of MoTER1 at telomeres that originally lacked MoTER elements, thereby demonstrating that MoTER1 is an active transposon. The structures of MoTER1 and MoTER2, and the way they are organized at the chromosome ends, are reminiscent of the retrotransposons TART and HeT-A, which are responsible for maintaining the Drosophila telomeres. Therefore, we hypothesize that M. oryzae strains from ryegrass have dual systems for telomere maintenance – one based on telomerase, and another which uses terminally targeted retrotransposons to repair degraded ends that arise during telomere crisis. Ironically, it appears that the presence of the MoTER elements causes the telomeres to be unstable in the first place – an apparent case of a selfish element working hard to ensure the security of its own job!


161. mRNA profiling revealed enrichment of orphan gene transcripts during asexual development of a filamentous ascomycete Neurospora crassa. T. Kasuga1, H. H. Wilkinson2, D. Ebbole2, B. Shaw2, G. Mannhaupt3, J. Taylor1 and L. Glass1. 1Dept. Plant & Microbial Biology, Univ. California, Berkeley, CA. 2Dept. Plant Pathology & Microbiology, Texas A&M Univ., College Station, TX. 3MIPS, Neuherberg, Germany.

We asked if there is any correlation between the pattern of expression and phylogenetic age for Neurospora crassa genes. N. crassa genes were classified into six mutually exclusive groups (Cai et al., J. Mol. Evol 2006): (1) Eukaryote/Prokaryote-core, a group of genes whose orthologs were identified in non-fungal Eukaryotes and/or Prokaryotes, (2) Basidiomycota-core, genes shared with basidiomycetes, (3) Hemiascomycete-core, (4) Euascomycete- specific, (5) N. crassa orphans, which lack sequence similarity to any of the genes in SIMPS database and (6) Remainder. We then examined the mRNA profiles obtained during vegetative growth and conidiation using N. crassa microarray representing all the predicted ORFs (c.a. 11,000). We found an over-representation of Euascomycete-specific genes in very young hyphae (0 to 3 hours old) and in mature hyphae engaged in conidiation (18 to 27 hours old; enrichment test, p <0.01). On the other hand, Eukaryote/Prokaryote-core genes showed peak expression pattern in 6 to 15 h old hyphae. The most notable finding was the expression timing of N. crassa orphan genes. There are c.a. 2,000 orphan genes in the N. crassa genome and 95% of them do not have functional annotation. N. crassa orphan genes were over-represented during development of aerial hyphae and conidiation, which coincided with development of the conspicuous and distinctive asexual morphology characteristic of this fungus.


162. Functional analysis of genes regulated by MAT1-2 in Gibberella zeae using a proteomics approach. Seung-Ho Lee1, Sung-Hwan Yun2, and Yin-Won Lee1. 1School of Agricultural Biotechnology, Seoul National University and Center for Agricultural Bio-materials, Seoul 151-921; 2Department of Biological Resources and Technology, Soonchunhyang University, Asan 336-745, Korea


The homothallic ascomycetes Gibberella zeae (anamorph = Fusarium graminearum) is an important pathogen with ubiquitous geographic distribution and causes severe diseases on cereals. Ascospores formed within the sexual fruiting body (perithecium) of G. zeae can overwinter and initiate the primary infection in the next spring. Thus, a greater understanding of sexual development in G. zeae is needed for a comprehensive control strategy of the diseases caused by this fungus. We have focused on identifying the protein specifically controlled by MAT1-2, a master regulator of sexual reproduction in G. zeae. To select differentially expressed proteins under control of MAT1-2 during the perithecial stage, we compared the protein profiles of a self-fertile G. zeae and its isogenic strain deleted for MAT1-2 using a two-dimensional electrophoresis. Total 384 protein spots were visualized by silver staining and 12 spots were differentially expressed during sexual reproduction mode; only two proteins were highly expressed in the MAT1-2 deletion mutant. We identified genes corresponding to these proteins from the genome database and determined their gene expression patterns and functional requirement for sexual development. Of 13 genes examined, 10 genes were essential for either perithecia or ascospore formation in G. zeae. This study is the first report on functional analyses of a set of genes controlled by MAT1-2 at the protein level.


163. Genetic questions that we expect to answer by sequencing the genome of the white-rot basidiomycete Pleurotus ostreatus. Antonio G. Pisabarro, Lucía Ramírez Public University of Navarre, Pamplona, Spain. E-mail: gpisabarro@unavarra.es


The sequencing of the P. ostreatus genome been scheduled for 2007 by the JGI. P. ostreatus has been studied as edible fungus, as model for lignin degradation and as source of poly-aromatic hydrocarbon degrading enzymes. However, the genetic questions that can be addressed using this organism as model are far beyond that. This organism is dikaryotic in all its life cycle except at the basidia. This allows separating the two nuclei present in a mature sexually competent individual to study both parental haplotypes. Electrophoretic analyses of P. ostreatus have revealed prominent length polymorphisms in homologous chromosomes. The sequence of the two sets of homologous chromosomes will make available the two haplotypes of a mature organism for the first time. This will permit to determine the minimal synteny between homologous chromosomes and to study the meiotic behaviour of structurally polymorphic chromosomes. In P. ostreatus various quantitative loci (QTLs) have been identified whose genetic architecture is poorly known. The sequence of the two haplotypes will permit to study this architecture and to evaluate the contribution of other genomic regions interacting with it. Finally, the study of the contributions of the two haplotypes to subcellular structures (nucleus, mitochondria) will be possible, and this will open the door to deeper analyses


164. Phenotypic and metabolomic analyses of the Fusarium graminearum snf1 mutant. Martin Urban, John Antoniw, William Allwood, Andrew Beacham, Jane Ward, Mike Beale and Kim Hammond- Kosack. Rothamsted Research, Herts, AL5 2JQ, UK; Kim.Hammond-Kosack@bbsrc.ac.uk


The Ascomycete fungus Fusarium graminearum (Fg) is a ubiquitous plant pathogenic fungus in all major cereal growing areas of the world. Infections, which occur during plant anthesis, lower grain quality and contaminate grains with trichothecene mycotoxins. The genome was sequenced by the BROAD Institute and completely aligned to the genetic map. Fg has 4 chromosomes and minimal repetitive DNA (http://www.broad.mit.edu/annotation/fungi/fusarium) . In an effort to identify the set of pathogenicity genes in Fg and other fungi, a database of known fungal and Oomycete pathogenicity genes was established at Rothamsted Research ( http://www.phi- base.org) . We have identified all the Homologues of verified pathogenicity and virulence (HvPV) genes in Fg, and created a chromosome visualisation software, called Mycomap to inspect visually the chromosomes for clustering of HvPV genes. This analysis identified a region on chromosome 1, where 5 HvPV genes are clustered together within 24 kb. This includes the S. cerevisiae SNF1 gene homologue, a regulator of catabolite derepression in yeast and a regulator of cell wall degrading enzymes in Cochliobolus carbonum and F. oxysporum. We deleted the snf1 gene in Fg as the first member of the HvPV gene cluster in chromosome 1 to characterise the importance of catabolite derepression during plant pathogenicity and mycotoxin production. The availability of the snf1 mutant, which exhibits highly reduced virulence on wheat ears, has allowed us to use 1H-NMR metabolomic analysis as a tool to identify fungal candidate metabolites with a role in plant pathogenicity. Rothamsted Research receives grant aided support from the Biotechnology and Biological Sciences Research Council.


165. Withdrawn


166. A whole genome oligo microarray approach to decipher Podospora anserina sexual development. Frederique Bidard 1, Sandrine Imbeaud 2, Sylvie Arnaise1, Anne Bourdais1, Karine Budin2, Evelyne Coppin1, Eric Espagne1, Olivier Lespinet1, Fabienne Malagnac1, Mathieu Paoletti3, Leonardo Peraza- Reyes1, Nancy Reymond2, Sven Saupe3, Eliette Sicault-Sabourin3, Denise Zickler 1, Philippe Silar1, Corinne Clave3, Herve Delacroix2, Veronique Berteaux-Lecellier1 and Robert Debuchy1. 1Institut de Genetique et Microbiologie, Univ Paris-Sud, UMR8621, F-91405 Orsay, France. 2Centre de Genetique Moleculaire, GODMAP, CNRS UPR 2167, F-91198 Gif sur Yvette, France. 3Institut de Biochimie et Genetique cellulaire, Univ Bordeaux2, UMR 5095, F-33077 Bordeaux, France.

The sexual development of the heterothallic euascomycete Podospora anserina has been analyzed for many years in terms of signal transduction pathways controlling the switch from vegetative to reproductive state, autophagy requirement for fruiting-body maturation, functions of MAT proteins and the developmental role of peroxisomal proteins. The completion of the P. anserina genome sequence allows all these research areas to benefit now from a microarray approach. Agilent 22K microarrays targeted against the 10,700 putative CDS of P. anserina will be used to compare wild type and the numerous mutants obtained in this model–system. This project is funded by Agence Nationale de la Recherche, grant n° ANR-05-0385-01.


167. Transcriptional Analysis of the Pathogenic Fungus Magnaporthe grisea during rice infection Cécile Ribot*, Arnaud Lagorce***, Aurélie Darchis, Fabien Munier, Jean-Benoit Morel**, Rick De Rose*, Roland Beffa* and Marc-Henri Lebrun*. *Bayer CropScience / CNRS, 69263 Lyon Cedex 09, France **CIRAD, TA73/09, 34398 Montpellier Cedex 05, France ***Institut de Genetique et de Microbiologie, Laboratoire de Genomique des Archaea, Université Paris XI / UMR8621 CNRS, 91405 ORSAY Cedex, France


Magnapothe grisea/Oryza sativa is a relevant model to investigate the molecular mechanisms underlying fungal infection of plants. M. grisea infections are responsible of the main foliar disease of rice and cause severe crop losses world-wide. This fungus is well suitable to molecular biology since most tools (genome sequence, EST databases, DNA chip, forward and reverse genetics) are available. Using Agilent microarrays representative respectively of 14 000 ORF from M. grisea, we performed a genome wide transcriptional analysis to highlight fungal genes expressed during the infection process. Leaves from the rice cultivar IR64 were infected by the virulent M. grisea strain PH14. Infected rice leaves were harvested 5 days after infection at the onset of lesions. This time point corresponds to the active invasion and destruction of plant tissues by M. grisea. Out of the M. grisea 14 000 genes, we could detect hybridization signals for 1800 genes that are expressed during infection. Among these 1800 genes, 240 genes are specifically expressed during infection, as no signal was detected in a mycelium array. The expression of a subset of genes that are either (1) highly expressed during infection or (2) are expressed during infection but not in the mycelium was monitored using real time RT-PCR. This strategy confirmed the expression during infection for 75% of the genes. Interestingly, bioinformatic analyses revealed an over representation of genes encoding for proteins putatively secreted. While only 5 % of the proteins encoded by the whole genome of M. grisea are predicted to be secreted, 43 % (105/240) of the protein specifically expressed during the infection process a signal peptide for protein secretion. These results suggest that M. grisea may produce numerous secreted effector proteins in host tissues that could be involved in the infection process. A systematic analysis of M. grisea proteins secreted during infection has been started to study this process.


168. Relative Protein Quantification through Stable Isotope Labeling by Amino Acids in Aspergillus flavus: Temperature Regulation of Aflatoxin Biosynthesis. D. Ryan Georgianna1,3, David C. Muddiman2, and Gary A. Payne1. 1Department of Plant Pathology and Center for Integrated Fungal Research. 2Department of Chemistry. 3Functional Genomics Graduate Program. North Carolina State University,Raleigh, NC 27606 USA.


Aflatoxin biosynthesis is inhibited at 37C, the optimum temperature for growth of Aspergillus flavus. Transcriptional analysis has shown that all aflatoxin biosynthetic genes except the pathway regulatory genes aflR and aflS are down regulated at 37C relative to 28C, suggesting that AFLR may be modified at 37C. To quantify the response of AFLR and other proteins to high temperature we adapted a stable isotope labeling by amino acids (SILAC) strategy for relative protein quantification in A. flavus. SILAC relies on the quantitative incorporation of labeled amino acids into proteins to provide a powerful mass spectrometry based proteomics tool useful for both the rapid quantification of proteins and identification of interactions. This technique has been used in several systems; including yeast, mammalian cells, and Arabidopsis cell culture. Samples were prepared using in-gel trypsin digestion of selected 1D-PAGE gel slices and analyzed on an ESI LTQ linear ion trap mass-spectrometer directly coupled to RP- HPLC. The A. flavus labeling strategy was optimized to provide a homogenously labeled sample with ~90% incorporation of [13C6] arginine. Furthermore, we found that the relative abundance of aflatoxin pathway enzymes compared between 28C and 37C is consistent with the relative abundance of their encoding transcripts at the respective temperatures. This is the first report of SILAC being used to quantify proteins in a filamentous fungus as well as a multi- cellular free-living prototrophic organism.


169. Comparative analysis of the cell wall sub-proteome from genetically-related strains of Cryptococcus neoformans with distinct pathogenic properties. Jamal Stie and Deborah Fox. Research Institute for Children/LSUHSC. Pediatrics and Microbiology, New Orleans, LA


Fungal infections are a frequent and growing cause of morbidity and mortality in the immuno-suppressed patient population, with effective treatment and continued antifungal drug development compromised by the conservation, at the molecular level, of many key physiological processes common to both humans and fungi. However, fungi elaborate one unique structure that is essential for their survival, the fungal cell wall, which acts as a physical barrier, and mediates events necessary for environment sensing, growth and morphogenesis. While the carbohydrate and protein composition of the cell wall has been extensively studied for many fungi, the cell wall of Cryptococcus neoformans, a significant opportunistic fungal pathogen that causes life-threatening meningoencephalitis is largely uncharacterized. Recent efforts to sequence and annotate the genome of this pathogen revealed the 99.5% genetic identity of two related strains of C. neoformans that possess markedly different virulence phenotypes, with the B3501A strain considerably more virulent than JEC21. Because the protein population within the cell wall plays an important role in the mediation of virulence in other pathogenic organisms, we hypothesize that differences in the cell wall protein composition could contribute to differences in virulence. Here we examined the hypothesis that B3501A and JEC21 differentially express proteins within the cell wall sub-proteome, contributing to the differences observed in the virulence properties of the two strains. Using a directed proteomics approach, we have performed a comparative analysis of the cell wall sub-proteomes from each strain, revealing a small subset of proteins that are unique to each, suggesting the presence of strain-specific cell wall-associated virulence determinants. Ongoing studies utilizing LC-MS/MS will permit the identification of both common and unique protein populations within the cell wall sub-proteome for each strain, leading to the elucidation of important surface-associated mediators of host interaction during pathogenesis in C. neoformans.


170. PhESTDB: An Integrated resource for Phytophthora and Soybean EST sequences. Sucheta Tripathy and Brett M. Tyler. Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA-24061 sutripa@vbi.vt.edu bmtyler@vt.edu


PhESTDB is an unique integrated EST data resource, containing 33,350 P.sojae ESTs, 99,319 P.infestans ESTs and 37,465 public Soybean unigenes. This database is the most complete resource for P.sojae EST sequences to date. The P.sojae sequences were quality trimmed followed by vector/ adaptor, poly A/T trimming. A total of 3769 Soybean EST sequences were separated from the P.sojae ESTs from an infection library using in silico methods. Subsequently, the sequences were clustered and assembled using a wrapper perl script TGICL.The clustering and assembly produced 7863 unigenes of P.sojae, 2292 soybean unigenes from the infection library, and 14754 unigenes of P.infestans. The unigenes were primarily annotated using NCBI- BLASTX against NR database accelerated with a Timelogic board.The unigenes were then passed through a modified algorithm of loglikelihood(McLachlan,1984) to separate the UTRs from the ORFs. The protein sequences were annotated for domains, motifs, profiles and fingerprints using InterproScan. Sequences were submitted to signalp and TMHMM servers to get secretory and trans-membrane domains. The resulting outputs were parsed and stored in the database. The database was designed in mysql and the interfacing was done with PHP and perl CGI. We have created an easy-to-use Perl GD-based browser which is distinct from Gbrowse. In the browser the genome to unigene alignments are shown in greater detail which also indicates any overlaps between the unigenes. Each EST unigene is made clickable to view detailed information about assembly and their primary annotation including various other related information such as NCBI links and links to gene models. Through the gene models this database is linked to the VBI microbial database. This database has a very powerful query page, that can retrieve any kind of information the user may need. Data can also be downloaded from the database in text format. The database is publicly available at http://phytophthora.vbi.vt.edu/EST .


171. Sequencing of the genome of the forest pathogen Heterobasidion annosum s.l. Jan Stenlid1, Matteo Garbelotto2, Ursula Kües3, James B Anderson4, Francis Martin 5, Halvor Soilheim6. 1Swedish University of Agrcultural Sciences, Uppsala, Sweden, 2 University of California, Berkeley, USA, 3 Georg August University of Göttingen, Germany, 4University of Toronto, Canada, 5 INRA-Nancy, France 6 Norwegian University of Life Sciences, Ås Norway


Heterobasidion annosum s.l. causes a devastating root rot in conifer plantations and natural forests throughout the northern hemisphere. The JGI- sponsored genome sequencing for H. annosum will provide the first comprehensive genetic information on a plant pathogenic homobasidiomycete allowing for new insights into plant- microbe interactions with trees, in particular conifers. It is important to broaden the taxonomic base for understanding the mechanisms of plant-microbe interactions, studying genes and proteins involved, and identifying pathogenicity determinants. Comparative genomics of plant pathogens with a gradient of taxonomic relatedness to H. annosum will help to understand the evolution of such factors. Comparisons can also be made in the response of plants to various types of trophic interactions; necrotrophic, biotrophic and mycorrhizal mutualists and the model tree Populus. Moreover, this project will also gain insights into fungal evolutionary history and biology including development, non self recognition, mating, wood degradationand secondary metabolism.


172. High throughput discovery of virulence factors among transcription factors of Alternaria brassicicola. Yangrae Cho, Mauricio La Rota, Kwang-Hyung Kim, Derrick Scott, Amanda Cronin, Meagan Callilhan, Graciela Santopietro, and Christopher Lawrence. Virginia Tech, Blacksburg, VA


Alternaria brassicicola is a ubiquitous necrotrophic fungus causing destructive rots among diverse Brassica family members. We have optimized relatively easy and economical PEG-mediated protoplast transformation methods for this fungus. In order to produce targeted gene knockout (KO) mutants, we have utilized unconventional linear minimal element constructs as well as conventional gene replacement constructs. Using these two methods, we produced knockout mutants for over 100 genes and investigated changes in virulence. Here we present the results related to mutants for 40 protein coding genes. These candidates were comprised of five kinases and four transcription factors (TFs) involved in six known signal transduction pathways. In addition 31 TFs from the partially annotated A. brassicicola genome were also selected (both randomly and based on interesting predicted functional domains) for KO and subsequent phenotypic analyses. Near loss of virulence was observed for mutants of the Amk1, AbSte12, and AbMps1 genes, representing two signal transduction pathways of the mating pheromone responses and hypotonic shock responses. Significant reduction of virulence without any other discernable phenotypes has been observed for mutants of the AbPro1 gene whose homolog is involved in sporulation in diverse filamentous fungi. In addition, we have discovered mutants for two of the 31 selected TF genes showed reduced virulence. The study results thus far show a few common as well as several unique aspects of virulence mechanisms in A. brassicicola.


173. The genome of the necrotrophic fungus Alternaria brassicicola. C. Mauricio La Rota1, Yangrae Cho1, Thomas Mitchell2, Brett Tyler1, Sandra Clifton3, Dennis Knudson4, Susan Brown4, Christian Haudenschild5, Blake Meyers6 and Christopher Lawrence1. 1 Virginia Bioinformatics Institute, Blacksburg, VA. 2 Fungal Genomics Lab, North Carolina State University, Raleigh, NC. 3 Washington University Genome Sequencing Center, St Louis, MO. 4 Colorado State University, Fort Collins, CO. 5 Solexa Inc, Hayward, CA. 6 Delaware Biotechnology Institute, Newark, DE.


The genomes of several fungi within the Dothideomycete are in the process of being sequenced at various centers internationally. These fungi are economically important plant pathogens infecting various important crops such as wheat, corn, and Brassicas, to name a few. All of these fungi are capable of leading a saprophytic lifestyle but have a necrotrophic stage in their lifecycle. Comparisons between their gene contents and genome structures are likely to reveal important details about their evolutionary history, production of toxins and the capability to switch from a saprophytic phase to a necrotrophic one in order to infect plant tissues. We present here the first draft assembly of the 30Mb genome of the Brassica pathogen, Alternaria brassicicola with a 6.4x coverage and provide details about the progress in annotating the genome, the manual curation and the functional genomics aspect of the project. Analysis of the genome features and structure will help to further the understanding of necrotrophic pathogen-plant interactions. This project is funded by USDA-CSREES (Proj. # VAR-2004-05551).


174. Comparative and functional genomics in identifying aflatoxin biosynthetic genes. Jiujiang Yu1,7, Jeffery Wilkinson2, Gary Payne3, Masayuki Machida4, Bruce Campbell5, Joan Bennett6, Deepak Bhatnagar1, Thomas Cleveland1, and William Nierman7,8. 1USDA/ARS, Southern Regional Research Center, New Orleans, LA 70124, USA; 2Mississippi State University, Mississippi State, MS 39762, USA; 3North Carolina State University, Raleigh, NC 27695, USA; 4National Institute of Advanced Industrial Science and Technologies (AIST), Tsukuba, Ibaraki, Japan; 5USDA/ARS, Western Regional Research Center, Albany, CA 94710, USA; 6Rutgers University, New Brunswick, NJ 08901, USA; 7The Institute for Genomic Research, Rockville, MD 20850, USA; 8The George Washington University School of Medicine, Washington, DC 20037, USA

Identification of genes involved in aflatoxin biosynthesis through Aspergillus flavus genomics has been actively pursued. A. flavus Expressed Sequence Tags (EST) and whole genome sequencing have been completed. Groups of genes that are potentially involved in aflatoxin production have been profiled using microarrays under different culture conditions and during fungal infection of corn. Preliminary annotation of the sequence revealed that there are about 12,000 genes in the A. flavus genome. Many genes in the genome, which potentially encode for enzymes involved in secondary metabolite production, such as polyketide synthases, non-ribosomal peptide synthases, cytochrome P450 monooxigenases, have been identified. Comparative analysis of A. flavus genome with food grade industrial fermentation organism A. oryzae can help understanding the mechanism of aflatoxin biosynthesis and solving the problem of aflatoxin contamination.


175. Validation of DNA microarrays derived from the Fusarium verticillioides Gene Index. Robert A.E. Butchko, Daren W. Brown and Robert H. Proctor. Mycotoxin Research Unit, National Center for Agricultural Utilization Research, 1815 N. University Street, Peoria, IL 61606


 Fusarium verticillioides is a pathogen of maize and it can produce the toxic polyketide derived secondary metabolites called fumonisins. Fumonisins have been shown to cause animal diseases and are epidemiologically correlated to esophageal cancer and neural tube defects in humans. In an effort to identify genes involved in regulation of fumonisin production and in pathogenicity on maize, a large scale EST sequencing project was conducted in collaboration with The Institute for Genomic Research. The Fusarium verticillioides Gene Index (FvGI) contains up to 11,126 sequences including 7198 tentative consensus (TC) and 3921 singleton EST sequences. DNA microarrays were designed based on the FvGI and were constructed by NimbleGen Systems, Madison WI. Each DNA microarray contains 12 different 24-bp oligonucleotide probes specific to each of the unique sequences in the FvGI. Here, we present data from an experiment where total RNA from a six-point time course of F. verticillioides cultured in a liquid medium conducive to fumonisin production was used to hybridize to the microarrays. Analysis of FUM gene expression patterns over time were used to validate the microarray. The expression of FUM genes determined by the microarray analysis is consistent with expression patterns determined by Northern analysis and RT-PCR. To help determine the orientation of singleton ESTs whose direction could not be determined from sequence data or BLAST analysis, probe sets to both plus and minus strand sequences were included for all of the singletons as well as TCs containing two open reading frames. Microarray analysis successfully resolved the orientation of a number of the singletons for which the orientation was not obvious and demonstrated the existence of two open reading frames in some TCs. Global patterns of gene expression under these conditions are also presented.


176. Measuring LOH rates in Candida albicans by fluctuation analysis. Anja Forche 1, Merima Helić 1, and Judith Berman1. 1University of Minnesota, Minneapolis, MN, USA e-mail: forche@umn.edu


Candida albicans is the most common human fungal pathogen. While it does not undergo meiosis, clinical strains exhibit high levels of genetic and genomic variability that can lead to the development of drug resistance. To explore the mechanisms that give rise to genetic and genomic changes, we have measured basal rates of recombination by following loss of heterozygosity (LOH) in fluctuation assays. We used two counter-selectable genetic markers, GAL1 and URA3, located on opposite alleles at the same locus on chromosome 1. Basal LOH rates were approximately 10-6 events/cell/generation for each marker, indicating that the assay is effective, that it is independent of the marker used and that both homologues of chromosome 1 undergo equal rates of LOH. SNP microarrays were used to study the extent and the mechanisms of LOH along chromosome 1 in strains that had undergone LOH at GAL1. Eighty-six percent of LOH events were caused by a single cross-over event that extended to the telomere, most likely due to break-induced replication. Shorter gene conversion tracts (9%) and LOH of whole chromosomes (5%) also were observed. We are currently testing if 1) the basal LOH rate is altered in the presence of anti-fungal drugs and other stresses such as temperature and toxic agents, and if 2) LOH rates differ at different chromosomal loci including telomeres, centromeres and mid-arm loci on all eight chromosomes. Preliminary results suggest that LOH is much higher at telomeres than at centromeres or internal chromosomal loci.


177. Creating a Critical Mass of Data for Genome Annotation and Comparative Analysis. Igor Grigoriev1*, Andrea Aerts1, Alan Kuo1, Asaf Salamov1, Diego Martinez2, Jean Challacombe2, Thomas Brettin2, Erika Lindquist1, Harris Shapiro1, Gregory Werner1, Susan Lucas1, Jane Grimwood3, Jeremy Schmutz3, Paul Richardson1, James Bristow1. 1US DOE Joint Genome Institute, Walnut Creek, CA 94598 2 US DOE Joint Genome Institute - LANL , Los Alamos, NM 87545 3 US DOE Joint Genome Institute - Stanford, Palo Alto, CA 94304 *ivgrigoriev@lbl.gov


The DOE Joint Genome Institute contributes to the rapidly growing number of fungal genomes available to the research community through high-throughput sequencing and annotation. We sequenced and annotated 9 fungal and 2 oomycete genomes (genome.jgi.doe.gov) and expect to nearly double this number in 2007. The broad and diverse set of genomes covers major branches of the fungal phylogenetic tree and samples some of the branches at higher depth, which allows analysis of differences between clades and within species on the genomic level. In addition to several basidiomycete and ascomycete genomes, we have annotated the genome of the zygomycete Phycomyces blakesleeanus and are currently annotating the chytridiomycete Batrachochytrium dendrobatidis. Many of these genomes are either finished or are in finishing, which enables effective analysis of genome organization and evolution, such as, for example, a conditionally dispensable chromosome in Nectria haematococca MPVI. Genome projects triggered development of additional resources of ESTs, proteomics and microarray data to assist with genome annotation and expression analysis. User Communities with over 100 biologists around the world actively participate in analysis and manual curation of predicted genes and functions using the JGI Genome Portal. Creating a critical mass of data improves genome annotation and enables efficient comparative analysis to address a broad spectrum of biological questions and to find important industrial applications.


178. A journey into the unknown: investigating temperature sensitive mutants in Neurospora crassa. Kevin McCluskey1, Sheera A. Walker1, Rachel L. Yedlin1, David Madole2 and Michael Plamann1,2. 1Fungal Genetics Stock Center, and 2University of Missouri- Kansas City, School of Biological Sciences.

In our continuing effort to bring added value to the materials in the FGSC collection, we have endeavored to identify the open reading frame associated with genetically mapped temperature sensitive lesions in Neurospora crassa. There are nearly 60 such ts lesions mapped in the N. crassa genome and because there is no function for most of these, they are known as 'unknown'. We used functional complementation of the temperature sensitivity to identify open reading frames for two such lesions, un-16 and un-4. When we identified un-16, we found that it had been previously identified by Sakai et al (2002) as part of their investigation of the near-by upr-1. un-16 encodes a ribosomal protein and we sequenced the two mutant alleles and found that in both there was a single leucine to arginine substitution. A search of the genome databases revealed that the leucines in question are very highly conserved- from archae to humans. The ability to complement the temperature sensitive lesion in strains carrying the un-16 lesion makes it possible to use this as a selectable marker. The second unknown to be complemented, un-4, encodes a mitochondrial import protein. Our next target is un-7 and currently we have identified 8 candidate ORFs from existing data. The FGSC is supported by grant 0235887 from the US National Science Foundation.


179. Optimised methods for proteome analysis of the encapsulated fungus Cryptococcus gattii. Jocelyne M D’Souza-Basseal1,Ben R Herbert2, Matthew Padula2, Cameron Hill2, Mark B Krockenberger3, Elizabeth J Harry4 and Dee Carter1. 1School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW Australia; 2Proteomics Technology Centre of Expertise, University of Technology, Sydney, NSW Australia; 3Faculty of Veterinary Science, University of Sydney, Sydney, NSW Australia; 4Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, NSW Australia Contact email: j.dsouza@mmb.usyd.edu.au

The fungal species Cryptococcus gattii is an environmental saprophyte capable of causing serious disease in people and animals, and is uniformly fatal without treatment. Like most systemic mycoses, cryptococcosis is difficult to treat and this is due to the scarce number of safe and efficacious antifungal drugs, and to our limited knowledge of the intricacies of fungal virulence. Proteome analysis enables the identification and characterization of the protein complement of a cell, tissue or organism at any one time, under specific conditions. The global nature of proteomics means areas of little knowledge, like fungal disease, can be addressed without any prior knowledge or assumptions about the role of differentially expressed proteins. Furthermore, as proteins are targets for drug interventions in microbial infections, proteome approaches are the most rapid, direct and powerful means of identifying novel candidate antimicrobial targets. The major virulence factor of C. gattii is its polysaccharide capsule, composed mainly of glucuronoxylomannan residues and mannoproteins. In our preliminary proteome analysis, an encapsulated C. gattii strain was extracted using the current best practice for two-dimensional electrophoresis of microorganisms. However, this gave very limited extraction of high molecular weight proteins and suggested that the capsule may be trapping the ‘missing’ proteins by ionic interactions. We have developed a novel method of protein extraction using high salt combined with acidic conditions. We present here studies using a range of different salts that greatly increased protein extraction from various encapsulated C. gattii strains.


180. Transcritome analyses of developmental processes associated with hypoxic stress in Aspergillus nidulans and A. fumigatus. Kap-Hoon Han1, Hyoun-Young Kim1, Jong Hwa Kim1, Yeong-Man Yu2, Pil-Jae Maeng2, Hee-Moon Park2, Suhn-Kee Chae3, Keon-Sang Chae4, Kwang-Yeop Jahng4, and Dong-Min Han5. 1Dept. of Pharmaceutical Engineering, Woosuk University, Wanju, 565-701, 2School of Bioscience & Biotechnology, Chungnam Nat'l University, Daejeon, 305-764, 3Dept. of Life Science & Technology, PaiChai University, Daejeon, 302-735, 4Div. of Biological Sciences, Chonbuk Nat'l University, Jeonju, 561-756, 5Dept. of Life Science, Wonkwang University, Iksan, 570-749, Korea.


Aspergillus fumigatus is the primary causative agent of an opportunistic fungal disease, aspergillosis. Unlike Aspergillus nidulans, A. fumigatus has no known sexual development process. Furthermore, in A. nidulans, hypoxic condition is the one of most important environmental factor for generating fruiting bodies. This condition is also important to A. fumigatus because of the environment of host cell is usually maintained as hypoxic condition. To study relationship between hypoxic stress, developmental process and virulence, comparative DNA microarray analyses were performed using A. fumigatus and A. nidulans chips. Comparisons of the two transcriptome provided important information of hypoxic stress response and sexual development process. This work was supported by the KOSEF grant (No. R1-2006-000-11204-0) and KRF (KRF-2005-070-C00123).


181. Identification of proteins involved in early phase of conidia germination by quantitative proteomics in Aspergillus nidulans. Young Taek Oh*, Chun Seob Ahn, Jae Won Kim, Chang-Won Lee**. Department of Microbiology, Research Institute of Life Science, Gyeongsang National University, 900 Gazwa-dong, Jinju 660-701, Korea *presenting author, **corresponding author


The asexual spore, or conidium of A.nidulans become competent for growth and nuclear division in a process called conidial germination, and that begins when the conidium breaks dormancy and growth isotropically. To find out critical proteins involved in early phase of conidia germination, we performed a global proteome analysis of differentially expressed protein between dormant conidia with germinating conidia. More than 1200 protein spots were detected on silver stained 2-D gel with IPG strip pH 4-7. Image analysis revealed that 97 protein spots showed quantitative variations that were significant (P <0.05) and reproducible in the 30minute germinating conidia. And 144 protein spots showed quantitative variations that were significant (P <0.05) and reproducible in the 60minute germinating conidia. Protein identification was carried out peptide mass fingerprinting by MALDI-TOF mass spectrometry. Under 30minute germinating conidia, the protein expression levels of 9 proteins including thioredoxin reductase are up-regulated, and 5 proteins including transketolase are down-regulated. Under 60minute germinating conidia, the protein expression levels of 13 proteins including peptidyl-prolyl cis-trans isomerase are up-regulated, and 12 proteins including mannitol-1- phosphate dehydrogenase are down-regulated. The identified proteins in this report enhance to understanding of the process of conidia germination.


182. Coding tandem repeats generate genetic diversity in Aspergillus fumigatus genes. Emma Levdansky1, Jacob Romano1, Kevin J. Verstrepen2, Gerald R. Fink2 and Nir Osherov1. Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA2, Department of Human Microbiology,Sackler School of Medicine1, Tel-Aviv University, Tel-Aviv, Israel.E-mail: nosherov@post.tau.ac.il


Genes containing multiple coding tandem repeats are highly dynamic components of genomes. In humans, expansion of coding repeats is associated with various diseases, including Huntington’s Disease and Fragile X Syndrome. In fungi, many repeat-containing genes encode for cell-wall proteins containing a leader sequence and glycophosphatidylinositol (GPI)- anchor. Several encode for known adhesins (ie. Saccharomyces cerevisiae FLO11, Candida albicans ALS1, ALS5, ALS7 and ALS9, Candida glabrata EPA1). They undergo frequent recombination-dependent expansion or contraction in size, creating size-variability between different yeast isolates and alterations in adhesion. This variation provides the functional diversity in cell-surface antigens which allows rapid adaptation to the environment. Aspergillus fumigatus is the most common mold pathogen in man, causing both deadly invasive diseases in immunocompromised patients and allergic diseases in patients with atopic immune systems. We analyzed the entire genome of A. fumigatus for open-reading frames (ORFs) containing putative repeats, using the ETANDEM software program, a numerical score for tandem repeats in a nucleotide sequence. Fourteen of 30 top-ETANDEM-scoring genes analyzed showed strain-specific size variation of repeat containing-regions. Four of these, (Afu3g08990, Afu2g05150 (MP-2), Afu4g09600 and Afu6g14090) are putative cell-wall proteins containing a leader sequence and GPI-anchor motif. All four are expressed genes and produce size-variable mRNA encoding a discrete number of repeat amino-acid units. Their expression during development and in response to cell-wall disrupting agents was analyzed. Our findings suggest that a subset of the A. fumigatus cell-surface proteins may be hyper-variable due to recombination events in their internal tandem repeats. We have created knock out strains in A.fumigatus 293 for two putative cell-wall coding genes (Afu3g08990 and Afu6g14090). We are currently studying the effect of the deletion of these genes on the phenotype. Preliminary results indicate that compared to the wild type strain, Afu3g08990-deleted strain exhibits early germination rates and reduced adhesion to ECM (extra cellular matrix) of alveolar lung cells (A549 cells). In addition, we are analyzing Afu3g08990 and Afu6g14090 cellular location by tagging the genes with a myc epitope.


183. Molecular analysis of the response of cultured A549 lung cells to A. fumigatus infection. Haim Sharon and Nir Osherov1. Department of Human Microbiology, Sackler School of Medicine,1 Tel-Aviv University, Tel-Aviv, Israel.E-mail: nosherov@post.tau.ac.il


 Aspergillus fumigatus is the most prevalent airborne fungal pathogen which causes fatal invasive aspergillosis in immunocompromised patients. Invasive pulmonary aspergillosis (IPA) is caused by inhalation of A. fumigatus spores and growth of the fungus inside the lungs, often spreading from the initial site of infection in the lungs to attack various organs in the body. Our main goal is to better understand the mechanisms controlling damage in infected lung alveolar epithelial cells. We assumed that lung alveolar cells infected with A. fumigatus undergo a specific and pre- programmed molecular response. Two approaches were used to analyze the response of infected cells: A) Kinome analysis, identification of specific phospho-proteins using a panel of 36 mono- specified antibodies. B) Transcriptional analysis using Affymetrix arrays. A549 lung epithelial cells were infected with A. fumigatus conidia or culture filtrate and compared to uninfected cells. Kinome analysis revealed five main proteins, Adducin gamma, PKR, Erk1/2, JNK and MEK1/2 which showed differences in phosphorylation patterns between infected and uninfected cells. These results suggest that the infected cells mount a strong JNK/MAPK pathway stress response to A. fumigatus infection. Interestingly, adducin, a cytoskeletal protein, was phoshphorylated more intensively in response to conidia than to culture filtrate, whereas PKR, which plays a role in mediating the activation of the MAPK pathways, was phoshphorylated only in response to culture filtrate. Affymetrix transcriptional analysis revealed 388 genes differentially expressed at least 2 fold (p <0.05) in response to infection by germinated conidia, and 478 genes in response to culture filtrate. Interestingly, the response to germinated conidia was characterized by strong (>5 fold) upregulation of 35 genes involved in cell death, immune response, cytokine activity (especially IL8) and response to wounding. A significant number of these genes is controlled by the transcription factor NFk-B. In contrast, the response of A549 cells to culture filtrate was mainly characterized by the down regulation of 277 genes involved in metabolism and biosynthesis. Our results demonstrate that A549 lung cells respond differentially to infection with A. fumigatus conidia or culture filtrate. We propose that during early conidial infection, infected cells mount a vigorous protective response. In contrast, during late infection, the accumulation of secreted culture filtrate elicits a marked inhibition of cellular metabolism. These results help clarify the progression of cellular infection by A. fumigatus at the molecular level, and suggest novel ways to interfere with this destructive process.


184. Metabolic network-driven analysis of genome-wide transcription data from Aspergillus nidulans. Helga Moreira David1, Gerald Hofmann2, Ana Paula Oliveira2, Hanne Jarmer3, and Jens Nielsen2. 1Fluxome Sciences A/S, Diplomvej 378, 2800 Lyngby, Denmark 2 Center for Microbial Biotechnology, Technical University of Denmark, 2800 Lyngby, Denmark 3 Center for Biological Sequence Analysis, Technical University of Denmark, 2800 Lyngby, Denmark


Aspergillus nidulans is a model organism for aspergilli, an important group of filamentous fungi that encompasses human and plant pathogens, as well as industrial cell factories. Aspergilli have a highly diversified metabolism and, because of their medical, agricultural and biotechnological importance, it is valuable to understand how their metabolism is regulated. We therefore performed genome-wide transcription analysis of A. nidulans grown on glucose, glycerol, and ethanol with the objective of identifying global regulatory structures. We furthermore reconstructed the complete metabolic network of this organism, which resulted in linking 666 genes to metabolic functions, as well as assigning metabolic roles to 472 genes that were previously uncharacterized. Through combination of the reconstructed metabolic network and the transcription data, we identified subnetwork structures that pointed to coordinated regulation of genes involved in many different parts of the metabolism. Thus, for a shift from glucose to ethanol, we identified a coordinated regulation of the complete pathway for oxidation of ethanol, as well as up-regulation of gluconeogenesis and down-regulation of glycolysis and the Pentose Phosphate (PP) pathway. Furthermore, upon a change in the carbon source from glucose to ethanol, the cells shift from using the PP pathway as the major source of NADPH for biosynthesis to use of the malic enzyme. Our analysis indicated that some of the genes are regulated by common transcription factors, making it possible to establish new putative links between known transcription factors and genes, through clustering.


185. The Leptosphaeria maculans genome initiative. M.-H. Balesdent1, B. J. Howlett2, P. Wincker 3, J. Amselem1,5, A. Cozijnsen2, I. Fudal1, L. Gout1,4, M. Meyer 1, A. Stachowiak 6, A. Van De Wouw 2, T. Rouxel1. 1INRA-PMDV, F-78026 Versailles,France; 2School of Botany, Uni-Melbourne, Parkville, VIC 3010, Australia; 3Genoscope, 91057 Evry Cedex, France ; 4Protection des Plantes, INA-PG, 78850 Thiverval-Grignon, France;

5INRA-URGI, 91000 Evry, France; 6IGR, Poznan, Poland


Genoscope (National Sequencing Centre), the French sequencing agency is involved in large-scale sequencing of the genome of the Dothideomycete Leptosphaeria maculans. Previous collaborative projects between INRA Versailles and Genoscope consisted in precise sequencing, assembly and finishing of a 1.1 Mb genomic region in isolate v23.1.3. These data enabled characterization of the first known retrotransposons and avirulence genes in L. maculans. These analyses also showed that the genome comprised regions of long A+T-rich regions reminiscent of heterochromatin, alternating with G+C-equilibrated, gene-rich regions. The goal of the genome initiative is to release an annotated assembly with 12-x genome coverage for L. maculans isolate v23.1.3. Additional sequencing of BAC-ends for physical mapping and ESTs from mycelia grown under three different conditions is underway. The first draft 5-x coverage indicated a high content of repeats in the genome. The first 12-x assembly was released Nov. 15, 2006, and comprised of 1277 super contigs of which 142 are more than 100 kb (mean : 39.78 kb; max : 866.65 kb). From these data, a bigger- than-expected genome size of ca. 50 Mb is hypothesised and a ca; 30% of repeats in the genome is suggested. First analysis from the final assembly (expected in January 2007) will be presented. A. Stachowiak was funded by Marie-Curie Fellowship HPMT-CT-2001-00395 ‘FUNGENE’


186. The cyclic AMP induced gene expression of Magnaporthe grisea during appressoria formation. Yeon Yee Oh, Shaowu Meng, Douglas E. Brown, Sean Coughlan1, Thomas K. Mitchell, Ralph A. Dean Fungal Genomics Lab, North Carolina State University, Raleigh NC. 1Agilent Technologies, Inc. Wilmington DE.


The cyclic AMP signaling pathway modulates appressoria development in rice blast fungus, Magnaporthe grisea. To identify the cAMP induced cellular processes for appressoria induction, we compared global gene expression patterns in cAMP elaborating appressoria verse germinating spores on non inductive hydrophilic surfaces. After 9 hours incubation, RNA was extracted from tissues of each condition and microarray experiments were performed using the M. grisea v2.0 oligonucleotide array, created in collaboration with Agilent Technology. 8.6 % of M. grisea genes were found to be differentially expressed by cAMP treatments. This included some previously known pathogenecity factors. Functional analysis suggests that melanin biosynthesis, secondary metabolism, protein degradation, protein synthesis and lipid degradation are downstream of cAMP signaling pathway and are necessary for appressoria morphogenesis. This was verified by gene knock out followed by phenotype analysis. We will present the results of gene expression studies, classification of differentially expressed genes and functional characterization of selected genes.


187. Natural history and evolutionary principles of gene duplication in fungi. Ilan Wapinski, Avi Pfeffer, Nir Friedman, Aviv Regev. Broad Institute, MIT, Cambridge, MA USA e-mail: ilan@eecs.harvard.edu


Gene duplication and loss is a powerful source of functional innovation. However, the general principles that govern this process are still largely unknown. With the growing number of sequenced genomes, it is now possible to examine these events in a comprehensive and unbiased manner. Here, we develop a novel procedure that resolves the evolutionary history of all genes in a large group of species. We apply our procedure to seventeen fungal genomes to create a genome- wide catalog of gene trees that determine precise orthology and paralogy relations across these species. We show that gene duplication and loss is highly constrained by the functional properties and interacting partners of genes. In particular, stress-related genes exhibit many duplications and losses while growth-related genes show selection against such changes. This dichotomy is relaxed following whole-genome duplication. Duplicated genes rarely diverge with respect to biochemical function, but typically diverge with respect to regulatory control. Surprisingly, paralogous modules of genes rarely arise, even following whole-genome duplication. Rather, gene duplication drives the modularization of functional networks through specialization, thereby disentangling cellular systems.


188. The effector secretome of Phytophthora infestans: Structure and function. Sophien Kamoun 1, Joe Win 1, William Morgan 1, Sang-Keun Oh 1, Carolyn Young 1, Rays Jiang 2, 3, Francine Govers 3, Michael C. Zody 2, Chad Nusbaum 2. 1 Department of Plant Pathology. The Ohio State University, OARDC, Wooster, OH, USA. 2 The Broad Institute of MIT and Harvard, Cambridge, MA, USA. 3 Laboratory of Phytopathology, Plant Sciences Group, Wageningen University, The Netherlands.


The oomycete Phytophthora infestans causes late blight of potato and tomato and is arguably the most destructive pathogen of solanaceous crops. P. infestans establishes parasitic colonization of plants by modulating host cell defenses through an array of disease effector proteins. The biology of P. infestans effectors is poorly understood, but tremendous progress has been made recently. Two classes of effectors target distinct sites in the host plant: apoplastic effectors are secreted into the plant extracellular space, while cytoplasmic effectors are translocated inside the plant cell, where they target different subcellular compartments. Of particular interest are the RXLR effectors that are characterized by a conserved motif following the signal peptide. The RXLR domain is functionally interchangeable with a malaria host targeting domain and appears to function in delivery into host cells. The recent completion of the genome sequence of P. infestans enables genome-wide cataloguing of the effector secretome. Using computational analyses, we identified up to 700 candidate RXLR effector genes. These were frequently organized in clusters of paralogous genes, many of which exhibit hallmarks of positive selection probably as a result of a coevolutionary arms race with host factors. Predictably, effector genes are typically expressed and often up-regulated during infection. We also utilized the discovered RXLR effectors in high-throughput in planta expression assays to screen for alteration of plant defense response and gain an insight into their function.


189. Withdrawn


190. Withdrawn


191. Proteome analysis of the response of Aspergillus fumigatus to iron limitation. André D. Schmidt1, Olaf Kniemeyer1, Hubertus Haas2 and Axel A. Brakhage1. 1Leibniz Institute for Natural Product Research and Infection Biology (HKI) / Friedrich-Schiller-University Jena, Germany 2Division of Molecular Biology/Biocenter, Innsbruck Medical University, Austria

The acquisition of iron is known to be an essential step in any microbial infection process due to iron-limiting conditions in the human host. This iron limitation is caused by high-affinity iron-binding proteins like transferrin or lactoferrin in the host. Since iron plays an essential role in key metabolic processes like DNA synthesis, oxidative phosphorylation or electron transport A. fumigatus has to overcome the iron deficiency by the synthesis of siderophores, which chelate iron. It was shown that an A. fumigatus strain unable to synthesize siderophores was attenuated in virulence in a murine infection model. To understand the cellular processes, induced by iron starvation, we analysed the proteome of A. fumigatus strain ATCC 46645 grown under iron-deficiency conditions. Under iron depletion, proteins involved in siderophore biosynthesis are upregulated, e.g. L-ornithine N5-oxygenase (SidA), and iron cluster-containing proteins as aconitase or 3-isopropylmalate dehydratase are down-regulated. In addition, proteins involved in the heme biosynthesis are less abundant under iron-deficiency. Further proteins analysed under different non- linear pH-scales will be presented and their putative role will be discussed.


192. Comparative Genomics of plant pathogenic Fusarium species. Li-Jun Ma1, Won-Bo Shim2, Manfred Grabherr1, Michael Koehrsen1, Reinhard Engels1, Sinead O’Leary1, Matter Pearson1, Sarah Young1, David DeCaprio1, Chinnappa Kodira1, James Galagan1, Seogchan Kang3, Charles Woloshuk4, Harold Corby Kistler5, Bruce Birren1. 1Broad Institute of MIT and Harvard. 2Texas A&M University. 3Penn State University. 4Purdue University. 5University of Minnesota.


The genus Fusarium collectively represents the most important group of fungal plant pathogens, causing various diseases on nearly every economically important plant species. Of equal concern is the health hazard posed to humans and livestock by the plethora of Fusarium mycotoxins. Our goal is to utilize the power of comparative genomics to improve gene annotation, to identify functional non-coding elements and to study the evolution and pathogenicity of this group of economicly important species. F. oxysporum and F. verticillioides have been selected as sequence targets to facilitate the comparative studies with the existing F. graminearum genome. Sequencing, assembly and automated annotation of F. verticillioides is complete and sequencing and assembling for F. oxysporum is ongoing. The released F. verticillioides assembly contains 41.7Mb with 14,206 predicted proteins. More than 99% of the assembly was anchored to the genetic map. A newly constructed optical map for F. oxysporum spans more than 60Mb, which is much bigger than either F. graminearum or F. verticillioides. Preliminary analysis of the sequence indicates that the increased genome size is mainly caused by large segmental duplications and the existence of many transposable elements. One of the focal points of our comparative analysis is to identify gene clusters associated with mycotoxin production and tracing their evolutionary history. We will report our recent progress and discuss the impact of comparative genomics on our understanding of genome dynamics, pathogenicity and mycotoxin production in these genomes. Funding for Fusarium comparative project was provided by USDA/NSF microbial genome sequencing program.


193. Aspergillus Comparative Database: a web-based tool for comparative analysis. Vinita Joardar, Jonathan Crabtree, Rama Maiti, Natalie Fedorova, Paolo Amedeo, Samuel Angiuoli, William Nierman, Owen R. White, and Jennifer R. Wortman. The Institute for Genomic Research, Rockville, MD, USA. vinita@tigr.org


Comparative genome analysis in the genus Aspergillus has been facilitated by the availability of genome sequences for multiple species. Ortholog clusters were computed based on the mutual best blastP hits between the Aspergillus proteomes. Syntenic blocks were identified by searching for collinear orthologs, with allowances for gaps and rearrangements, along the full chromosomes and/or supercontigs. The results of the genome and proteome level computes for the Aspergillus genomes were stored in the Aspergillus Comparative Database (asp), a chado relational database. Sybil, a web-based software package developed at TIGR, was used for visualization and analysis of comparative genomics data. Sybil uses a graphical user interface to present and navigate the information stored in asp. The interactive graphical displays allow the user to navigate from global genome views down to specific protein reports. Protein cluster reports, lists of singletons, comparative sequence displays and publication-quality figures can be customized based on user specifications. The Sybil package also leverages the comparative data for annotation improvement. We present an overview of the Sybil package applied to the comparative analysis of aspergilli ( http://www.tigr.org/sybil/asp/index.html ). The Sybil visualization software is freely available for download from http://sybil.sf.net


194. Using comparative genomics to identify virulence characteristics in pathogenic yeast. David Fitzpatrick and Geraldine Butler. School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland.


Due to their increasing clinical importance, nine Candida and closely related species have been sequenced. The wealth of genomic data now available, allows us to investigate the differences that account for increased pathogenicity in particular Candida species. In order to identify specific metabolic activity associated with pathogenesis, we have assigned KEGG orthology identifiers to all available Candida genome data, using a bidirectional best BLAST strategy against the manually curated KEGG GENES database. All annotated Candida data has subsequently been mapped onto reference metabolic pathways from the KEGG PATHWAY database. Using the information gleaned from our metabolic pathway analysis we have attempted to correlate species-specific components with virulence, by noting which are specific to pathogenic Candida such as Candida albicans, Candida parapsilosis and Candida tropicalis but absent in closely related non-pathogenic species such as Debaryomyces hansenii and Lodderomyces elongisporus. Pathways that are present in numerous pathogenic Candia species are of special interest as they have the potential to yield broad-spectrum targets for novel antifungal drugs. To help visualize genotypic and metabolic differences between species, we have also developed the Candida Gene Order Browser, an online tool that displays the syntenic context of genes from all available Candida genomes.


195. The grass endophyte Epichloe festucae: genome, unigenes, and gene expression. U. Hesse, P. Maynard, S. Macmil, G. Wiley, K. Andreeva, W. E. Beech, C.D. Van Horn, V.C. Bumgardner, V. Gopal- Puram, J. Wiseman, J. Webb, L. Gill, S. Alluri, E. Arnaoudova, M.L. Farman, J. W. Jaromczyk, B.A. Roe, C.L. Schardl. University of Kentucky, Plant Pathology Dept.


The ascomycete, Epichloe festucae, is a model endophyte that 1) switches between mutualistic and antagonistic states, 2) is seed transmissible, 3) has a sexual state amenable to genetic analysis, and 4) is rich in bioprotective alkaloids. This fungus grows systemically and intercellularily throughout the life of its host plant. On each reproductive tiller the fungus either infects benignly and transmits clonally in seeds, or produces its sexual state (stroma) and chokes inflorescence development. The E. festucae genome was estimated at 29 Mb in 6 chromosomes. The genome sequence was assembled from cloned insert end reads (4.2 x coverage) and preassembled pyrosequencing reads (454-sequencing: 20 x raw, 1.7 x assembled), giving 3967 supercontigs, of which 1004 larger than 2 kb covered ca 92% of the genome. Gene prediction (FGENESH) identified 9912 putative genes. We sequenced 25,000 ESTs from each of two normalized libraries — one of choked inflorescences, the other of benignly infected inflorescences — yielding 5077 E. festucae unigenes annotated by BLAST and InterPro. Additionally, ca 700,000 pyrosequencing reads were obtained from non-normalized cDNA derived from four biological replicates each of asymptomatic and choked inflorescences. Of these, 118,865 were identified in the E. festucae genome sequence, 23,046 in the unigene set, and 21,703 in the predicted gene set, allowing analysis of gene expression and in symbio regulation. Sequences are BLASTable at http://www.genome.ou.edu/blast/ef_blastall.html, Sequence data and annotations are stored in a MySQL database, soon to be published in GBrowse.


196. Analysis of TOX2, the HC-toxin biosynthetic gene cluster in Cochliobolus carbonum. Kouhei Ohtani, John S. Scott-Craig, and Jonathan D. Walton. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA.


HC-toxin is a cyclic tetrapeptide host-selective toxin produced by the filamentous fungus Cochliobolus carbonum, a pathogen of maize. HC-toxin production in C. carbonum is controlled by a single locus, Tox2, which is composed of at least seven duplicated and coregulated genes involved in HC-toxin biosynthesis, export, and regulation. The Tox2 locus was earlier partially characterized by mapping, sequencing, and gene disruption but we still didn't know its overall structure nor how many additional genes are required for HC-toxin biosynthesis. To understand more thoroughly the structure of Tox2 and to find new genes involved in HC-toxin biosynthesis, we constructed a bacterial artificial chromosome (BAC) library from a C. carbonum toxin-producing strain and screened it with known Tox2 genes. More than six BAC clones were identified and shotgun sequenced to at least 4x coverage. To date we have identified several new putative Tox2 genes. One encodes an alpha subunit of fatty acid synthase, which is predicted to complex with the product of TOXC (encoding a beta subunit of fatty acid synthase) to synthesize the backbone of Aeo (2-amino-9,10-epoxi-8-oxodecanoic acid). Other new genes encode three P450s and an isoamyl alcohol oxidase. Like the known Tox2 genes, all of these new genes are absent in toxin non-producing isolates of C. carbonum. All have plausible roles in HC-toxin biosynthesis, which is being tested by targeted gene disruption.


197. The Genome Sequence of the Brown Rot Fungus Postia placenta. Jean F. Challacombe1, Diego Martinez1, Thomas S. Brettin1, Susan Lucas2, Paul Richardson2, Eddy Rubin2, and Dan Cullen3. 1Los Alamos National Laboratory and DOE Joint Genome Institute, Los Alamos, NM. 2DOE Joint Genome Institute, Walnut Creek, CA. 3 USDA, Forest Products Laboratory, Madison, WI.


The brown rot fungi, a group of filamentous basidiomycetes, rapidly depolymerize the cellulose in wood without significant lignin removal. This type of decay differs from white rot fungi, such as Phanerochaete chrysosporium, which simultaneously degrade lignin and cellulose. Both white and brown rot fungi are common inhabitants of forest litter where they play an important role in carbon cycling. Brown rot fungi are most commonly responsible for the destructive decay of wood in buildings and other structures, and it has been estimated that 10% of the U.S. timber harvest decays in service each year. Irrespective of their economic importance, the mechanism of brown rot wood decay is poorly understood. To better understand the biology of this type of wood degradation, the Department of Energy’s Joint Genome Institute has sequenced the entire 33Mb genome of the model brown rot, Postia placenta. Using genomic DNA from dikaryotic strain MAD-698, the JGI generated 571,000 reads that assembled into 1243 haplotype scaffolds. Our investigation into the genome will provide an invaluable mechanistic perspective on cellulose depolymerization, and the unique haplotype assembly will allow us to gain insights into the complexities of genome evolution.


198. Withdrawn


199. Whole genome sequencing of the fungal plant pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Sabine Fillinger1,François Artiguenave2, Alain Billault3, Mathias Choquer1, Arnaud Couloux2, Christina Cuomo4, David DeCaprio4, Martin Dickman5, Elisabeth Fournier1, James Galagan4, Corinne Giraud1, Chinnappa Kodira4, Linda Kohn6, Fabrice Legeai2, Caroline Levis1, Evan Mauceli4, Cyril Pommier2, Jean-Marc Pradier1, Emmanuel Quevillon2,8, Jeffrey Rollins, Béatrice Ségurens3, Adeline Simon1, Muriel Viaud1, Jean Weissenbach3, Patrick Wincker3, and Marc-Henri Lebrun8. 1UMR BIOGER, INRA, Versailles, France; 2URGI,INRA, Evry, France;3Génoscope- CNS, Evry, France; 4 The Broad Institute of Genome Research, Cambridge MA, USA; 5 Inst. for Plant Genomics and Biotech, College Station TX, USA; 6 University of Toronto, Mississauga, Canada; 7Department of Plant Pathology,University of Florida, Gainesville, FL, USA 8 Plant and Fungal Physiology, CNRS-BayerCropScience, Lyon, France


Botrytis cinerea and Sclerotinia sclerotiorum are destructive polyphageous pathogens of many economically important crops provoking grey and white mould respectively. Both fungi are leotiomycetes. In 2005, the Broad Institute released the assembly of a 4-5 x genomic sequence from B. cinerea strain B05-10 (TMRI/Syngenta) as well as the 7-8 x genomic sequence of S. sclerotiorum. At the same time, the French national sequencing center (Genoscope) started the genome sequencing of grapevine and two of its pathogens: Stolbur phytoplasm and B. cinerea strain T4. In addition to WGS 50.000-60.000 ESTs have been sequenced for both species. Automatic gene prediction with ab initio and similarity softwares has been applied to the three genomes suggesting 14.000 to 16.000 potential gene calls for both species. A manual annotation process involving an international consortium of 20 research groups will validate and correct the data. Comparative analyses of both B. cinerea strains on one hand and B.cinerea > vs. S. sclerotiorum on the other hand revealed a common set of 8400 gene calls, 1200 of which are absent from other fungal genomes. In addition both species present a high degree of sequence similarity (76 % average sequence identity in orthologous proteins), and of synteny at the contig level. S. sclerotiorum has twice as much repetitive elements (7.7 %) than B. cinerea (3 - 4 %) with an enrichment of all transposable elements and tandem repeats. RIPping also occurred at higher levels in S. sclerotiorum. The comparison between B. cinerea and S. sclerotiorum offers the first opportunity to compare the genomes of two closely related necrotrophic plant pathogens helping understanding evolutionary trends that have shaped their genomes.


200. Global Gene Expression Profiles of Phytophthora ramorum strain Pr102 in response to plant host and tissue differentiation. Caroline Press and Niklaus Grunwald, USDA, 3420 NW Orchard Ave., Corvallis, OR 97330. pressc@science.oregonstate.edu


The release of the draft genome sequence of P. ramorum strain Pr102, enabled the construction of an oligonucleotide microarray of the entire genome of Pr102. The array contains 344,680 features (oligos) that represent the transcriptome of Pr102. P. ramorum RNA was extracted from mycelium and sporangia and used to compare gene expression across tissue types and in the presence of the host (Rhododendron sp.). The purpose of the experiment was to identify genes where expression was responsive to tissue types and upon exposure to the host plant for further study. Gene expression studies were performed using a Nimblegen microarray. Genes were determined to be differentially expressed between tissue types if they were statistically significant (P = 0.05 after FDR) and resulted in a greater than 20-fold change in gene expression. In the comparison between mycelia and sporangial tissues, 263 genes demonstrated a greater than 20 fold change in gene expression. Of those genes, 52 genes were significantly downregulated in sporangia as compared to mycelium and 214 genes were significantly upregulated in sporangia as compared to mycelium. Several of the differentially expressed genes appear to be of the same type as those in a similar study in Phytophthora infestans by Kim and Judelson (2003) and include genes involved in cell wall restructuring, cell division and signaling functions. Not surprisingly, several genes involved in energy production, electron transport chains and growth are reduced in sporangia. 50% of the genes with differential expression have not yet been classified as to function in the current annotation. Further experimentation and analysis is ongoing.


201. Computational Resources for Fungal Genomics. Gangman Yi1, Jaehee Jung1, Serenella A. Sukno2, and Michael R. Thon1,2. Texas A&M University, 1Department of Computer Science, and 2Department of Plant Pathology & Microbiology, College Station, TX USA


As the number of sequenced fungal genomes continues to grow, there is an increasing need for effective algorithms for whole genome analyses. We present two applications that are aimed at improving our knowledge of gene function and genome evolution. First, we present C-Hunter, an algorithm and software application designed to identify clusters of functionally related genes in genomes. C-Hunter utilizes functional categories defined in graph-based vocabularies such as the Gene Ontology (GO). Clusters identified in this manner need only have a common function and are not constrained by gene expression patterns or other properties. C-Hunter source code and example report files are freely available from our web site. We also present AAPFC (Automated Annotation of Protein Functional Class). AAPFC addresses the need for high quality, automated functional annotation of proteins in fungal genomes. The method utilizes data mining and statistical pattern recognition techniques to learn models for assigning Gene Ontology functional categories to unannotated proteins. In its current implementation, AAPFC is trained using fungal GO-annotated proteins obtained from the UniProt database. The AAPFC web site provides users with GO annotations and protein features in graphical and textual formats. Users can annotate small sets of proteins via a web interface and large sets of proteins by contacting the authors.


202. Transcriptional patterns in the extramatrical mycelium of the ectomycorrhizal fungus Paxillus involotus in response to various sources of nitrogen. Derek P. Wright, Dag Ahren, Anders Tunlid & Tomas Johansson Microbial Ecology, Ecology Building, Lund University, Sweden


The positive effects of ectomycorrhizal (ECM) fungi on plant nutrition have traditionally been attributed to the quantitative effect of the extramatrical mycelium on uptake of dissolved nutrients, such as nitrogen. In forest soils nitrogen is present either in inorganic (mainly as ammonium) or organic (amino acids, peptides and proteins) forms and nitrogen mobilization by hyphae from soil is directly linked to hyphae uptake capacities. Transcriptional programs for assimilation of various sources of inorganic and organic forms of nitrogen were investigated in the ECM association between Paxillus involotus and birch. Nitrogen sources of various degrees of complexity, ammonium phosphate, ammonium sulphate, glutamine, chitin, and bovine serum albumin were provided as patches in peat microcosms for fungal in growth. After fungal establishment in these patches total RNA was isolated and used for global transcriptional analyses. We used a custom cDNA microarray containing approx. 4,900 gene representatives. By bioinformatic means and using EST sequence information we could among those identify 462 reporters representing putative enzymes. From a metabolic reconstruction of these 462 enzymes we found them potentially involved in 901 enzymatic reactions and 173 metabolic pathways. By combining this information with transcriptional data we could observe major shifts in a number of metabolic pathways as responses to the nitrogen source provided.


203. Comparative Genomic Hybridization in the Genus Neurospora: Assessing the Ability to Recover Evolutionary Relationships. Luz B. Gilbert*, Lee Chae, Takao Kasuga, Louise Glass, and John W. Taylor Department of Plant and Microbial Biology, UC Berkeley, *lgilbert@berkeley.edu


Comparative Genomic Hybridization (CGH) using DNA microarrays is becoming a popular method of determining phylogenetic relationships among individuals and even species. Although researchers praise the large-scale genome information output as an advantage of this technology for use in phylogenetic analysis, few have questioned the reliability of CGH to correctly determine evolutionary relationships. With the tools at my disposal for the filamentous fungus Neurospora: a genome sequence, a DNA microarray, and a well supported phylogeny for this genus, I had the ability to rigorously address the utility of CGH for phylogenetic studies using both experimental and simulated data. The results suggest constructing phylogenetic trees from this type of data requires careful consideration of which tree-building method is used and which taxon is printed on the array.


204. Analysis of the Magnaporthe grisea secretome by liquid IEF fractionation and mass spectrometry. Gerardo Gutierrez-Sanchez, Punit Shah, James Atwood III, Denise Lennon, Estee-Lina Tran, Peter Albersheim, Alan Darvill, Ron Orlando and Sheng-Cheng Wu. Complex Carbohydrate Research Center,University of Georgia, 315 Riverbend Road, Athens, GA 30602-4712, USA.

Magnaporthe grisea, a fungal plant pathogen, is a major threat to rice production globally. The M. grisea genome encodes many secreted proteins and in response to various growth conditions it secretes extracellular proteins (ECPs) which play indispensable roles in host pathogen interactions. For many years, plant pathologists have concentrated on elucidating the relation between environmental factors and field development of the disease, developing fungicides, or breeding resistant cultivars. However, molecular basis of the resistance mechanisms in rice plants against M. grisea or of host specificity for M. grisea has not been clearly elucidated. A more extensive catalog of the secretome would therefore represent a valuable resource to elucidate processes such as pathogenesis and disease resistance. The aim of this study is to gain new insights into the dynamics and complexity of the proteins that are secreted by both Magnaporthe grisea and its rice host (Oryza sativa L.) during infection. To date, most of the proteomics studies in plant pathogenic fungi have been limited to 2-D gel analysis. Here we present a simple and effective “non-gel”-based fractionation method to separate the secreted proteins from M. grisea and rice plant, followed by LC-MS/MS analysis for protein identification and statistical validation with the PROVALT algorithm."


205. Preliminary GO annotation of a blast fungus Magnaporthe grisea. Shaowu Meng, Douglas E. Brown, Thomas K. Mitchell, Ralph Dean*. Center for Integrated Fungal Research, North Carolina State University, Campus Box 7251, 851 Main Campus Drive, Raleigh, NC 27695


The Magnaporthe grisea genome assembly Version 5 was released in the spring of 2006 along with automatic annotations. The next step is a curated community annotation effort. Gene ontology (GO) based systems are evolving into a reliable and rapid means of assigning annotations. Here, we report our preliminary GO annotation of M. grisea genome. This was done in two steps. Firstly, we identified orthologs between M. grisea and Saccharomyces cerevisiae through cataloging reciprocal best hits between translated ORFs of the two organisms using blastp. The cataloging was iteratively performed on the remaining un-annotated genes until the last reciprocal best hit was found. The result was 4,464 unique M. grisea ORFs annotated using 2,581 unique S. cerevisiae GO terms totally at 26,966 times. Next, for those M. grisea ORFs which did not have orthologs in the S. cerevisiae database, we applied the cataloging process using the GO proteins databases, which included multiple organisms. In this step, 1,760 unique M. grisea ORFs were annotated using 1,263 unique GO terms totally at 5,381 times. The GO annotation of M. grisea covered 50.9% of predicted genes. Our GO annotation provides a base for further functional genomics analysis of M. grisea, and for assigning newly created terms from the PAMGO community that are specific to plant pathogens. *Corresponding author.


206. Conserved Synteny in Trichoderma ressei with Related Filamentous Ascomycetes. Diego Martinez1,2, Mary Anne Nelson2 and Margaret Werner-Washburne2. 1Los Alamos National Laboratory/Joint Genome Institute, Los Alamos, NM 87018 2Department of Biology, University of New Mexico, Albuquerque, NM 87131


The current pace of whole genome sequencing has made it almost trivial to produce nearly complete fungal sequences. This now gives us the ability to perform whole genome comparisons of fungi to investigate biological questions and genome evolution. To this end, syntenic regions between Trichoderma reesei and four other filamentous fungi (Fusarium graminearum, Neurospora crassa, Magnaporthe grisea and Aspergillus nidulans) were identified. An algorithm was designed that takes homologs from two fungi and places them side by side, optimizing homolog density, and minimizing gaps between homologs and the number of homologs in a syntenic region. As would be expected, the percent coverage of synteny between T. reesei and the other four ascomycetes declines with time since the last common ancestor, with F. graminearum having the highest synteny and A. nidulans the lowest. This approach enabled the identification of regions in the T. reesei genome that were conserved in multiple genomes. Finally, for genes within these highly conserved regions, GO terms and Enzyme Commission codes were assigned. These results were used to assess the possibility that there is selective pressure to force genes that are in the same biochemical pathway, have similar function or are involved in similar processes to maintain proximity.


207. Comparative proteomics of the in vitro and in planta secretomes of Fusarium graminearum. Janet M. Paper, John S. Scott-Craig and Jonathan D. Walton. DOE-Plant Research Laboratory, Michigan State University, E. Lansing MI 48824


F. graminearum (Fg) is the cause of head blight and stalk rot of wheat and maize. Its sequenced genome is predicted to encode ~12,000 proteins. Secreted fungal proteins have several known functions related to pathogenesis, including phytotoxicity, degradation of cell wall polymers (penetration, ramification, and nutrition), and inhibition of plant defense proteins. We are using peptide mass fingerprinting to analyze the secretome of Fg in vitro and in planta. Secreted proteins from Fg grown in vitro on different media or extracted from infected wheat heads by vacuum infiltration were digested en masse with trypsin and analyzed by LC-MS/MS. From culture filtrates of Fg grown on sucrose, carrot or maize cell walls, collagen, xylan, pectin, or oat bran, ~230 proteins could be identified with high statistical probability. Because many secreted proteins are catabolite-repressed and/or substrate-induced, the spectrum of proteins was influenced by the medium, e.g., collagen induced the most proteases and sucrose yielded the fewest hydrolases. Despite the preponderance of host proteins in infected wheat heads, we could identify 120 fungal proteins. Many of these (25) were predicted to interact with host cell walls (e.g., hydrolases, sugar oxidases, cellulose-binding proteins, acetylxylan esterases). Some (42) in planta proteins were not found any of the in vitro analyses. About 27% of the in planta proteins were of unknown function or predicted to be non-enzymatic. Although >85% of the in vitro proteins were predicted to have signal peptides, only 61% of the in planta proteins were. Among the many predicted non-secreted proteins found in planta were 13 housekeeping enzymes including enolase, triose phosphate isomerase, phosphoglucomutase, calmodulin, aconitase, and malate dehydrogenase. The presence of these proteins in the in planta secretome might be due to fungal lysis in planta, but several of them have been reported to be immunogens secreted by animal pathogenic fungi.


208. Simple Sequence Repeats in Neurospora crassa : distribution, polymorphism, and marker potential. Tae-Sung Kim1, James Booth2, Hugh G. Gauch, Jr.3, Qi Sun4, Jongsun Park5, Kwangwon Lee1. 1Department of Plant Pathology, 2Department of Biological Statistics and Computational Biology, 3Department of Crop and Soil Sciences, Cornell University, Ithaca, NY 14850 USA 4Cornell Theory Center, Ithaca NY 14850 USA 5Fungal Bioinformatics Laboratory and Department of Agricultural Biotechnology Seoul National University, Seoul 151-921, Korea.


Simple sequence repeats (SSRs) have been successfully used as molecular markers for various genetic studies in eukaryotic systems. Despite the growing number of sequenced genomes, a thorough statistical examination on the distribution of the SSRs and experimental verification of the usefulness of SSRs in filamentous fungi for genetic and genomics studies are lacking. We identified and characterized the 2749 SSRs of 963 SSR types in the whole genome of the model fungal system Neurospora crassa. The tri-nucleotide (nt) repeats were the most common SSRs in N. crassa. The distribution of the tri-nt SSRs was significantly biased in exons. We further characterized the distribution of 18 abundant SSR types, which account for 71% of total SSRs in the N. crassa genome. We tested whether the occurrence rate of SSRs is random across chromosomes using Poisson log linear model. Based on our analyses, we concluded that there are systematic differences in SSR occurrence rates between chromosomes as well as between SSR types. We attempted to explain the non-randomness of the SSR occurrence between the chromosomes using factors such as chromosome size and gene density. We also characterized the size variation of SSRs among accessions using Polymorphic Index Content (PIC). We characterized the PIC scores of abundant SSR types using randomly selected 162 SSR loci in seven N. crassa accessions. Finally, utilizing the polymorphic SSRs, we built three linkage maps from three intra-species specific populations. The genetic orders of SSR markers along the chromosomes from the three linkage maps were highly consistent with the position in the physical map suggesting that the genetic architectures of the six natural accessions are similar to each others.


209. Annotation and analysis of the genome of Phycomyces blakesleeanus, a model photoresponsive zygomycete. Alan Kuo1*, Asaf Salamov1, Jasmyn Pangilinan1, Erika Lindquist1, Harris Shapiro1, Scott Baker2, Luis Corrochano3, and Igor Grigoriev1. 1DOE Joint Genome Institute, Walnut Creek, CA, USA. 2Pacific Northwest National Laboratory, Richland, WA, USA. 3Departamento de Genetica, Universidad de Sevilla, Spain. *akuo@lbl.gov


Light induces in P. blakesleeanus multiple developmental and biochemical responses (sporangiophore growth and development, beta-carotene synthesis). P. blakesleeanus is an intensively studied, experimentally tractable model organism, and whole-genome analysis is expected to further elucidate the signaling pathways underlying its photoregulation. To this end the genome was sequenced to 7.49X depth and assembled into 475 scaffolds totaling 56Mbp, and 47847 ESTs were assembled from cDNAs of light and dark cultures. We combined into a single annotation pipeline a variety of gene modeling methods (homology-based, EST-based, and ab initio), and predicted 14792 protein-coding genes. Many of these gene predictions are supported by homology in nr (68%), by Pfam domains (44%), or by ESTs (35%). We next assigned GO terms to 41% of the proteins and EC numbers to 16%. We then distributed these annotations to the Phycomyces consortium, along with tools to curate them manually. We expect that the annotation will provide a solid platform for expression analysis. In addition to its value as a model organism, P. blakesleeanus is the second zygomycete with a sequenced genome, after the related Rhizopus oryzae. We therefore will present preliminary results of comparative analysis between the two zygomycetes. LBNL-62091 ABS This work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231 and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396.


210. Development of wheat leaf rust, Puccinia triticina, ESTs from various spore stages. Guus Bakkeren1, Junhuan Xu1, Rob Linning1, Yehoshua Anikster2, Brent McCallum3, Travis Banks3, Sarah Munro4, Michael Mayo4, Brian Wynhoven4, Johar Ali4, Richard Moore4. 1Agriculture & Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, BC, V0H 1Z0, Canada; 2Tel Aviv University, Israel; 3 Agriculture & Agri-Food Canada, Cereal Research Centre, Winnipeg, MB, R3T 2M9; 4 Michael Smith Genome Sciences Centre, Vancouver, BC, V5Z 4S6, Canada. bakkereng@agr.gc.ca


We are in the process of increasing genomic resources for the obligate wheat pathogen, the leaf rust fungus P. triticina. Approximately 17,000 EST reads covering a teliospore stage from senescent wheat and pycnio- and aeciospore stages from an alternate host infection on Thalictrum speciosissimum (meadow rue), were added to our previously established, 30,000 reads-large EST database (holding ESTs from urediniospore, appressorium and other wheat infection stages). These new spore stages contributed approximately 65%, 80% and 45% new unigenes to the existing database, respectively. From initial analyses it appears that aeciospores might express gene complements more similar to the urediniospore stages from wheat we sampled previously; this might illustrate the possible resemblance of function of these spores, i.e., infecting wheat. The pycniospores stage, which represents the sexual stage, displayed the largest pool of new genes. Other trends and comparisons will be presented.


211. The secretome of Coprinopsis cinerea. Andrzej Majcherczyk, Dorothea Fragner, Ravi Chandra Dwivedi, Mojtaba Zomorrodi, and Ursula Kües. Institute of Forest Botany, Georg-August-University, Göttingen, Germany


Basidiomycetes secrete many different proteins for degradation of complex substrates. In the complex fungal secretomes, only few proteins can be studied by direct detection of enzymatic activities and changes in substrate composition. To understand and to follow degradation processes, we use mass spectrometry for the analysis of tryptic digests of proteins with a Mascot database containing known protein sequences, annotated genomic sequences and ESTs from Coprinopsis cinerea, Trametes versicolor and Pleurotus ostreatus, respectively. Analysis of the C. cinerea secretome showed an increasing complexity with rising age of cultures. Several different glucosidases, oxidative enzymes (e.g., laccases, manganese-dependent peroxidases, glyoxal and glucose oxidases), lectin-like proteins and multiple proteolytic enzymes have been identified in liquid culture. Other enzymes effective in substrate degradation localize to the fungal cell wall and methods are developed to identify their individual nature. Part of this work is supported in frame of a Common Lower-Saxony-Israel Project (VW-Vorab). The Deutsche Bundesstiftung Umwelt financially supported our laboratory.


212. Identification of pathogenicity factors among secondary metabolite-related genes in Alternaria brassicicola. Kwang-Hyung Kim1, Yangrae Cho1, Mauricio La Rota1, Robert Cramer2, and Christopher Lawrence1. 1 Virginia Bioinformatics Institute and Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. 2 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Duke University, Durham, NC 27708-9902


Very little information is currently available concerning the pathogenic determinants produced by Alternaria brassicicola, the causal agent of black spot disease of Brassica plants. Production of secondary metabolites with host and non-host specific phytotoxicity has been proposed to play a role during the infection process of plants by fungi, especially in necrotrophic fungal pathogens. Gene clusters containing Polyketide Synthase (PKS) and Non-Ribosomal Peptide Synthetase (NPS) coding genes have been shown to be a major source of toxic secondary metabolites in fungi. To test the hypothesis that secondary metabolites produced by A. brassicicola contribute to pathogenicity, 8 NPS and 9 PKS genes were first identified by mining the A. brassicicola genome sequence database in our lab. Knockout (KO) mutants, constructed by gene disruption technology developed in our lab, were obtained for all 17 genes and evaluated for changes in fungal development and pathogenicity. Results of studies so far indicate some of the KO mutants of PKS and NPS genes showed interesting phenotypes and/or reduced virulence. Here we present functional characterizations of two interesting NPS genes, AbNPS2 and AbNPS4. As a result of NCBI BLAST searches, AbNPS2 and AbNPS4 showed high similarity (E-value 0.0) to NPS4 and NPS12 of Cochliobolus heterostrophus, respectively. To further characterize the abnps2 and abnps4 transformants, we are conducting chromatographic and histomorphological analyses. Future studies include further characterization of other PKS and NPS genes, KO mutagenesis and plant pathogenicity analyses, and structural analyses (LC-MS and NMR) of the in vitro-produced metabolites identified thus far.


213. Contributions of aneuploidy to fluconazole resistance in Candida albicans. Anna Selmecki, Anja Forche, Judy Berman. University of Minnesota, Minneapolis, MN selme003@umn.edu


Genomic instability and aneuploidy are common in Candida albicans. Using comparative genome hybridization (CGH) microarrays, we found a strong correlation between aneuploidy and azole resistance1. Furthermore, we identified a segmental aneuploidy of chromosome 5L (Ch5L) DNA that was due to the formation of an isochromosome (a chromosome with two identical arms), in many fluconazole resistant (FluR) strains. Isochromosome 5L (i(5L)) formation is often accompanied by loss of heterozygosity (LOH) along Ch5L and while i(5L) is relatively stable under rich culture conditions, loss of i(5L) does occur and always correlates with a loss of FluR. Microarray analysis of i(5L) strains show that most genes on Ch5L exhibit increased expression and may contribute to FluR. Several well characterized genes involved in drug transport, ERG11 and TAC1, are present within the isochromosome and thus present in increased gene copies. We found that deletion of one or two extra copies of these genes in an i(5L) strain background has only partial effects on FluR, suggesting that increased copies of these genes alone are not responsible for the high level of resistance seen in the i(5L) strain. We are currently using telomere-mediated chromosome truncations of Ch5L to determine the degree to which different gene regions convey resistance. The effects of aneuploidy on gene copy number, gene expression level, LOH, and acquired azole resistance will be discussed. 1. Selmecki et al. (2006) Science 313: 367-370.


214. Initial analyses of the transposable elements of Rhizopus oryzae. Margi Butler1, Manfred Grabherr2, Tim Goodwin1, Russell Poulter1, Michael Koehrsen2, Reinhard Engels2, Sinead O’Leary2, Chinnappa Kodira2, Bruce Birren2 and Li-Jun Ma2. 1 University of Otago, NZ. 2Broad Institute of MIT and Harvard, Cambridge MA. margi.butler@otago.ac.nz


Rhizopus oryzae is the first Zygomycete fungus to be sequenced and differs from all previously sequenced fungal genomes in being highly repetitive. Repeat sequences account for approximately 35% of the genome. Such a heavily repetitive genome provides the unique opportunity to study the origin of the repeats and their impact on genome evolution. Analyses show that more than half of the identified repeats are derived from transposable elements. Representatives of all the mobile elements, including class I retrotransposons (LTR retrotransposons, non-LTR retrotransposons and DIRS-like elements) and class II DNA transposons (DDE transposons, Helitrons and Cryptons) are found in the genome assembly. Remarkably, many of the mobile elements are apparently functional. No evidence of RIP (repeat induced point mutation) was detected among copies of transposable elements. The presence of abundant, apparently functional, elements will provide a unique resource for analysing transposon and retrotransposon replication. We will describe the frequency, diversity and distribution of these elements in the genome and will discuss the their impact on the genome dynamics of organism. Funding for the Rhizopus oryzae genome sequencing was provided by NHGRI


215. Identifying Divergent Genes in the Genus Neurospora and Related Species Using Comparative Genomic Hybridizations. Luz B. Gilbert*1, Takao Kasuga1, Jeff Townsend 2, Louise Glass1, John W. Taylor1. 1Department of Plant and Microbial Biology, UC Berkeley, 2Department of Ecology and Evolutionary Biology, Yale University, *lgilbert@berkeley.edu


Array based Comparative Genomic Hybrization or CGH is a useful tool to identify candidate genes that differ between species. Using the 70mer expression array designed for Neurospora crassa we compared seven species of Neurospora, as well as Podospora anserina and Sordaria macrospora in an effort to detect divergent genes. A second whole-genome tiling array was constructed by Nimblegen Systems for Neurospora crassa for a more comprehensive CGH analysis in both genic and intergenic regions of the genome. Two of the nine species used in the 70mer study were competitively hybridized to the Nimblegen tiling array. Genes found divergent using both arrays were classified according to functional category and a subset were selected for sequencing analysis.


216. Whole transcriptome analysis of Coprinus cinereus meiotic development: a nosey look at mushroom sex. Claire Burns1; Jason Stajich2; Jason Lieb3; Walt Lilly4; Allen Gathman4; Mimi Zolan1; Pat Pukkila3. 1Dept. of Biology, Indiana University, Bloomington, IN, USA; 2Dept. of Plant Biology, University of California, Berkeley, CA, USA; 3Dept. of Biology, University of North Carolina, Chapel Hill, NC, USA; 4SE Missouri State University, Cape Girardeau, MO, USA


Coprinus cinereus, a basidiomycete mushroom fungus ideally suited to meiotic studies due to its synchronous development, has a near-completed genome. EST information is available for ca. 2500 genes, and gene prediction algorithms indicate a total of ca. 12,500 genes. We have designed and fabricated a 13,230 element 70-mer oligonucleotide microarray using ArrayOligoSelector. Design criteria and array formulation will be presented. These arrays are to be made available to the wider Coprinus community. The tightly-regulated light and temperature dependent life cycle of the mushroom allows sampling of gill tissue in which cells are at a specific meiotic stage at a certain time. Meiosis occurs synchronously and takes ca. 12 hours. Our initial analyses using the new Coprinus arrays concern a broad 15-hour timecourse through the meiotic process, describing events prior to fusion of dikaryotic nuclei, nuclear fusion, condensation and alignment of meiotic chromosomes, first meiotic division, and completion of the second division. Analysis of resultant data using several algorithms, including ArrayLod and SAM, will be presented. This work is supported by NIH, and by the Indiana METACyt Initiative of Indiana University, which is funded in part through a major grant from the Lilly Endowment, Inc.


217. Genome sequence of Batrachochytrium dendroabatidis. Christina Cuomo1, Manfred Grabherr1, Lucia Alvarado1, David DeCaprio1, Chinnappa Kodira1, James Galagan1, Timothy James2, Joyce Longcore3, and Bruce Birren1. 1Broad Institute of MIT and Harvard, Cambridge, MA 2Uppsala University, Uppsala, SWE 3University of Maine, Orono, ME


Batrachochytrium is a pathogen of amphibians implicated as a primary causative agent of amphibian declines. This recently emerging pathogen was identified in 1998 as the cause of amphibian deaths in Australian and Central America. More recently, B. dendrobatidis has been implicated in population declines of frog species in North America, South America, Europe and Africa. We chose to sequence strain JEL423, isolated from a sick Phylomedusa lemur frog from Panama. We produced a 7.4X whole genome shotgun assembly, which contains 23.4Mb of sequence in 348 contigs, which are linked into 69 scaffolds. The sequence is highly repetitive

 (~24%), but no significant similarity is found to previously characterized transposons. Progress in automated gene prediction will be described. This is the first representative of the Chytrid order to have its genome sequenced, and will provide the opportunity for comparisons across the fungal clade along with the sister animal clade.


218. Withdrawn


219. Fungal Comparative Genomics – Progress under the Fungal Genome Initiative. Christina Cuomo, Matthew Henn, Li-Jun Ma, Kurt Labutti, Sean Sykes, Manfred Grabherr, Evan Mauceli, Sante Gnerre, Matthew Crawford, Michael Koehrsen, Reinhard Engels, Lisa Larson, Phil Montgomery, Matthew Crawford, Jared White, Matthew Pearson, Clint Howarth, Qiandong Zeng, Chandri Yandava, Sinead O’Leary, Lucia Alvarado-Balderrama, Sarah Young, Dave DeCaprio, Chinnappa Kodira, David Jaffe, Chad Nusbaum, James Galagan, and Bruce Birren. The Broad Institute of MIT and Harvard, Cambridge MA, bwb@broad.mit.edu


The Fungal Genome Initiative was launched to generate genomic resources to promote research on organisms across the fungal kingdom. The FGI has been creating fungal genome sequences, annotations, and analytic tools to support studies of individual organisms and their evolutionary origins. To date the FGI has released genome sequence for 28 fungi, including fungi from all four major branches of the fungal tree. In addition, 35 approved fungi are currently in the sequencing queue. A primary focus of the FGI has been to promote comparative studies by sequencing clusters of related fungi and phenotypic variants of species to help interpret the biology of key models or pathogens. Recently initiated projects include major sequencing efforts around human pathogenic fungi, Candida albicans (5 species), dermatophytes (5 species), Coccidioides (14 new strains), and other dimorphic pathogens (9 strains of 4 species). Comparative sequencing projects around plant pathogenic Fusarium and Verticillium species are also underway. We will describe recent progress on sequencing as well as new tools and web sites to help make these comparative data most useful to the community.


220. Sequencing and Comparative Analysis of Candida Genomes. Christina Cuomo1, Matthew Rasmussen2, Manfred Grabherr1, David DeCaprio1, Lohith Kini2, Chinnappa Kodira1, Esther Rheinbay2, Radek Szklarczyk2, Geraldine Butler3, Neil Gow4, Joseph Heitman5, Michael Lorenz6, Andre Nantel7, Manolis Kellis1,2, and Bruce Birren1. 1Broad Institute of MIT and Harvard, Cambridge MA 2MIT Computer Science and Artificial Intelligence Laboratory, Cambridge, MA 3 University College Dublin, Dublin, Ireland 4 University of Aberdeen, Aberdeen, United Kingdom 5Duke University Medical Center, Durham, NC 6University of Texas Health Science Center, Houston, TX 7National Research Council of Canada, Montreal, PQ, Canada


Analysis of an individual fungal genome such as Candida albicans can be greatly enhanced by comparison to genomes of related species. Comparative genomics highlights sequences conserved between species and can inform such basic questions as how genomes evolve, what genes make a species unique, and what genes and noncoding sequences are shared among species. As part of the Fungal Genome Initiative, we have sequenced five related Candida species: C. albicans (strain WO-1), C. tropicalis, Lodderomyces elongisporus, C. guilliermondii, and C. lusitaniae. We have released assemblies and preliminary gene sets from each genome. Additionally we have generated comparative genomic resources including whole genome alignments, protein families, species specific genes, and Candida specific genes. These resources are being utilized by a community based comparative analysis project which is currently underway. The growing set of Candida genome sequences allows comparisons across a range of evolutionary distances, enabling many different approaches to study the conservation of genes and regulatory elements as well as the evolution of these elements and genomic architecture within Candida species.


221. DelsGate, a robust and rapid gene deletion construction method for functional genomics. María D. García-Pedrajas1†, Laura B. Kapa1, Michael H. Perlin2, and Scott E. Gold1. 1Department of Plant Pathology, University of Georgia, Athens, GA 2, Department of Biology, University of Louisville, Louisville, KY Estación Experimental del Zaidín, CSIC, Granada, Spain


With increasing availability of fungal genome sequences there is great demand for fast, simple methods to generate gene deletion constructs. We describe a method, named DelsGate, that combines PCR and Gateway cloning technology together with the I-SceI homing endonuclease to generate precise deletion constructs in a simple, universal and robust manner in just two days. These constructs may then be used to produce deletion mutants in the organism of interest. The initial version (DelsGate?) involved PCR of the entire ORF and 1 kb of 5’ and 3’ flanks, followed by self-ligation, inverse PCR and Gateway cloning to generate the final construct. A simplified improved version (DelsGate) was later devised consisting of PCR of only the 5’ and 3’ 1 kb gene flanks followed directly by in vitro Gateway cloning and final circular deletion construct generation by in vivo recombination in E. coli. For use in DeslGate we have produced vectors for transformation of Ascomycetes and the Basidiomycete fungus Ustilago maydis. We have tested the feasibility and reproducibility of the DelsGate approach by generating deletion constructs for 12 genes in U. maydis. Although not tested here DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species.


222. High throughput gene knockouts in Neurospora. Patrick D. Collopy1, Hildur V. Colot1, Gyungsoon Park2, Carol Ringelberg1, Liubov Litvinkova2, Susan Curilla1, Lorena Altamirano2, Norma Gorrochotegui-Escalante1, John Jones2, Katherine A. Borkovich2, and Jay C. Dunlap1. 1Department of Genetics, Dartmouth Medical School, Hanover, NH, USA 2Department of Plant Pathology, University of California, Riverside, CA


Gene disruptions have been made in predicted open reading frames in the annotated Neurospora crassa genome using a high throughput procedure as part of an NIH-funded Program Project (P01)*. Knock-out (KO) cassettes were created by yeast recombinational cloning techniques and electroporation of N. crassa conidia can be performed in a 96-well format. In order to expedite molecular confirmation of KO mutant strains, we have developed a program ( http://borkovichlims.ucr.edu/southern/ ) that allows automated identification of the appropriate restriction enzyme to use during Southern analysis. Additionally, we have developed and implemented a Laboratory Information Management System (LIMS; http://borkovichlims.ucr.edu/php/sLIMS.php ) to keep track of the various steps of our gene KO processes. All plates and tubes used during the knockout procedure are labeled with barcodes and managed systematically. Completed N. crassa KO mutant strains are submitted to the Fungal Genetics Stock Center after targeted gene replacements are confirmed by Southern analysis. The list of submitted strains is available at the Neurospora genome project website ( http://www.dartmouth.edu/~neurosporagenome/knockouts_completed.html ). Use of these tools and our current progress in creating KO mutants will be presented. *Colot and Park et al., 2006. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. PNAS 103:10352-10357


223. High-throughput mutation procedure for Neurospora genes. Lorena Altamirano1, Luibov Litvinkova,1, Gyungsoon Park1, John Jones1, Hildur V. Colot2, Patrick D. Collopy2, Norma Gorrochotegui- Escalante2, Carol Ringelberg2, Jay C. Dunlap2 and Katherine Borkovich1. 1 Department of Plant Pathology, University of California, Riverside, CA 2 Department of Genetics, Dartmouth Medical School, Hanover, NH, USA


Gene deletions have been made in predicted open reading frames and genes in the annotated Neurospora crassa genome using a high throughput procedure as part of an NIH-funded Program Project (P01). Many steps of the process utilize a 96-well format, including construction of knock-out (KO) cassettes (using yeast recombinational cloning techniques), electroporation of N. crassa conidia and subsequent analysis of transformants. In order to expedite molecular confirmation of KO mutant strains, we have developed a program ( http://borkovichlims.ucr.edu/southern/ ) that allows automated identification of the appropriate restriction enzyme to use during Southern analysis. Additionally, we have created and implemented a Laboratory Information Management System (LIMS; www.borkovichlims.ucr.edu/php/sLIMS.php ) to track the various steps in the gene KO process. All plates and tubes used during the knockout procedure are labeled with barcodes and managed using the LIMS. Completed N. crassa KO mutant strains are submitted to the Fungal Genetics Stock Center after the gene replacements are confirmed by Southern analysis. The list of submitted strains is available at the Neurospora genome project website ( http://www.dartmouth.edu/%7Eneurosporagenome/knockouts_completed.html ). To date, we have constructed knockout mutants for1480 Neurospora genes and a total of 2233 mutant strains have been submitted to the FGSC. Another 960 genes are in various stages of the knockout procedure.


224. The Comparative Fungal Genome Websites of the Broad Institute. M. Crawford, M. Koehrsen, R. Engels, L. Larson, P. Montgomery, J. White, M. Pearson, C. Howarth, T. Ledlie, D. Park, D. DeCaprio, J. Galagan, B. Birren. Broad Institute, Genome Biology, Cambridge, MA


Efforts at the Broad's Fungal Genome Initiative focus on the sequencing of clusters of related organisms. To make these data as useful as possible we are developing a set of comparative group web sites to analyze multiple genomes. These sites feature redesigned statistics pages that highlight differences and similarities between different assemblies, genomes, and gene predictions. BLAST and feature searches can be performed simultaneously on the genomes of all the organisms in the group, with the results tabulated for meaningful comparison. The process for downloading has also been adjusted: sequences and other files generated for several organisms can be conveniently downloaded together. For group sites where the corresponding genes between organisms have been mapped, a set of views and tools facilate the analysis of related genes, including a Tree View and an Alignment view. The Argo browser now incorporates a comparative view that can assist in analyzing two or more genomes simultaneously. To make these tools as widely useful as possible we are integrating published genomes generated by others to give users a consistent analysis platform.


225. Tagging target genes of the MAT1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A). Anita Keszthelyi1, Apor Jeney1, Zoltán1, Odette Mendes2, László Hornok1 and Cees Waalwijk2. 1Agricultural Biotechnology Center, Szent-Györgyi A. u. 4, H-2100 Gödöllõ, Hungary 2Business Unit Biointeractions and Plant Health, Plant Research International BV, P.O. Box 16, 6700 AA Wageningen, The Netherlands cees.waalwijk@wur.nl


Mating type in filamentous ascomycetes is controlled by idiomorphic alleles, named MAT1-1 and MAT1-2, which contain one to three genes. Of these genes MAT1-1-1 and MAT1-2-1 encode putative transcription factors and are thus considered to be the major regulators of sexual communication and mating. Fungi with no known sexual stage may also have fully functional mating type genes therefore it was plausible to hypothesize that the MAT products may also regulate other types of genes not involved directly in the mating process. To identify putative target genes of these transcription factors in Fusarium verticillioides, DMAT1-2-1 knock out mutants were produced and transcript profiles of mutant and wild type were compared by means of differential cDNA hybridization. Clones, either up- or down- regulated in the DMAT1-2-1 mutant were sequenced and a total of 248 sequences were blasted against the NCBI database as well as the G. zeae and G. moniliformis genomes. Fifty-five % of the clones were down-regulated in the mutant, indicating that the MAT1-2-1 product positively affected these tagged sequences. On the other hand, 94 sequences (~45%) were found to be up-regulated in the mutant, suggesting that the MAT1-2-1 product also exerted a negative regulatory function on this set of genes. Sequences involved in protein synthesis and metabolism occurred more frequently among the clones, up-regulated in the mutant, whereas genes belonging to cell signalling and communication were especially frequently tagged among the sequences, down-regulated in the mutant.


226. Computational Predictions of Gene Structure in the Filamentous Fungus. Aspergillus niger. Christine Chee1, Diego Martinez1,2 and Mary Anne Nelson1. 1Department of Biology, University of New Mexico, Albuquerque, NM 87131; cchee@unm.edu, manelson@unm.edu 2Los Alamos National Laboratory/Joint Genome Institute, Los Alamos, NM 87018; admar@lanl.gov


The Department of Energy‘s Joint Genome Institute has recently sequenced the genome of the filamentous fungus Aspergillus niger. Like all eukaryotic organisms, A. niger has many genes with complex structure. To assist in the discovery of this large number of genes, the JGI utilizes a gene prediction pipeline to automatically predict the intron – exon structure of genes. All information is displayed on an interactive website, The JGI Genome Portal (http://genome.jgi-psf.org/index.html). Due to the complexities of gene structure, it is necessary to manually verify and correct about 25% of the predicted gene models; the most common errors are truncated 5’ and/or 3’ ends. The interactive Genome Portal allows for comparison of A. niger predicted proteins against the proteins of many other species simultaneously, as well as known partial cDNAs and mRNA information. If editing is required, the curator can adjust the gene model structure or create an entirely new gene model. The combination of sequence comparisons and editing ability allows the researcher to create the most accurate gene models currently possible.


227. An update on the Neurospora crassa community genome annotation project. Heather M. Hood1, Matthew R. Henn2, Dave DeCaprio2, James E. Galagan2, Alan Radford3, Bruce W. Birren2, Jay C. Dunlap4, & Matthew S. Sachs1. 1Oregon Health & Science University, Beaverton, OR 97006; 2Broad Institute of MIT & Harvard, Cambridge, MA 02141; 3University of Leeds, Leeds, UK; 4Dartmouth Medical School, Hanover, NH 03755.


To produce a more accurate Neurospora protein-coding gene catalog, we need to include the wealth of information about gene structure and function that is contained within the scientific community. A web-based community annotation resource was developed and released in March 2006 to help accomplish this (http://www.broad.mit.edu/annotation/genome/neurospora/CAHome.html). Community annotators can use this site to add a variety of information to genome features, including both genetic and functional data. They can also correct gene models and add alternative transcripts. Immediately following submission, community annotations are appended to the gene detail page and are publicly available. Each submitted annotation is inspected for accuracy by curators; once curated, the annotation is scheduled to be incorporated into the official gene detail page as part of an updated genome release. A new release of Neurospora gene predictions (Version 3) included community input. As of December 2006, the community annotation project has 57 registered participants that have contributed over 500 annotations, including 130 citations. Over 1100 E-compendium entries were also incorporated as community annotations, further enriching the data set. Finally, a Textpresso database was implemented for Neurospora literature. This database contains over 2500 searchable full-text articles related to Neurospora and fungal biology. Taken together these new community resources extend the utility of the genome sequence data.


228. Almost finished: the complete genome sequence of Mycosphaerella graminicola. Gert H.J. Kema1, Igor Grigoriev2, Andrea Aerts2, Asaf Salamov2, Hank Tu2, Harris Shapiro2, Jim Bristow2, Jane Grimwood3, and Stephen B. Goodwin4. 1 Plant Research International B.V., P.O. Box 16, 6700 AA Wageningen, The Netherlands. 2 Department of Energy, Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA, 94598, USA. 3 Stanford Human Genome Center, 975 California Avenue, Palo Alto, CA 94304, USA. 4 USDA-ARS, Purdue University, 915 West State Street, West Lafayette, Indiana, 47907-2054, USA.


Mycosphaerella graminicola is a haploid ascomycete causing septoria tritici blotch of wheat. An 8.9x shotgun sequence of bread wheat strain IPO323 was generated through the Community Sequencing Program of the U.S. Department of Energy’s Joint Genome Institute (JGI), and was finished at the Stanford Human Genome Center. The finished genome of M. graminicola contains 39.6 Mb. All available ESTs (37,747) were placed on the finished assembly to assess completeness. In addition, 1793 Diversity Array Technology markers were sequenced at JGI and aligned with the physical map. The genetic and physical maps showed near-perfect colinearity, demonstrating the high quality of both the genetic linkage map and the genome assembly. Fifteen scaffolds are complete and represent entire chromosomes with sizes from 548 kb to 6 Mb. In addition, six scaffolds (ranging from 21 kb to 2.4 Mb) contain a single telomere and could connect in any combination. Two additional scaffolds do not contain telomeres and only 17 gaps remain to be closed. Our sequence data suggest that isolate IPO323 contains 18-20 chromosomes, which represents the highest number of chromosomes reported among ascomycetes. This conclusion is corroborated by high-density genetic linkage maps and detailed pulsed-field gel analyses. The complete mitochondrial sequence is a circular genome of 43,947 bp. The machine annotation predicted 13,413 genes, of which 1126 have been annotated manually (http://www.jgi.doe.gov/Mgraminicola). Gene annotations generally indicate that M. graminicola has fewer genes per family than other plant-pathogenic filamentous fungi, which may reflect its initially extracellular biotrophic and subsequently necrotrophic lifestyle. This genome represents the first nearly finished genome of any filamentous plant pathogen and is of great importance for comparative genomics involving other Dothideales, such as the destructive banana Black Sigatoka pathogen Mycosphaerella fijiensis that currently is sequenced at the ~7.1x level.


229. Improvements in Aspergillus fumigatus annotation. Paolo Amedeo, Natalie Fedorova, Rama Maiti, Vinita Joardar, Jonathan Crabtree, Samuel Angiuoli, William Nierman, Owen White, Jennifer Russo Wortman. The Institute for Genomic Research 9712 Medical Center Dr., Rockville, MD 20850


Aspergillus fumigatus was among the first fungal genomes to be fully sequenced and annotated. At the time, few characterized genes in closely-related organisms were available in the public databases, which had a negative effect on the quality of annotation. Since then annotation tools have improved and several other Aspergilli have been fully sequenced and annotated. We have revised the annotation of Aspergillus fumigatus leveraging comparative analysis techniques and the newly-available data from related organisms. Here we describe these processes and discuss improvements made to the annotation.


230. Withdrawn


231. Mitochondrial Genomics in the Genera Pythium and Phytophthora. Frank N. Martin1 and Paul Richardson2. 1USDA-ARS, Salinas, CA, 2 Joint Genomics Institute, Walnut Creek, CA.


Mitochondrial genomics can be useful for investigating the processes contributing to evolutionary divergence at a mtDNA sequence level and hence, provide insight to the phylogenetic relationships of the organisms under study. While the mitochondrial genomes of the related genera Pythium and Phytophthora encode a similar suite of genes, they differ from each other by the presence of a large inverted repeat (IR) that is found in Pythium (it can represent approximately 80% of the genome size). In an effort to gain a better understanding of the evolutionary forces responsible for sequence divergence in genomes with and without an IR, as well as to clarify the phylogenetic relationships within the individual genera, the mitochondrial genomes of 15 Pythium and 10 Phytophthora species were sequenced. Comparative genomics among species within a genus indicated that certain regions of the genome were more polymorphic than others. In Pythium, the most polymorphic region was the small unique region and adjacent IR sequences. In Phytophthora genomic inversions were observed with many of the rearrangements corresponding to phylogenetic groupings. Two closely related species (P. ramorum and P. hibernalis) also were found to have small IRs (1.1-1.5 kb in size). While the IR in Pythium appeared to stabilize the genome from rearrangements, the data suggests that the rate of evolutionary divergence was more dependent on the specific gene rather than its location within the IR.


232. Withdrawn


233. Transcriptome of conidium and ascospore development in Fusarium graminearum. Kye-Yong Seong1, Matias Pasquali1, Jin-Rong Xu2, and H. Corby Kistler1,3. 1University of Minnesota, St. Paul, MN, USA; 2Purdue University, West Lafayette, IN, USA; 3USDA ARS Cereal Disease Laboratory, St.Paul, MN, USA.


To understand the infection cycle of the head blight pathogen F. graminearum, gene expression profiles were monitored during both ageing and germination for conidia and ascospores. Ascospores and conidia were treated in a similar manner, either aged under desiccating conditions or suspended in liquid germination medium. RNA was extracted from cultures and used to query the 18K feature F. graminearum Affymetrix GeneChip. Overall, a slightly greater number of probe sets corresponding to genes were detected in ascospores (9,207) than in conidia (8,815; detection p value <0.001) but the majority of probe sets (8,068) were shared between conidia and ascospores. While a similar number of genes were detected at most stages of development, the biggest difference among spore types was upon desiccation where the number of probe sets detected in ascospores (6,801) was more than twice the number detected in conidia (2,916). These results indicate that ascospores remain more metabolically active than conidia upon ageing. Peroxisomal proteins and genes involved in lipid beta-oxidation are strongly up-regulated both in fresh conidia and in ascospores. After suspending conidia or ascospores in liquid germination medium, numerous genes involved in transcription, RNA splicing, protein synthesis, and amino acid and nucleotide metabolism were highly induced. Up-regulation of proteasome components and secretory proteins were observed as spores established polarized growth after 8h of incubation. Comparing gene expression in spores with expression in hyphae under a variety of environment regimes indicates that a total of 328 probe sets were specific for ascospores and another 150 were specific for conidia. Spore-specific gene expression may be used to develop hypotheses concerning spore maturation, dormancy and initiation of germination. This work is supported by the National Research Initiative of the USDA CSREES, award 2004-35604-14327.


234. Restriction-site Associated DNA (RAD) mapping can be used to rapidly map mutations using Neurospora microarrays. Zachary A. Lewis, Anthony Shiver, Michael Miller, Eric A. Johnson, and Eric U. Selker. University of Oregon, Eugene, Oregon


Many important insights into fundamental biological processes have come from the study of filamentous fungi. The wealth of sequence information currently available for this group has facilitated evolutionary analysis, genome level expression analysis, and reverse genetic studies. However, the ability to carry out forward genetic screens and selections continues to drive discovery in the fungi. Whereas isolation of mutants is relatively easy, mapping and identification of the mutant genes is often the "rate limiting" step in genetic studies. We have performed a selection for genes that are defective in DNA methylation (dim). In order to identify the mutant genes, we have adapted microarray-based RAD mapping for use with Neurospora oligo arrays. We demonstrate that this technique can be used to detect genome wide restriction site polymorphisms from two polymorphic Neurospora crassa strains, Mauriceville and Oak Ridge. Moreover, we demonstrate that this technique can be used to rapidly map a mutation. RAD mapping can be used to identify restriction site polymorphisms in any two polymorphic strains without a priori knowledge of the polymorphisms. Therefore, in addition to forward genetics, we propose that RAD mapping will useful for analysis of phenotypic variation in natural populations (e.g. QTL analysis).




Industrial Biology and Biotechnology


235. Targeted Introduction of Point Mutations in Epichloë festucae. Kurtis Knapp, Richard Johnson, Christine Voisey, and Gregory Bryan. AgResearch Ltd.


AgResearch has a number of proprietary endophytes that increase plant yield and provide resistance to abiotic stress. However, these strains can not currently be commercialized because they produce mammalian toxic alkaloids. We will eliminate production of these alkaloids using a point mutagenesis technique called Rapid Trait Development System (RTDS). In RTDS conversions, one transforms an oligonucleotide that hybridizes with the gene of interest but contains a single base mismatch. The cell then alters the mismatched base in the genome. This technique is attractive to agricultural biotechnology because it avoids the untoward changes of random mutagenesis and the regulatory issues of transgenics. We will present progress toward implementation of RTDS to fungal endophytes of grasses. Initial work will be on mutations with a selectable phenotype in the Epichloë festucae strain FL1.


236. New screening approaches for fungal strain development. Peter J. Punt1, Xavier O. Weenink2, Marc van der Maarel1, Jan Jore1, Arthur Ram2, Cees van den Hondel2. 1TNO Quality of Life, Zeist, the Netherlands,2Leiden University, Leiden, the Netherlands


Since the development of recombinant DNA technologies for yeast and filamentous fungi, a considerable part of the strain development programs was diverted to the use of molecular genetic tools. Whereas these approaches have exerted considerable success, recent developments in our laboratory have shown that new developments in classical biological screening approaches, or a combination of both, can still be very useful. A first purely classical approach is based on the discovery of a so-called suicide (SUI) substrate, which we have successfully used for the selection of protease deficient fungal host strains. These protease deficient strains show an increased resistance to the SUI substrate allowing their selection. The advantage of this non-GMO approach is that it can be applied to new and already established production strains. A combination of a molecular and classical approach is based on the use of the so-called glucoamylase carrier approach. Combining this approach with fungal strains unable to use starch as a carbon source allowed us to select for hyper secretive fungal strains generated by classical mutagenesis (Weenink et al., 2006). Moreover, the same approach also allows for selection of the highest producers in a collection of primary transformant strains expressing a glucoamylase-fusion gene.


237. Oxidative protein folding in Aspergillus niger. Anna Harvey1, Andrew Plumridge1, Mick Ward2, Huaming Wang2 & David B Archer1. 1School of Biology, University of Nottingham, University Park, Nottingham, NG7 2RD 2Genencor Intl., 925 Page Mill Road. Palo Alto, CA 94304, USA plxarh@nottingham.ac.uk


The formation of protein disulphide bonds is one of several processes that occur within the endoplasmic reticulum (ER) of eukaryotes, which can be vital for the correct folding of secreted proteins. The pathway for the formation of protein disulphide bonds has been characterised in the yeast model organism, Saccharomyces cerevisiae. In S. cerevisiae, two proteins, Ero1p and Pdi1p are essential for the correct formation of protein disulphide bonds, and their expression is regulated by ER stress through the unfolded protein response. In the filamentous fungus, Aspergillus niger, the presence of a Pdi1p homologue, PDIA, has already been demonstrated. We have identified a homologue of the Ero1p protein and we show that its expression is transcriptionally regulated by ER stress. The functions of eroA and the product EROA are being further characterised by investigating the effects of deletion and over- expression.


238. Chrysonilia sitophila: a Case Study for Ascomycete Biodegradation and Bioremediation Potential. Carvalho, MB1,a, Martins, I1,a, Garcia, H2, Spencer-Phillips, P3, Gil, A2, San Romão, MV 1,4 and Silva Pereira, C 1,* 1ITQB-UNL/IBET, Instituto de Tecnologia Química e Biológica - Universidade Nova de Lisboa / Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal. 2Universidade de Aveiro, Departamento de Química,Aveiro, Portugal 3University of the West of England, Bristol, UK 4EVN, Estação Vitivinícola Nacional, Dois Portos, Portugal. * Corresponding author, spereira@itqb.unl.pt a Equal contributing authors


Persistent organic pollutants, such as pentachlorophenol (PCP), are globally recognized as a major environmental concern. PCP constitutes an acute risk to health considered, by the WHO (2004), as one of the most hazardous substances. PCP use is nowadays wide restricted but is still frequently detected in all environments. Whole fungal cell biocatalysis represents an elegant way for elimination of extreme toxic products. However, fungi exploitation for biodegradation is often sub-optimal and rarely reliable. One of the underlying reasons for the fragility of many approaches is the lack of scientific knowledge concerning the diversity, activity and dynamics of the microbial communities responsible for the biodegradation processes. There is a close relation between fungi biodegradation and bioremediation abilities because the aromatic matrix of lignin resembles the basic molecular skeleton of PCPs. During cork stopper manufacturing process, cork slabs are initially colonised by Chrysonilia sitophila and latter by other fungal species, such as Penicillium glabrum and Trichoderma longibrachiatum. These fungal species are able to perforate the complete thickness of the cork cell wall (lignin 20%, suberin 40%), suggesting their active biodegradation ability and were able to degrade a less, relative to PCP, chlorinated chlorophenol. Fungi biodegradation ability was screened following cork decay by spectroscopy (FITR). C. sitophila degrades lignin and suberin, tolerates concentrations of PCP above 20 mg/L and can apparently grow solely in the presence of PCP. Growth media supplementation with lignin/suberin enriched composite apparently reduces PCP toxicity. PCP degradation pathway by C. sitophila is being analysed following PCP degradation intermediates formed and the fungi proteome during growth on different substrates (presence and absence of PCP).


239. Bottlenecks in Bacterial Transglutaminase Production in Trichoderma reesei. Marja Paloheimo1, Susanna Mäkinen1, Kim Langfelder2 and Jari Vehmaanperä1. 1Roal Oy, Rajamäki, Finland. 2AB Enzymes GmbH, Darmstadt, Germany.


Transglutaminases catalyse cross-linking in proteins via an acyl transfer reaction between peptide-bound glutamine and lysine. TGases are widespread in mammals and they have also been found in microorganisms, plants, invertebrates, fish and birds. The cross-linking activity of TGases is exploited in several food applications, e.g. in improving the texture of processed meat and fish products. There is a growing interest to use TGases in technical applications, such as use in the textile industry. TGases could be used in modification of silk and wool fibres. For technical applications a cheap source of TGase must be available. For this reason a preliminary study was performed to determine whether Trichoderma reesei is a suitable host for production of a bacterial transglutaminase. The S. mobaraensis tg gene was used as a test gene. The TGase is activated after secretion by two-step cleavage of the pro-peptide from the precursor. Constructs encoding the full-length and mature and the corresponding inactive TGase forms were expressed in T. reesei. In addition the pro-sequence was expressed separately in strains already expressing the mature form of TGase. The TGase encoding sequences were expressed in T. reesei as 3´-fusions to a sequence encoding a carrier polypeptide. The results obtained will be shown and discussed.


240. Targeting cellular stress response systems with natural compounds to enhance antifungal activity. Jong Kim1, John Beck1, Shen-Chieh Chou2, Noreen Mahoney1, Kathleen Chan1, Russell Molyneux1 and Bruce Campbell1*. 1 Plant Mycotoxin Research, USDA, Albany, CA 94710 USA. bcc@pw.usda.gov. 2 School of Pharmacy, University of Colorado Health Science Center, Denver, CO 80262.


A series of natural phenolic compounds (i.e., benzoic/cinnamic acid derivatives, etc.) were applied to various gene deletion mutants of Saccharomyces cerevisiae in order to identify gene targets for fungal control. Four groups of molecular targets were identified, as follows: transcription regulators of pH response or glutathione transferase; vacuolar H(+)- ATPase; mitogen-activated protein kinase kinase (MAPKK); and antioxidative enzymes. Treatments with 2,3- dihydroxybenzoic acid disrupted fungal glutathione homeostasis. Several compounds targeted mitochondrial superoxide dismutase (Mn-SOD). Functional complementation analysis using Aspergillus flavus sodA, encoding Mn-SOD verified increasing oxidative stress improved antifungal activity of commercial fungicides. Activity of strobilurin was greatly elevated on A. fumigatus and A. nidulans by co-application with berberine or veratraldehyde. These compounds also prevented A. fumigatus MAPK mutants from escaping toxicity of fludioxonil. Esterification of dihydroferulic acid (DFA) with methyl, ethyl, or propyl side chains greatly enhanced antifungal acitivity. We conclude that targeting fungal antioxidative stress systems with phenolic agents improves fungal control.


241. Withdrawn


242. Ceramidase CerA of Aspergillu oryzae promotes degradation of biodegradable plastic containing urethane bonds. Shinsaku Ohtaki1, Hiroshi Maeda2, Toru Takahashi1, Youhei Yamagata1, Katsuya Gomi1, Fumihiko Hasegawa2, and Keietsu Abe1, 2*. 1Grad. Sch. of Agricul. Science, 2New Industry Creation Hatchery Center, Tohoku University, Japan.


When the industrial fungus Aspergillus oryzae is grown in a medium containing a biodegradable plastic polybutylene succinate-co-adipate (PBSA) as a sole carbon source, the fungal cells secret several proteins including cutinase CutL1 as a PBSA degrading enzyme and several biosurfactant proteins that bind to the surface of PBSA and recruit CutL1 to promote PBSA degradation. From the culture broth, we found and purified another novel protein having a molecular mass of 100 kDa. Because the purified protein is predicted to be a neutral ceramidase by using A. oryzae genome information, the gene encoding the 100 kDa protein is designated cerA. We cloned the cerA gene from A. oryzae and constructed a recombinant A. oryzae strain highly expressing cerA. Purified recombinant CerA hydrolyzed C12-NBD-ceramide. Although CerA scarcely cleaved ester bonds between succinate and 1,4-butanediol in PBSA, CerA promoted degradation of PBSA in combination with CutL1. Since PBSA contains urethane bonds in every 200-300 butylene-succinate ester units and the urethane bonds are similar to the amide bonds in ceramides, CerA seems to degrade PBSA endogenously at the urethane bonds and to be applicable degradation of ester polymers containing urethane bonds.


243. Characterization of novel thermostable fungal cellobiohydrolases. Terhi Puranen1, Sanni Voutilainen2, Matti Siika-aho2, Jarno Kallio1, Satu Hooman2, Liisa Viikari2, Anu Koivula2, and Jari Vehmaanperä1. 1Roal Oy, Rajamäki, Finland, 2VTT Technical Research Centre of Finland, Espoo, Finland


Cellulose degradation requires sequential or simultaneous synergic action of three types of hydrolytic enzymes: cellobiohydrolases, endoglucanases and beta-glucosidases. Various filamentous fungi produce these enzymes in order to hydrolyze insoluble cellulose into glucose. Cellulases are currently extensively studied in enzyme industry for cellulosic biomass conversion to ethanol. Here, molecular cloning of three different cellobiohydrolase genes from thermophilic ascomycetes is presented together with their heterologous expression in Trichoderma reesei. The recombinant cellobiohydrolases that belong to the glycosyl hydrolase (GH) family 7 were purified and characterized in terms of pH optimum, thermal stability and kinetic parameters. Thermostable cellulases such as described here have been proposed to improve the overall process economy of the biomass conversion with favourable impact on enzyme need, hydrolysis performance and flexibility of the process.


244. Identification and characterisation of the Trichoderma reesei homologue of Aspergillus nidulans creB. Jai Denton, and Joan Kelly. University of Adelaide, Adelaide, Australia.

Trichoderma reesei is an industrially significant organism, particularly in the cellulase production industry. The industrial hyper cellulase producing strain RUT-C30 was found to contain a mutation within the coding region of the T. reesei homologue of the carbon catabolite repressor, CreA. The carbon catabolite repression gene (CCR) creB has been previously implicated in the regulation of genes encoding enzymes involved in cellulose utilisation. In Aspergillus nidulans, disruption of the creB gene results in deregulation of cellulase encoding genes normally subject to strong CCR, without the severe reduction in mycelial growth that is present in creA disruptions. The T. reesei homologue of creB is an ideal candidate for disruption for the development of a commercially exploitable strain, since no extreme effects on morphology are predicted. A homologue, labelled cre2, was identified within the T. reesei genome. In order to test for complementation of an A. nidulans creB mutant strain, the T. reesei cre2 gene was amplified and used to transform an A. nidulans strain containing a creB null mutation. The T. reesei cre2 gene complemented the effect of the creB mutation on utilisation of quinate and proline, and sensitivity to allyl alcohol in the presence of glucose. A disruption of cre2 was generated through the introduction of a selectable marker within the coding region. The disrupted strain was found to deregulate genes normally subject to CCR.


245. Withdrawn


246. Optimization of cellulase expression in Trichoderma reesei for the production of cellulose ethanol. Rebecca D. Taylor1 (taylorr@agr.gc.ca), Anne Johnston1, Daneille Schneiderman1, Jiro Hattori1, Nick Tinker1, Linda Harris1. 1 Agriculture and Agri-food Canada, Eastern Cereal and Oilseeds Research Center, Ottawa, Ontario Canada K1A 0C6. Alex Sobko2, Wei Ling Tan2, Jason Edwards2, Chris Hindle2, Theresa White2. 2 IOGEN Corporation, 310 Hunt Club Road East, Ottawa, Ontario, Canada K1V 1C1.


The fungus Trichoderma reesei is used by IOGEN Corporation to produce cellulase enzymes for the production of cellulose ethanol from farm silage (wheat straw, corn stalks) and other biomass. The goal of this project was to analyze gene expression changes in T. reesei upon overexpression of cellulase proteins. To this end, we acquired 9298 high quality expressed sequence tags (ESTs) from 10 cDNA libraries constructed from fungal cultures grown under varied conditions. We also directly PCR amplified additional genes of interest not present in our EST population. In total, we have 2979 unique sequences including 899 contig and 2080 singleton sequences representing approximately one third of predicted T. reesei genes. cDNA microarrays were constructed using these unique sequences. Biological samples for array hybridizations were harvested from 14L T. reesei batch fermentation cultures in the presence of soluble cellulase inducer. The results from these experiments will be discussed.

 

247. Engineering of a novel protein secretion pathway in Aspergillus niger. Robbert Damveld1, Miranda Hartog1, Peter ten Haaft1, Inge Minneboo1, Cees Sagt1, Han de Winde1, Thibaut Wenzel1. 1DSM Food Specialties, P.O. Box 1, 2600 MA Delft, The Netherlands.


Production of enzymes on an industrial scale is often limited to extracellularly enzymes. When enzymes are produced intracellular, the downstream processing is costly and process robustness is not guaranteed. We have developed a technology which enables the secretion of enzymes which are normally localized in the cell. The folding conditions of intracellular proteins differ from those of secreted proteins. In the cytosol proteins fold under relative reducing conditions compared to the oxidizing environment in the ER. Moreover, many secreted proteins undergo extensive N- glycosylation and disulphide bridge formation. The set of folding enzymes and chaperones in the secretory pathway and in the cytosol are different as well. Therefore the simple solution of forcing intracellular proteins through the secretory pathway does in many cases fail to result in biologically active secreted enzymes. We have developed a method to secrete intracellular enzymes in an active form. This technology allows the intracellular enzymes to be folded in their native environment in the cytosol using cytosolic folding enzymes and chaperones. After folding the intracellular proteins are translocated to a modified cell compartment. After translocation in this modified cell compartment the content is released into the medium by a specific process. Using controlled fermentations we have established that the physiology of cells containing this novel secretory system is not dramatically different form non- modified cells. We will show the recently obtained proof of principle of this concept with the Green Fluorescent Protein (GFP). In addition we have demonstrated the concept for intracellular enzymes, which are secreted in an active form using this novel approach. Moreover we also have preliminary evidence that this technology can be used to secrete metabolites, which are normally localized intracellular. This is the first report that describes the introduction of a completely novel secretory pathway in eukaryotic cells, which allows the production of intracellular enzymes in a secreted active form enabling industrial application on an economically feasible scale.


248. A transiently disrupted non-homologous end joining pathway in Aspergillus nidulans allows simple and efficient gene targeting. Jakob Blæsbjerg Nielsen, Michael Lynge Nielsen, and Uffe Hasbro Mortensen. Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, Denmark jbn@biocentrum.dtu.dk


Gene targeting was developed more than a decade ago for high-throughput gene deletions in the unicellular Saccharomyces cerevisiae owing to the fact that DNA double strand breaks are effectively repaired by the homologous recombination (HR) pathway. In multicellular eukaryotes, from filamentous fungi to man, DNA double strand breaks are preferentially repaired by the non-homologous end joining (NHEJ) repair pathway. When performing gene targeting in such multicellular organisms, the influence of NHEJ compromises the efficiency by causing random integration of the gene targeting DNA. Elimination of NHEJ by deleting one of the main genes in the pathway, e.g. ku70, ku80 or lig4 has been done in several fungal species including Aspergillus fumigatus, A. nidulans, Sordaria macrospora and Neurospora crassa. In such strains, the gene targeting efficiency is often as high as 100%. The phenotype of NHEJ strains includes altered colony morphology and an increase in sensitivity to various genotoxins. This raises the concern that phenotype of NHEJ deficient strains may produce synthetic effects with other mutations introduced in this background. This may hamper direct interpretation of the phenotype of this new mutation. In line with this, authors often recommend as a safety measure to reconstitute the NHEJ pathway, e.g. by retransforming with the functional gene or sexual crossing with wild-type strains, before analyzing the effects of novel mutations. In both cases, strain reconstruction constitutes a bottle neck in large scale gene-targeting experiments. To bypass these problems, we have developed an effective alternative in A. nidulans. The system employs a strain with a transiently disrupted ku70 homolog as the starting point for efficient gene targeting. The ku70 mutation in this strain can via a simple selection scheme be reverted to wild type after the desired genetic manipulation(s) have been carried out. The system can easily be adapted to other filamentous fungi.


249. Applied genome-scale modelling of Aspergillus niger. Mikael Rørdam Andersen, Michael Lynge Nielsen, Jens Nielsen. Center for Microbial Biotechnology, Build. 223, Technical University of Denmark, DK-2800 Lyngby, Denmark, jn@biocentrum.dtu.dk


The filamentous fungus Aspergillus niger has through the years fascinated academic and industrial researchers alike due to its innate ability to produce a large number of enzymes and high concentrations of acids. However, the regulatory mechanisms governing these processes are not thoroughly understood. We are combining genome-scale stoichiometric modeling, transcriptomics and novel tools for graphical analysis to elucidate these mechanisms. A genome-scale model was constructed based on literature of A. niger and related aspergilli. Completion of pathways was performed using pathway databases. Genomic information was added based on the sequencing and annotation provided by DSM Food Specialities (Pel et al, manuscript accepted). The model comprises 1230 biochemically unique reactions (isoenzymes not included). 850 are supported by literature and 873 are backed by the genomic evidence of 1024 open reading frames. The remaining reactions are transport reactions and other reactions added for connectivity. Using a digital pathway map of the model, the computed metabolic fluxes were analysed for a large number of substrates. The results are in accordance with the available literature. Using a fast PCR-based cloning-free approach (Nielsen et al, 2006) adapted for A. niger, deletion mutants of carbon metabolism regulators of A. niger have been constructed. These are to be analysed using steady-state cultures and DNA arrays for A. niger.


250. Withdrawn


251. Coprinopsis cinerea as a host for recombinant laccase production. Sreedhar Kilaru1,2, Martin Rühl1, Patrik J. Hoegger1, Aletta Grimrath1, Karin Lange1, Mojtaba Zomorrodi1, Andzrej Majcherczyk1, and Ursula Kües1. 1Institute of Forest Botany, Georg-August-University, Göttingen, Germany 2 School of Biological Sciences, University of Bristol, Bristol, UK


Laccases (EC 1.10.3.2) from basidiomycetes are used for various industrial applications, e.g. in paper pulp bleaching, bioremediation, textile dye decolourization, and wood composite production. For overproduction of laccases, we developed a recombinant expression system using the basidiomycete Coprinopsis cinerea as a host. Various homologous and heterologous laccase genes have by now been expressed in C. cinerea under control of the Agaricus bisporus gpdII promoter. Yields of enzyme activities differ from gene to gene. Alteration of growth conditions (media, temperature, aeration) improved yields with 20fold increases in the best cases. At our current best cultivation conditions and our best gene, laccase activities of up to 30 U/ml are obtained. Individual enzymes are characterized in their properties and found to differ from each other, making them attractive for different biotechnological applications. Our laboratory is financially supported by the Deutsche Bundesstiftung Umwelt (DBU).


252. Multivalent Influenza antigens produced in N. crassa, using a novel application of heterokaryon fusion technology. Edward B. Cambareri, Yuliya Brudnaya and W. Dorsey Stuart Neugenesis Corporation, 849 Mitten Rd, Burlingame, CA 94010


Neugenesis Corporation has adapted patented technologies, developed to produce multimeric subunit proteins such as human IgGs, for use in the production of multivalent vaccines. Individual glycoprotein antigens or antigen variants can be expressed in different strains of the fungus, and then fused to create a heterokaryon strain that can produce either a mixture of secreted soluble antigens or high molecular mass particles that display the antigens. New production strains can be generated in as little as six weeks, and different antigens can be mixed and matched using the inherent combinatorial properties of fungal strain fusion. Commercial levels of tailored antigen mixtures can be rapidly isolated from simple, defined growth medium. Data will be presented demonstrating production from heterokaryons of a pandemic influenza vaccine candidate; a virus-like particle (VLP) displaying active H5 Hemagglutinin and N1 Neuraminidase glycoproteins.


253. Isolation of isoosmotic up-regulated genes in Aspergillus oryzae and use of its promoters for protein expression system. Ken Oda1, Kazutoshi Sakamoto2, Toshihide Arima3, Yuka Okita2, Dararat Kakizono2, Osamu Yamada2, Shinichi Ohashi1, Osamu Akita5, Kazuhiro Iwashita2,4. Kanazawa Institute of Technology1,3-1 Yatsukaho, Hakusan, Ishikawa, Japan tel:D+81(76)274-7500, fax+81(76)274-7511, e-mail:Fodaken@neptune.kanazawa-it.ac.jp National Research Institute of Brewing2, Prefecture Univ. of Hiroshima3, Hiroshima Univ.4, Jissen Women's Univ.5, Japan


The mechanism of response to osmotic pressure exists in eukaryotes. In A. orzyae, response to osmotic pressure is significant factor for environment recognition in koji-making (solid-state culture), and in fact it was suggested that osmotic pressure regulation component, such as AtfA, or HogA, is important. We analyzed the genes which respond to isoosmotic pressure (0.8M NaCl and 1.2M sorbitol) by microarray analysis. In isoosmotic condition 96 genes in 3000 spots array were 2-fold up- regulated from 30 to 90 min after induction. Some genes that are needed to respond to osmotic stress, such as atfA, , gpd, , and srk1, were isolated. 19 genes were over 5-fold up-regulated, and named as Isoosmotic Up-regulated Genes (IUG). 5'-upstream region of 10 genes in IUG were cloned and applied to GUS reporter assay to evaluate induction ability in isoosmotic condition. The promoters of two genes (IUG2 and IUG9) were strictly regulated. To construct protein expression system the promoter region of high expression vector pNGA142 was substituted with IUG2 and IUG9 promoters. EGFP as a model of heterologous protein was expressed after 2hr only in induction conditions, suggesting that response speed of this system is fast and control of expression is strict. We constructed new protein expression system which protein expression was induced by osmotic pressure and their level of expression are controlled by NaCl concentration.


254. Chemostat cultivation of Aspergillus niger for C-source mediated induction of the unfolded protein response and transcriptome analysis. Theo Goosen1,2, Thomas Jørgensen1,3, Jens Iversen1,3, Arthur Ram1 and Kees van den Hondel1. 1Leiden University, Institute of Biology Leiden, Fungal Genetics Research Group, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands, 2BioCentre, HAN University, Laan van Scheut 2, 6525 EM Nijmegen, The Netherlands, 3Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark.


Folding of nascent proteins in the endoplasmic reticulum (ER) has been identified as a major bottleneck in production of heterologous proteins. Accumulation of unfolded proteins in the ER elicits a stress response called the unfolded protein response (UPR). We have used C-limited chemostat cultures of Aspergillus niger to investigate UPR resulting from expression of a gene encoding a mammalian antibody fragment (scFv). Chemostat cultivation provides well-defined growth conditions and reduces biological sample variation among replicates, which make it an excellent method for both physiological and molecular studies, like transcriptomics. Two strains were cultivated for comparison. Strain ABGT1026 expresses lama scFv under transcriptional control of the glaA promoter from a single targeted gene. Strain AB94-85 is an isogenic control strain lacking the scFv gene-fusion. Both strains were cultured in chemostat cultures, at a dilution rate of 0.16 h-1, initially with xylose-limitation. After five retention times the medium was changed to maltose-limitation (inducing conditions for the glaA promoter). Cultures were performed in triplicate. There was no apparent physiological difference between the two strains. Culture profiles and yield coefficients of all six cultures were almost identical. Mycelium obtained from fifth retention time on xylose and maltose was subjected to expression analysis using Affymetrix arrays with probe sets based on the A. niger genome. Among 14554 probe sets, glaA had highest expression-level on maltose. It was also expressed on xylose; but at a level four times lower than on maltose. ANOVA of triplicate expression results of four conditions (two strains and two C-sources) revealed that transcription of around 290 genes were >2-fold up- or down-regulated (p 4-fold). A single gene was identified as differentially expressed, when comparing the two strains. Interaction analysis showed this was independent of C-source. Expression of scFv did not result in increased UPR at steady state on maltose, however expression of some genes associated with UPR and protein secretion was enhanced (1.6-2-fold) on maltose compared to xylose. The investigation has yielded one new lead regarding the low yield production of heterologous proteins, and a number of clues on the differences in expression of genes involved metabolism of xylose and maltose.



Biochemistry and Secondary Metabolism



255. Chemogenomics of antioxidant inhibition of aflatoxin biosynthesis. Jong Kim1, Jiujiang Yu2,3, Noreen Mahoney1, Kathleen Chan1, Deepak Bhatnagar2, Thomas Cleveland2, Russell Molyneux1, William Nierman3,4 and Bruce Campbell1*. 1Plant Mycotoxin Research, USDA, Albany, CA 94710 USA. bcc@pw.usda.gov. 2Food and Feed Safety Research, USDA, New Orleans, LA 70124 USA. 3The Institute of Genomic Research, Rockville, MD 20850 USA. 4The George Washington University, Washington, DC 20006 USA.


Natural compounds were used to probe the functional genomics (chemogenomics) of aflatoxin biosynthesis. Caffeic acid (12mM) treatment of Aspergillus flavus inhibited aflatoxin biosynthesis without affecting growth. Microarray analysis showed genes in the aflatoxin biosynthetic cluster were completely down-regulated (log2 ratio –0.04 to –3.13). However, aflatoxin pathway regulator genes, aflJ or laeA, and sugar utilization cluster genes showed only minor changes in expression (log2 ratio 0.08 to –0.58). Genes in amino acid biosynthetic and aromatic compound metabolism were up-regulated (log2 ratio > 1.5). Most notable was up-regulation (log2 ratio 1.08 to 2.65, qRT-PCR) of four peroxiredoxin genes orthologous to the AHP1 gene (alkyl hydroperoxide reductase) of Saccharomyces cerevisiae. Antioxidants trigger induction of ahp genes in A. flavus to protect the fungus from oxidizing agents, e.g., lipoperoxides, reactive oxygen species, etc. This detoxification attenuates upstream oxidative stress-response pathway signals, and suppresses aflatoxigenesis.


256. BDM-1, a phosducin-like protein required for G-protein signaling in Cryphonectria parasitica, is a phosphoprotein targeted by casein kinase II. Joanna Salamon-Kozubowska1 and Angus L. Dawe 1,2. 1Department of Biology and 2Molecular Biology Program, New Mexico State University, Las Cruces, NM 88003


G-protein signaling modulates many responses in fungi, including virulence of plant and animal pathogens. Previous studies with the plant pathogen C. parasitica have identified components that include a phosducin-like protein (BDM-1). Perturbation of the G-protein signaling pathways has been shown to affect virulence, pigmentation and sporulation. Deletion of the beta subunit CPGB-1 or BDM-1 affected these characteristics in an identical manner and caused similar alterations in transcriptional profile. G-proteins levels were reduced in accumulation following infection with virulence-attenuating hypoviruses. In this study we have shown by western blotting that CPGB-1 protein levels are undetectable in the absence of BDM-1 and by phosphatase treatment that BDM-1 exists in a phosphorylated form. Re- phosphylation of BDM-1 was accomplished in the presence of total protein lysates, but this activity was inhibited by a casein kinase II inhibitor, indicating that BDM-1 is a substrate for this kinase. With these tools it is now possible to explore the effects of phosphorylation on BDM-1 function and determine if this modification is altered by the presence of hypoviruses, thereby providing a model for exploring the molecular events associated with viral-mediated changes of cellular signaling pathways.


257. Functional characterization of two cytochrome P450 monooxygenases, PaxP and PaxQ, involved in the biosynthesis of the indole-diterpene, paxilline. Sanjay Saikia1, Emily Parker2,3 and Barry Scott1. 1Institute of Molecular Biosciences and 2Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand. 3Current address: Department of Chemistry, Canterbury University, Christchurch, New Zealand.


Indole-diterpenes are a large, structurally and functionally diverse group of secondary metabolites produced by filamentous fungi. Biosynthetic schemes have been proposed for these metabolites but until recently none of the proposed steps had been validated by biochemical or genetic studies. Using Penicillium paxilli as a model experimental system to study indole-diterpene biosynthesis we previously showed by deletion analysis that a cluster of seven genes is required for paxilline biosynthesis. Two of these pax genes, paxP and paxQ (cytochrome P450 monooxygenases), are required in the later steps in this pathway. Here we describe the function of paxP and paxQ gene products by feeding proposed paxilline intermediates to strains lacking the pax cluster but containing ectopically integrated copies of paxP or paxQ. Transformants containing paxP converted paspaline into 13-desoxypaxilline as the major product and beta-PC-M6 as the minor product. beta-PC-M6, but not alpha-PC-M6, was also a substrate for PaxP and was converted to 13-desoxypaxilline. paxQ-containing transformants converted 13-desoxypaxilline into paxilline. These results confirm that paspaline, beta-PC-M6 and 13- desoxypaxilline are paxilline intermediates and that paspaline and beta-PC-M6 are substrates for PaxP, and 13- desoxypaxilline a substrate for PaxQ. PaxP and PaxQ also utilized beta-paxitriol and alpha-PC-M6 as substrates converting them to paxilline and alpha-paxitriol, respectively.


258. LaeA, a methyltransferase regulating secondary metabolite production in Aspergillus spp. Perrin, Robyn M; Bok, Jin Woo; Keller, Nancy P. 1630 Linden Dr., University of Wisconsin, Madison, WI USA 53706


Filamentous fungi are unique organisms – rivaled only by Actinomycetes and plants - in producing a wide range of secondary metabolites. These compounds often have obscure or unknown functions in the producing organism but have tremendous importance to humankind, displaying a broad range of useful pharmaceutical activities as well as less desirable phyto- and mycotoxic activities. The biosynthetic genes of most fungal secondary metabolites are clustered in genetic loci. LaeA is a nuclear methyltransferase that transcriptionally regulates secondary metabolite gene clusters in the filamentous fungal genus Aspergillus. Deletion and over- expression of laeA either silences or increases, respectively, the production of multiple secondary metabolites and expression of genes for their biosynthesis. Mutation of a canonical amino acid motif found in all methyltransferases results in a loss of function phenotype, indicating that methytransferase activity is critical for LaeA regulatory activity (Bok et al 2006 Mol Microbiol 61:1636). In order to understand the mechanism of LaeA function, we are using an A. nidulans strain expressing epitope-tagged LaeA and anti-LaeA antibodies to identify LaeA methylation targets and interacting proteins. Further analysis of biochemical function is being conducted using a series of mutated and truncated versions of recombinant LaeA. Elucidation of LaeA biochemical function is critical to understanding its role as a global regulator of secondary metabolism.


259. Which “alphabet” do zygomycetes use for their chemical language? Mareike Richter, Doreen Schachtschabel, Ute Neugebauer and Wilhelm Boland. Max Planck Institute for Chemical Ecology, Jena, Germany, mrichter@ice.mpg.de


During sexual interaction, zygomycete fungi communicate via carotene-derived compounds, namely trisporic acid and its precursors. The first sexually determined structures of Mucor mucedo are specialised aerial hyphae, called zygophores. Proteins are distributed uniformly in these hyphae. Carotenoids, in contrast, accumulate in the hyphal tip, shown by 2D Raman spectroscopy. The same technique also depicts the possible presence of trisporoids in the zygophore tips. The formation of zygophores is an important parameter to assess the bioactivity of trisporoids. Various synthetic trisporoid analogues exhibit a differential influence on zygophore formation in Mucor mucedo. Moreover, trisporoid production and carotene biosynthesis are linked via feedback regulation. The early precursor beta- carotene can be readily quantified by HPLC. After culture stimulation the correlation of morphological responses with beta-carotene concentration is allowed. The mechanistic basis of the induced regulation mechanisms remains to be established. The impact of trisporoids on gene transcription necessary for carotene synthesis and degradation is studied by real-time PCR. The early carotene biosynthesis is not under the control of trisporoids. But later enzymatic transformations, e.g. by phytoene desaturase and carotene dioxygenase respond to stimulation with synthetic trisporoids. In the future we want to investigate the consequences of different stimulating trisporoid analogues.


260. Agrobacterium tumefaciens-mediated disruption of a polyketide synthase gene in Cryphonectria parasitica. Keiichi Sudo1, Heather L. McLane2, Stuart B. Krasnoff4, Sandra L. Anagnostakis3, Donna M. Gibson4, and Alice C.L. Churchill1(acc7@cornell.edu). 1Department of Plant Pathology, Cornell University, Ithaca, NY, 2Boyce Thompson Institute, Ithaca, NY, 3Connecticut Agricultural Experiment Station, New Haven, CT, 4USDA-ARS, Plant Protection Research Unit, Ithaca, NY.


Cryphonectria parasitica is the causal agent of chestnut blight, which decimated American chestnut populations during the first half of the 20th century. Infection of the fungus with naturally occurring Hypovirus can reduce virulence, sporulation, and the production of fungal pigments, as well as perturbing signal transduction pathways. We cloned fragments of eight C. parasitica genes predicted to be involved in the synthesis of small molecule natural products. Most are polyketide synthase (PKS) genes, which encode key enzymes for the synthesis of linear and aromatic polyketides. We identified and sequenced a ~6.0 kb gene, PKS1, which is a member of a predicted secondary metabolite gene cluster containing at least 13 other putative regulatory, modifying and efflux genes within a ~37 kb genomic region. The PKS1 predicted protein is highly similar to several other uncharacterized fungal PKSs (E value of 0.0; greater than 60% identity). We disrupted PKS1 in C. parasitica by Agrobacterium tumefaciens-mediated transformation and have begun characterization of the targeted gene knockout (KO) strains. There are no significant differences between the KO and ectopic control strains in colony growth rate on an agar medium or in Golden Delicious apple fruits, which are used as an alternate virulence assay. Virulence and mating assays on dormant chestnut stems are in progress, as are efforts to identify potential differences in development or nutritional requirements.


261. Genetics, biosynthesis and evolution of the polyketide T-toxin produced by the fungus Cochliobolus heterostrophus race T. Patrik Inderbitzin and B. Gillian Turgeon. Cornell University, Department of Plant Pathology, Ithaca, NY, USA. Emails: pri2@cornell.edu, bgt1@cornell.edu.

The polyketide virulence determinant, T-toxin, is produced by race T of the filamentous ascomycete Cochliobolus heterostrophus, the cause of Southern Corn Leaf Blight. The genetics, biosynthesis, and evolution of the ability to produce T-toxin differ significantly from those of other polyketides (PK). Unlike genes required for biosynthesis of the PKs, lovastatin and aflatoxin, for example, T-toxin genes are not clustered but are distributed at two loci encompassing ca. 1.2 Mb of AT rich, highly repetitive DNA present in race T but absent in non-T-toxin producing C. heterostrophus race O. Prior to this study, three genes were known to be required for T-toxin synthesis: two polyketide synthases (PKS) and the decarboxylase DEC1. We have identified six additional genes, all unique to race T. These include LAM1, OXI1 and HYD1, encoding proteins similar to a 3- hydroxyacyl-CoA dehydrogenase, an oxidoreductase, and a serine hydrolase, respectively, and three genes encoding putative reductases, RED1, RED2, and RED3. Regarding function, some of the PKS reducing domains are degenerate and we speculate that enzymes encoded by the new genes assume these roles. Regarding gene distribution, T-toxin genes are not present in race O, in other Cochliobolus species, or in neighboring genera. An ortholog of one of the PKSs genes, however, is found in the corn pathogen, Didymella zeae-maydis and in the distantly related dung fungus, Delitschia winteri. This discontinuous distribution of the T-toxin genes, together with their physical location within AT rich, repetitive DNA at chromosomal breakpoints, suggests a complex evolutionary history.


262. Identification of a compound that stimulates perithecial formation in Giberella zeae. Seunghoon Lee1, Jianming Jin1, Sung-Hwan Yun2, and Yin-Won Lee1. 1School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. 2Dpartment of Biological Resources and Technology, Soonchunhyang University, Asan 336-745, Korea


Giberella zeae, a homothallic ascomycete, develops a fruiting body called perithecium during sexual stage. Many studies suggested that fungal sexual development is induced by specific changes in both gene expressions and metabolism. G. zeae is well known as a producer of many secondary metabolites such as mycotoxins and pigments. Recently, several genes required for production of metabolites such as polyketides and non-ribosomal peptides have been reported in this fungus; some are specifically related to perithecial development. To investigate the enhancement of sexual development by specific fungal metabolites, we searched and identify such compound from carrot agar culture of G. zeae. Exogenous supply of the purified compound to fungal culture caused increase in both the number of perithecia and black pigment production accumulated on the agar surface where peritheica were formed. NMR and MS analyses estimated that it is a long unsaturated compound with a formula C38H66O5, Mw 604.57. Detailed characterization of the compound is in progress. Further studies will be performed to identify the genes responsible for the biosynthesis of this compound.


263. Identification of AatB, a new component of the penicillin biosynthesis pathway of Aspergillus nidulans. Petra Sproete 1, Michael J. Hynes 2, and Axel A. Brakhage 1. 1 Leibniz Institute for Natural Product Research and Infection Biology (HKI) / Friedrich-Schiller- University Jena, Germany 2 Department of Genetics, University of Melbourne, Australia


The acyl coenzyme A:isopenicillin N acyltransferase (IAT) of A. nidulans, which is encoded by the aatA gene, catalyzes the final step of the penicillin biosynthesis, i.e., the exchange of the hydrophilic L-alpha-aminoadipic acid side chain of isopenicillin N for a hydrophobic acyl group. By analyzing a GFP-IAT protein fusion it could be shown that in A. nidulans – as in Penicillium chrysogenum – the IAT and therefore the enzymatic reaction is located in the peroxisomes. Further studies indicated a PTS1 dependent transport of the enzyme since the deletion of the rather untypical putative peroxisomal targeting sequence 1 (PTS1) Ala-Asn-Ile at the C terminus of the protein led to cytoplasmic localization of the IAT. Nevertheless, unlike the IAT of P. chrysogenum, such a mislocated enzyme seems to be functional because both, an A. nidulans strain lacking the PTS1 transporter and a strain possessing a mislocated IAT still produced about 50% and 80% of penicillin, respectively, compared to wild-type levels. Because an aatA disruption strain still was able to produce small amounts of an inhibitory substance, the A. nidulans database was searched for a putative redundant protein. A gene displaying a very similar exon distribution and a 58% similarity with the aatA gene but lacking the PTS1 encoding sequence was named aatB. First analyses of an aatB disruption strain indicated a participitation of AatB in penicillin biosynthesis of A. nidulans.


264. Identification of all chitin synthase genes in Rhizopus oryzae genome. Ayumi Abe and Teruo Sone. Research Faculty of Agriculture, Hokkaido University, Sapporo, JAPAN


Chitin and chitosan are main components of cell wall of zygomycetes. Chitin synthases in zygomycetes are thought to be the most important factor for the cell morphogenesis, but they have not been studied well. We aimed to identify all chitin syanthase genes in R. oryzae genome sequence data and to collect the information about the expression of those genes under the various culture conditions. We identified 26 chitin synthase genes from R. oryzae genome sequence, using conserved amino acid sequences by BLASTP analysis.

Phylogentic analysis resulted in the clustering of chitin synthases in zygomycetes independent from those of ascomycetes or basidiomycetes on which current classification is based. This indicates that a new classification of chitin synthases including the data of zygomycetes'

enzyme is appropriate. Notably, two genes in R. oryzae made a distinct clade, thus a new class of the enzyme, class VIII was proposed. Almost all of R. oryzae chitin synthase genes were expressed during at least one culture condition. Seven genes were specifically expressed during later period in solid culture, indicating the relationship to sporangiospore formation. These results may reflect the fact that zygomycetes' cell wall consists mainly of chitin and chitosan, and thus they conserved many isozymes of chitin synthases with different roles, in the genome.


265. Analysis of the cyanase-like gene cyn1 from the filamentous fungus Sordaria macrospora. Skander Elleuche* and Stefanie Pöggeler. Department of Genetics of Eukaryotic Microorganisms - Institute of Microbiology and Genetics, Georg-August-University Göttingen, Germany

The enzyme cyanase has been found in several organisms such as plants, bacteria and archae. However, so far this enzyme that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide has not been characterized in fungi. In nature the substrate cyanate is formed from the spontaneous dissociation of urea in aqueous solution. Structural analysis of the Escherichia coli cyanase revealed that the enzyme is a homodecamer of 17 kDa subunits. Although cyanate at high concentrations can be fairly toxic to E. coli, cyanate can serve as the sole source of nitrogen for growth of E. coli as a result of cyanase-catalyzed decomposition to ammonia. In this study, we investigated the role of the cyanase-like gene cyn1 from the filamentous ascomycete Sordaria macrospora. BLAST searches of a S. macrospora genomic sequence revealed a high degree of homology to cyanase encoding genes from various bacteria. The S. macrospora cyn1 gene encodes a putative protein of 164 amino acids with a predicted molecular mass of 18.7 kD. Homologues of the S. macrospora cyn1 gene were identified within the genomes of filamentous ascomycetes and basidiomycetes but not in the genomes of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. To better understand the function of the S. macrospora cyanase, we generated a deltacyn1 gene disruption mutant. Analysis of the mutant strain, cellular localization of the cyanase and enzyme activity will be presented.


266. An efficient gene targeting system to generate knockout mutants in the penicillin producer Penicillium chrysogenum. Birgit Hoff and Ulrich Kück. Department of General and Molecular Botany, Ruhr-University Bochum, Universitaetsstr. 150, 44801 Bochum, Germany, birgit.hoff@rub.de


Penicillium chrysogenum is the main producer of the pharmaceutical relevant beta-lactam antibiotic penicillin. DNA-mediated transformation of this filamentous fungus results mostly in ectopic integrations of the molecules. For a functional analysis, it is however desirable to obtain gene substitutions by homologous recombination, a process which occurs with a rather low frequency in this fungus. A cellular feature that decreases homologous recombination is the non- homologous end-joining (NHEJ) pathway, a mechanism that involves the binding of the highly conserved Ku heterodimer at the ends of a DNA double-strand break. Integration events mediated by NHEJ do not rely on homologous sites and therefore attenuate homologous recombination, which results in decreased frequencies of a gene knockout in a given transformation experiment. To improve gene targeting efficiency in P. chrysogenum, we successfully deleted the P. chrysogenum Pcku70 gene. No impairment in vegetative growth, sporulation and penicillin production could be assessed for the corresponding mutant strain but relative frequencies of homologous recombination were drastically increased to about 50- 80 % as deduced from targeting several different genes. To test the applicability of the Pcku70 knockout strain, genes for global regulators were substituted by the phleomycin resistance gene. From the sum of our investigations, we can conclude that the Pcku70 knockout strain is an ideal recipient for targeting of genes involved in regulation of secondary metabolism and fungal morphogenesis, and can therefore be useful in strain improvement programs.


267. Regulation of genes coding for synthesis of beta-lactam antibiotics in Acremonium chrysogenum and Penicillium chrysogenum. Birgit Hoff, Jacqueline Dreyer, and Ulrich Kück. Department of General and Molecular Botany, Ruhr-University Bochum, Universitaetsstr. 150, 44801 Bochum, Germany, birgit.hoff@rub.de


The filamentous fungi Acremonium chrysogenum and Penicillium chrysogenum are the main producer of the pharmaceutical relevant beta-lactam antibiotics cephalosporin C and penicillin. Expression of genes coding for the biosynthesis enzymes is known to be controlled by a complex network of global regulators such as CRE1, PACC, and CPCR1 etc. Using gene disruption approaches, we have generated knockout strains of both fungi to study the homologue of the Aspergillus nidulans velvet gene (veA). VeA co-ordinates asexual and sexual development in this homothallic fungus and was also found to control secondary metabolism. Expression and HPLC analyses have clearly indicated that the veA homologue acts as a regulator of beta-lactam biosynthesis in both fungi. In addition, detailed microscopic analyses have shown that deletion of veA caused alterations in hyphal morphology and fragmentation. From the sum of our investigations, we can conclude that the veA homologue in the two fungi controls both antibiotic biosynthesis and fungal morphogenesis.


268. Use of the DsRed reporter gene to establish an RNAi system for the beta-lactam-antibiotic producer Acremonium chrysogenum. Danielle Janus, Birgit Hoff, and Ulrich Kück. Department of General and Molecular Botany, Ruhr-University Bochum, Universitaetsstr. 150, 44801 Bochum, Germany, danielle.janus@rub.de


In filamentous fungi, RNA-silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We have adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level expression of the DsRed gene were constructed, resulting in red-colored fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by northern hybridizations, real-time PCR, and spectrofluorometric measurements of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of this system was demonstrated by investigating co-silencing of DsRed together with pcbC, encoding the isopenicillin N synthase (IPNS), an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can easily be detected by a bioassay, measuring the antibiotic activity of individual strains. In addition, the presence of IPNS was investigated by western blot hybridization. All transformants having a colorless phenotype show a simultaneous downregulation of the pcbC gene. The RNA-silencing system presented here will be a powerful tool for strain improvement and genome-wide analysis of this biotechnically important filamentous fungus.


269. A novel gene cluster in Fusarium graminearum expressed under mycotoxin induction conditions. L. Harris1, A. Saparno1, N. Alexander2, B. Blackwell1, S. McCormick2, D. Schneiderman1, A. Desjardins2, N. Tinker1, J. Hattori1, K. Seifert1, T. Ouellet1. 1Eastern Cereal & Oilseed Research Centre, Agriculture & Agri-Food Canada, Ottawa, ON, Canada; 2Mycotoxin Research Unit, National Center for Agricultural Utilization Research, ARS, USDA, Peoria, IL. Email: harrislj@agr.gc.ca

We have identified a cluster of eight genes (gene loci fg08077 – fg08084) that is concomitantly up-regulated (Northern and qPCR analysis) under growth conditions that promote mycotoxin production. Proteomics experiments (iTRAQ analysis) have confirmed the up-regulation of proteins encoded by two of these genes under toxin induction conditions. Gene disruption and add-back experiments followed by metabolite analysis of the transformants indicated that one of the genes, fg08079, is directly involved in the synthesis of butenolide, a mycotoxin derived from glutamic acid. As expression of these genes can be detected very early during plant infection, butenolide may play a role in plant infection. However, greenhouse testing for FHB (Fusarium Head Blight) using disruption mutants of fg08079 showed that this gene did not contribute significantly to virulence in wheat heads. The mycotoxin butenolide is produced by several Fusarium species and has been suggested to be associated with tall fescue toxicoses in grazing cattle. We are exploring interspecies conservation of this gene cluster by PCR screening and sequencing in other Fusarium genomes.


270. Identification of a biosynthetic gene cluster for a secreted glycolipid with antifungal activity in Ustilago maydis. Beate Teichmann, Lidan Liu and Michael Bölker. Department of Biology, University of Marburg, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany. E-mail: teichma3@staff.uni-marburg.de


Under conditions of nitrogen starvation, U. maydis secretes large amounts of the cellobiose lipid ustilagic acid. This amphiphatic glycolipid acts as biosurfactant and displays antibacterial and antifungal activity. Ustilagic acid consists of a cellobiose moiety O-glycosidically linked to the terminal hydroxyl group of di- or trihydroxypalmitic acid, respectively. We have identified a large gene cluster comprising 12 open reading frames that is responsible for UA biosynthesis. By mutational analysis and mass spectrometry we could characterize several enzymes that are involved in hydroxylation, glycosylation and secretion of UA. This allows us to propose a biosynthetic pathway for ustilagic acid. The cluster contains a putative Zn-finger DNA-binding protein, which is involved in regulation of cluster genes. We could show that expression of this transcriptional factor is necessary for UA production. Further we could demonstrate, that U. maydis has biocontrol activities against the plant pathogenic fungus Botrytis cinerea, which causes gray mold on tomato leaves. Coinoculation of haploid U. maydis cells with B. cinerea spores suppress all symptoms of disease. UA production is critical for the antagonistic activity of U. maydis cells against B. cinerea spores, since mutants defective for UA biosynthesis are unable to interfere with B. cinerea infection.


271. Secondary metabolites: fundamental and niche-specific roles in fungal development and cell-cell interactions. B. Gillian Turgeon and Shinichi Oide. Cornell University, New York


An exhaustive characterization of non-ribosomal peptide synthetase (NPS) genes of the corn pathogen, Cochliobolus heterostrophus, and the small peptides produced by the enzymes they encode, has been undertaken to ascertain what peptide secondary metabolite products are doing for the fungal cell. To date, the NRPS method of peptide biosynthesis has been described for filamentous ascomycetes (and to a limited extent, for basidiomycetes) and bacteria, only. NRPs have a broad spectrum of biological activities, many are useful in medicine, agriculture, industry, and biological research. Despite the fact that activities of the peptide products with respect to interactions with other organisms are well-documented, to suggest that this is their primary function is likely incorrect. For example, only one of the 12 C. heterostrophus NPSs is required for virulence. In fact, the physiological significance of these small peptides to the producing fungi is largely unknown. We document that NRPS enzymes are purveyors of small molecules for both basal metabolism and for specialized environmental niches. Certain NPSs are required for sexual reproduction, asexual reproduction, virulence, as nutrient ‘gatherers’ (such as iron-gathering siderophores), for responses to oxidative, nitrosative, nutrient, pH stress etc, for growth, and for hydrophobicity. Eight of the 12 C. heterostrophus NPSs are not conserved, while four are conserved in other ascomycetes. We have examined functional conservation of conserved genes in the wheat pathogen, Fusarium graminearum, and the Arabidopsis pathogen, Alternaria brassicicola. Comprehension of secondary metabolite function, from the perspective of the fungal cell itself, impacts our understanding of evolution of fungal pathogenicity mechanisms by addressing how pathogens become pathogens.


272. CoA-Transferase from Aspergillus nidulans is required to forward the CoA-moiety from propionyl-CoA to acetate. Christian Fleck, Matthias Brock. Leibniz Institute for Natural Product Research and Infection Biology e.V. – Hans-Knöll-Institute (HKI) christian.fleck@hki-jena.de


Metabolism of amino acids and propionate leads to the toxic intermediate propionyl-CoA. Efficient removal via the methylcitrate cycle is guaranteed by a functional methylcitrate synthase. Deletion of the coding region of this enzyme leads to the inability to grow on propionate or ethanol/propionate but not acetate/propionate. This led to the assumption that the level of propionyl-CoA may become reduced by direct transfer of the CoA-moiety to acetate. Therefore, we purified a CoA-transferase from Aspergillus nidulans and characterised the enzyme biochemically. The CoA-transferase was specific for the CoA-esters succinyl-CoA, propionyl-CoA, and acetyl-CoA as CoA-donors and the corresponding acids as acceptors. No other donors or acceptors suited as substrates. To elucidate the role of the enzyme under in vivo conditions the corresponding gene was deleted. The deletion mutant only showed mild phenotypes when tested for growth and development on propionate containing media. Therefore, a double mutant with a methylcitrate synthase deletion strain was created by sexual crossing. In contrast to the methylcitrate synthase deletion strain, the double mutant was neither able to grow on ethanol/propionate nor on acetate/propionate media. Thus, CoA-transferase is essential for removal of toxic propionyl-CoA in the presence of acetate as a CoA-acceptor.


273. The aflatrem biosynthetic genes in Aspergillus flavus and A. oryzae are clustered in two separate regions of the genome. Matthew J. Nicholson1, Brendon J. Monahan1, Bethan Pritchard2, Gary A. Payne2, and Barry Scott1. 1Massey University, New Zealand. 2North Carolina State University, USA.


Aflatrem is a potent tremorgenic toxin produced by Aspergillus flavus and is a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Using Penicillium paxilli as a model experimental system for investigating indole-diterpene biosynthesis, we have shown that the products of four genes (paxG, paxM, paxB and paxC) are necessary for the production of paspaline, the first stable indole- diterpene intermediate, and that two P450 monooxygenases (encoded by paxP and paxQ) are required for the subsequent conversion of paspaline to paxilline. We previously identified a cluster of genes in A. flavus NRRL6541 proposed to be involved in aflatrem (atm) biosynthesis, that included homologues of paxG (atmG), paxM (atmM) and paxC (atmC) (Zhang et al., 2004). Here we report the identification of a homologous atm gene cluster in the sequenced genomes of A. flavus NRRL3357 and A. oryzae RIB40, and the identification of a second gene cluster, containing homologues of paxB, paxP and paxQ in these genomes. Comparative analysis of sequences from all three genomes revealed total syntenic conservation and high nucleotide identity both within and between genes. In both species the aflatrem biosynthetic genes are clustered in two discrete genomic regions. In A. oryzae, the first cluster (containing homologues of paxG, paxC, and paxM) is located subtelomerically on chromosome 7, whilst the second cluster (containing homologues of paxB, paxP and paxQ) is located on chromosome 5. Comparison with the paxilline gene cluster of P. paxilli provides important insights into the evolutionary history of these aflatrem gene clusters.


274. Epigenetic regulation of the Aspergillus chemical arsenal. Elliot Schwab and Nancy Keller. University of Wisconsin- Madison, Plant Pathology. Madison, WI.


Bioactive small molecules are critical in Aspergillus species during their development and interaction with other organisms. Genes dedicated to their production are encoded in clusters that can be located throughout the genome. We show that deletion of hdaA, an Aspergillus nidulans histone deacetylase (HDAC), causes transcriptional activation of two subtelomeric gene clusters - and subsequent increased production of the corresponding molecules - but not of a telomere distal cluster. Furthermore, treatment of other fungal genera with HDAC inhibitors results in overproduction of several metabolites. The regulatory role of gene clustering and subtelomeric positioning of clusters will also be examined. Chromatin regulation of small molecule gene clusters may enable filamentous fungi to successfully exploit environmental resources by modifying chemical diversity.


275. Characterization of a putative fusarin mycotoxin biosynthetic gene cluster in Fusarium. Robert H. Proctor, Robert A.E. Butchko and Daren W. Brown. USDA-ARS National Center for Agricultural Utilization Research, Peoria, Illinois.


Fusarium verticillioides is a stalk and ear rot pathogen of maize and can produce the polyketide mycotoxins fumonisins in infected kernels. Although the genetics and biochemistry of fumonisin biosynthesis are relatively well understood in F. verticillioides, little is known about the biosynthesis of other secondary metabolites produced by this fungus and whether the regulatory mechanisms that affect fumonisin biosynthesis also affect biosynthesis of other secondary metabolites. To begin to address such issues, we are using microarray analysis to determine whether 15 previously identified F. verticillioides polyketide synthase (PKS) genes (Kroken et al, 2003 PNAS 100:15670- 15675) are located in clusters of coordinately regulated genes. To date, the analysis indicates that three of the PKS genes are located in clusters. For example, PKS10, which is required for the biosynthesis of fusarin mycotoxins, and eight contiguous genes adjacent to it exhibit similar changes in expression over time in liquid GYAM medium. These same nine genes also exhibit relatively high expression on whole maize kernels and low expression on embryo tissue. The predicted functions of some of these genes are consistent with enzymatic activities predicted to be required for fusarin biosynthesis. Similar arrangements of PKS10 and at least some of the adjacent genes are present in the genomes of F. graminearum and Nectria haematococca. Together, the data suggest that PKS10 and some of the genes next to it constitute a fusarin biosynthetic gene cluster.


276. Characterization of a Fumonisin Biosynthetic Gene Cluster in Fusarium oxysporum strain O-1890. Robert H. Proctor1, Jeong-Ah Seo2, Mark Busman1, Yin-Won Lee2 and Ronald Plattner1. 1 USDA-ARS National Center for Agricultural Utilization Research, Peoria, Illinois, USA. 2 Seoul National University, Seoul, Korea.


Fumonisins are carcinogenic mycotoxins produced by some Fusarium species. Most species, including F. verticillioides, produce predominantly B fumonisins (FBs), but F. oxysporum strain O-1890 produces predominantly C fumonisins (FCs). FBs have an alanine-derived methyl function that is absent in FCs. In this study, we determined the nucleotide sequence of the fumonisin biosynthetic gene (FUM) cluster in strain O-1890. Genes in the cluster were present in the same order and orientations as in the previously described F. verticillioides cluster. The FUM gene coding and intergenic regions were 89 – 92 % and 40 – 77% identical, respectively, in F. oxysporum and F. verticillioides. In contrast, the sequences flanking the cluster were completely different in the two species. As previously reported, the F. verticillioides FUM cluster gene FUM8 encodes an oxoamine synthase and fum8 mutants cannot produce fumonisins. In the current study, transformation of a fum8 mutant with a wild-type F. verticillioides FUM8 restored FB production while transformation with a F. oxysporum O-1890 FUM8 resulted in FC production. These data indicate that the ability to produce FBs versus FCs is determined by different FUM8 homologues.


277. Functional analysis of the Hypocrea jecorina lxr1 demonstrates that Lxr1 encodes a D-mannitol dehydrogenase and is not involved in L-arabinose catabolism. Benjamin Metz, Stefan Polak, Christian P. Kubicek and Bernhard Seiboth. Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Austria.


The pentose L-arabinose is one of the main compounds of plant hemicelluloses and pectins. Most genes of the fungal L- arabinose pathway have been cloned for the industrial important fungi H. jecorina (anamorph: Trichoderma reesei). Cloning of an H. jecorinai L-xylulose reductase gene (lxr1) was reported previously by heterologous expression in Saccharomyces cerevisiae which enabled this yeast to grow on L-arabinose. A functional analysis of lxr1 in our lab has now questioned the role of Lxr1 in L-arabinose catabolism: Growth of a Δlxr1 strain was not affected on L-arabinose and total L-xylulose reductase activity remained unchanged. In addition Northern analysis showed that lxr1 was not L-arabinose inducible. In summary, our results clearly show that Lxr1 cannot be the enzyme which is active in the L-arabinose catabolic pathway. Our data suggest now that Lxr1 is a D-mannitol 2-dehydrogenase which is involved in developmental processes such as sporulation and germination. The polyol D-mannitol is accumumlated intracellularly by many fungal species and during spore germination D-mannitol is metabolized rapidly by D-mannitol 2-dehydrogenase. This is supported by our data that D-mannitol dehydrogenase activity was reduced in Δlxr1 strains and transcription levels were up-regulated during sporulation or germination.


278. Search for genes involved in the neoechinulin A biosynthetic pathway in Aspergillus spp. Masahiro Takeno, Momoko Horiuchi, Mayu Ohishi, Koji Kuramochi, Fumio Sugawara, and Takashi Kamakura. Tokyo University of Science, Noda, Japan.


Neoechinulin A is a member of the family of indole alkaloids comprised of tryptophane and alanine. It was isolated in 1973 from Aspergillus rubber by Yamazaki and co-workers. It was reported that the neoechinulin A was an antioxidant compound which suppresses lipid peroxidation in dried bonito flakes, Katsuobushi, and results of many studies suggested that Neoechinulin A might be useful as a protectant against neuronal cell death in neurodegenerative diseases. Although the chemical synthesis method of neoechinulin A is already established, the biosynthetic pathway and genes involved in the biosynthesis of neoechinulin A are not determined yet. Therefore we have tried to find the genes by phage display method. Phage display method is one of the ways to find the binding protein(s) or peptide(s) to a particular compounds and at least some proteins involved in the neoechinulin A biosynthesis must have affinity with the neoechinulin A molecule. Since we expect that genes for biosysthesis of neoechinulin A may form gene cluster(s) in the fungal genome similar to other gene clusters for secondary metabolites, the gene isolated by phage display could be the guide for us to access the gene cluster for neoechinulin A biosynthesis. As a result, we found a candidate of biosynthesis gene of neoechinulin A. The candidate had homologous sequence with non-ribosomal peptide synthetase (NRPS).


279. Sweet 'n' greasy: glycolipid biosynthetic gene clusters from Ustilago maydis. Sandra Hewald, Beate Teichmann, Lidan Liu, Uwe Linne and Michael Bölker. University of Marburg, Dept. of Biology, Karl-von Frisch-Strasse 8, D-35032 Marburg, Germany. e-mail: boelker@staff.uni-marburg.de


Under conditions of nitrogen starvation, Ustilago maydis produces two classes of extracellular glycolipids that act as biosurfactants. Ustilagic acid (UA) consists of a cellobiose moiety glycosidically linked to the omega-hydroxyl group of 15,16-dihydroxypalmitic acid. In addition, U. maydis cells secrete mannosylerythritol lipids (MELs), which consist of the disaccharide mannosylerythritol esterified with acyl and acetyl groups at the mannosyl group. We have identified two large gene clusters, which are responsible for extracellular glycolipid production. Database comparisons and mutational analysis allowed us to propose biosynthetic pathways for both of these unusual secondary metabolites. Since U. maydis is very amenable to genetic analysis this organism can be used for metabolic engineering of these extracellular glycolipids. Deletion of a MEL-specific acetyl transferase resulted in the production of deacetylated derivatives of this glycolipid. By targeted disruption of UA-specific hydroxylases we were able to create strains that secrete novel variants of ustilagic acid.


280. Integrated database for functional analysis in Aspergillus flavus. C. P. Smith1, C. P. Woloshuk2, N. P. Keller3, J. Yu4, and G. A. Payne5. 1North Carolina State University, Raleigh, USA. chris@statgen.ncsu.edu. 2 Purdue University, West Lafayette, IN, USA. woloshuk@purdue.edu. 3University of Wisconsin, Madison, WI, USA. npk@plantpath.wisc.edu. 4USDA/ARS/SRRC, New Orleans, LA, USA. jiuyu@srrc.ars.usda.gov. 5North Carolina State University, Raleigh, USA. gary_payne@ncsu.edu.


Aspergillus flavus is a plant and animal pathogen that also produces the carcinogen, aflatoxin. Because of its economic importance and well characterized pathway of aflatoxin biosynthesis, several labs are studying the development, metabolism, ecology, and pathogenicity of this fungus. To facilitate the research efforts in these areas and to identify potential genes and pathways for functional analysis, we are developing a database to integrate multiple categories of data. This database resource will serve two important functions: 1) it will provide a platform for the deposition of data from individual experiments; and 2) it will permit the ready analysis of composite data from all experiments enabling researchers to mine a larger data set. It will include phenotypic measurements, gene expression data from microarrays, and metabolic profile information and will be flexible enough to allow the addition of new types of measurement in the future. Users will interact with the database through a web based interface and will be able to: describe experiments; upload data gathered during those experiments; run analyses on the data; select and download raw data; select and download the results of analyses. Recently acquired lab and field data will be used to highlight the structure and utility of this database. These measurements will include fungal growth, aflatoxin concentrations, and gene expression data acquired from lab studies and infected developing maize seeds. This research was funded by USDA/NRI/CGP 2006-35604-16666.


281. Analysis of Methylene Tetrahydrofolate Reductases in filamentous fungi. Rasmus J.N. Frandsen and Henriette Giese, Department of Ecology, Section for Gen and Micro, Copenhagen University, Denmark. raf@kvl.dk and heg@kvl.dk


A. tumefaciens mediated random mutagenesis of Fusarium pseudograminearum led to the isolation of a mutant which accumulated rubrofusarin instead of the normal mycelium pigment, aurofusarin. Determination of the T- DNA integration site and a blast analysis against F. graminearum showed that a gene (Fg-met13) with homology to methylene tetrahydrofolate reductases (MTHR) was affected. Analysis of all fully sequenced fungi showed that they all had two MTHR genes. Multiple sequence alignment of the identified Met12 and Met13 sequences shows that the sequences clustered in two groups, suggesting distinct evolutionary histories and functions. Deletion of met12 and met13 in S. cerevisiae have earlier showed that Sc-met13 is required for methionine biosynthesis, while no function or phenotype have been assigned to Sc-met12. Targeted replacement of Fg-met12 and Fg- met13 showed that Fg-met12, and not as expected Fg-met13, is required for methionine biosynthesis in Fusarium. Complementation of the Sc-met13 deletion strain with Fg-met12 and Fg-met13 showed that both are functional MTHRs. We are currently testing the expression patterns to see if this can account for the differences between the two species. The situation in other fungi is also under investigation. We are currently exploring the possibility that Fg-met13 is indirectly involved in aurofusarin formation, possibly by synthesising a co-factor required by one of the enzymes known to participate in the pathway.


282. FvVE1 Differentially Regulates the Biosynthesis of Fumonisins and Bikaverin in Fusarium verticillioides. Kyung Myung 1, Shaojie Li1, Hamed K. Abbas2 and Ana M. Calvo1. 1Department of Biological Sciences, Northern Illinois University, 1425 W Lincoln Hwy., Dekalb, Illinois. 60115, USA. 2Crop Genetics and Production Research Unit, USDA-ARS, Stoneville, Mississippi 38766, USA.


The veA homologous genes positively regulate sterigmatocystin and aflatoxin production in Aspergillus nidulans and in Aspergillus parasiticus and Aspergillus flavus, respectively. Whether veA homologs have a role in regulating secondary metabolism in other fungal genera is unknown. In this study, we examined the role of the veA homologous gene FvVE1 on the production of two different types of toxins in the plant pathogen Fusarium verticillioides. We found that deletion of FvVE1 completely suppressed fumonisin production on two natural substrates, corn and rice, but did not completely eliminate fumonisin production in fumonisin-inducing complex liquid medium GYAM. In contrast, deletion of FvVE1 reduced the yield of bikaverin in GYAM while it increased bikaverin production in corn. The effects of FvVE1 deletion on toxin production were found to be the same in the two different mating types. Complemented strains restored the production of both fumonisins and bikaverin to wild-type levels. Our results strongly suggest that FvVE1 differentially regulates fumonisin and bikaverin biosynthesis in a medium-dependent manner.

                

283. Characterization of putative protein phosphatase gene in Fusarium verticillioides that is associated with fumonisin biosynthesis, cellular development, and conidiation. Yoon-E Choi and Won-Bo Shim, Dept. Plant Pathology and Microbiology, Texas A&M University, TX 77843-2132


Fusarium verticillioides is known to cause maize ear rot and produce fumonisin B1 (FB1). To date, our understanding of the regulatory mechanisms associated with FB1 biosynthesis is incomplete. In this study, we present the isolation and characterization of a putative protein phosphatase gene (CPP1) in F. verticillioides and its role in FB1 biosynthesis. CPP1 expression study suggested that CPP1 is associated with FB1 biosynthesis in a negative manner. CPP1 knock-out mutants were generated, and we select a mutant strain PP179 for further analysis. PP179 showed elevated expression of FB1 biosynthetic genes, including FUM1 (PKS gene), and in turn produced a higher level of FB1 than the wild-type progenitor. Other significant PP179 phenotypes included reduced radial growth on solid media, reduced conidia germination efficiency, increased macroconidia development, and hyphal swelling. To verify that these phenotypes were directly due to CPP1 deletion, we complemented PP179 with the CPP1 gene. The complement strain, PPC4, showed the wild-type level FUM1 expression and restored the FB1 production level to that of wild type, providing molecular evidence that CPP1 is negatively associated with FB1 biosynthesis. Phenotypes such as growth defect, macroconidia production, and hyphal swelling were also restored in PPC4. The results suggest that CPP1 is a negative regulator of FB1 biosynthesis, however, the pleotropic effects of CPP1 deletion precluded definitive conclusions. Further study is necessary to identify a signaling pathway downstream of CPP1 that is directly associated with FB1 biosynthesis in F. verticillioides.


284. Investigating polyketide production in fungi: Tenellin and other PKS/NRPS genes. Andy Bailey, Kirstin Eley, Laura Halo, Thomas Thomas, Kate Harley, Deirdre Hurley, Zongshu Song, Russell Cox, Colin Lazarus and Thomas Simpson. School of Biological Sciences and School of Chemistry, University of Bristol, Bristol, UK.


Type I Polyketide Synthases (PKS) are large multidomain, multifunctional enzymes. In Fungi, these enzymes are iterative, but how they are programmed is mysterious, meaning that it is impossible to predict the chemical made even if the gene sequence has been determined. Using class-specific degenerate PCR-primers, we have isolated fragments from several PKS and PKS/NRPS hybrid genes from different fungi. We have sequenced the gene-cluster responsible for synthesis of the PKS/NRPS Tenellin produced by the insect pathogen Beauveria bassiana. Confirmation of the identity of this cluster was achieved by targeted disruption of the PKS/NRPS, with knockout strains failing to produce Tenellin. The gene shows the expected architecture with KS-AT-DH-cMeT-KR-ACP for the polyketide component, followed by a single NRPS domain including the C-A-T&R domains required for addition of the aminoacid moiety. The disrupted strain was still fully virulent on the insect host, showing that tenellin is not essential for the disease process. We hope to gain more understanding about how PKS proteins are programmed to make their specific product, particularly how the number of extension cycles is regulated and how the levels of reduction are controlled, by using domain-swaps between different PKS genes. These are expressed in Aspergillus oryzae and coupled with chemical analysis of any resulting new secondary metabolites.


285. Site-Directed Mutagenesis Studies of Pheromones of Schizophyllum commune. James L. Mackey and Thomas J. Fowler, Southern Illinois University Edwardsville, Edwardsville, Illinois. tfowler@siue.edu

Mating of homokaryons and maintenance of the dikaryon in S. commune are controlled in part by pheromone- responsive G protein-coupled receptors (GPCRs) and short, lipopeptide pheromones. Pheromone receptor activation initiates a presumed signal transduction cascade that leads to nuclear migration and fusion of hook cells. Pheromones from one subset of S. commune mating pheromones have a central cluster of three amino acids that vary between the pheromones. These pheromones can activate more than one receptor and these sets of receptors differ for each pheromone studied, but the sets have some receptors in common. Site-directed mutagenesis experiments focused on changing these three amino acids. Signal initiation was studied by heterologous expression of pheromones and receptors in Saccharomyces cerevisiae. Single amino acids substitutions have shown no change in receptor specificity for the four pheromones studied in this group. This is different compared to previous studies of a different subset of S. commune mating pheromones where single amino acid residues controlled the specificity of the pheromone. The current data suggest that this subset of pheromones does not rely on single amino acid residues for specificity of interaction with receptors. Double and triple amino acid substitutions should determine the key to specificity for this pheromone group. This research is funded by NSF.


286. Withdrawn


287. AREA in Fusarium verticillioides Affects Growth and Fumonisin Biosynthesis In Colonized Maize Kernels. Hun Kim and C. P. Woloshuk. Purdue University, West Lafayette, IN. hunkim@purdue.edu


Fumonisins are nitrogen-containing, secondary metabolites produced by Fusarium verticillioides. Evidence indicate that regulation of fumonisin biosynthesis is governed by carbon source, nitrogen source and pH. Previous studies have indicated that fumonisin B1 (FB1) biosynthesis is repressed in cultures containing ammonium phosphate (AP) as the nitrogen source. The fungus also fails to produce FB1 when grown on blister kernels, the earliest stages of kernel development. We generated a strain of F. verticillioides with a disrupted AREA gene, a regulator of nitrogen metabolism. The growth rate of the areA mutant on defined media containing either AP or casein hydrolysate was similar to the wild type. In contrast, the mutant grew poorly on media containing a nitrogen source of sodium nitrate or BSA. When the mutant was grown on mature maize kernels, it did not produce FB1, most likely because of poor growth. When AP was added to kernels, the mutant grew as well as the wild-type strain but neither strain accumulated FB1. This result indicates that the AREA gene is needed for growth on mature kernels and possibly for FUM gene expression. The areA mutant also grew well on blister kernels indicating that the elevated levels of free amino acids known to be present in early kernel development are sufficient to support growth of the mutant. We will present results from additional experiments that demonstrate how different nitrogen and carbon sources as well as pH affect FB1 biosynthesis in a strain that over-expresses AREA.


288. Comparative analysis of secondary metabolism gene clusters from two strains of Aspergillus fumigatus and closely related species Neosartorya fischeri and Aspergillus clavatus. Natalie Fedorova1, Vinita Joardar1, Jonathan Crabtree1, Rama Maiti1, Paolo Amedeo1, David Denning2, Jennifer Wortman1, Geoffrey Turner3, and William Nierman1. 1The Institute for Genomic Research, Rockville, MD, USA; 2School of Medicine and Faculty of Life Sciences, The University of Manchester, Manchester, UK; 3Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK. Email: natalief@tigr.org


Genes responsible for biosynthesis of secondary metabolites are often referred to as the most variable segment of fungal genomes. Comparative analysis of secondary metabolism gene clusters from A. fumigatus and the two closely related species confirmed that these organisms are indeed relentlessly updating their repertoire of mycotoxins. Out of 23 clusters identified in A. fumigatus, only seven have putative orthologs in the other two aspergilli. Most of these ‘core’ clusters are highly conserved and appear to be under strong negative selection. The rest of the clusters, however, are species- or even strain-specific, implying that the aspergilli must utilize specific mechanisms to ensure a quick divergence and turnover of clusters in the course of evolution. These mechanisms may involve de-novo assembly, segmental duplication, translocation, accelerated differentiation, and differential loss of clusters in various lineages.


289. Impact of methionine synthase deletion on metabolism of the phytopathogenic fungus Magnaporthe grisea. Oceane Frelin, Marie Emmanuelle Saint-macary, Marie Joseph Gagey, Christelle Barbisan, Roland Beffa, Marc-henri Lebrun, Geraldine Mey and Michel Droux. UMR2847 CNRS/Bayer CropScience, Physiologie des plantes et des champignons lors de l’infection, Bayer CropScience, 14-20 rue Pierre Baizet 69009 LYON


In organisms capable of sulfate assimilation, the reduced sulfur atom is incorporated in organic molecules to generate the two sulfur amino acids, cysteine and methionine. In filamentous fungi their biosynthetic pathways consist in features reported in both plant and bacteria as well as those described in the yeast Saccharomyces cerevisiae. Studies on sulfur metabolism in the phytopathogenic fungus Magnaporthe grisea were developed to understand its role during fungal development. With this aim a mutant of the methionine synthase (Met6) was obtained using gene replacement. The met6 null mutants were auxotrophic for methionine, reduced in sporulation and were unable to penetrate into host leaves. Our goal is to further understand the impact of absence of the methionine synthesis step on general metabolism in M. grisea. We performed targeted metabolite analysis through reversed chromatography using HPLC. In parallel, a targeted gene expression analysis through qPCR was also realized. Results show drastic differences in the sulfur containing compounds, amino acid profiles and sulfur-linked gene expression level between wild type strain and the met6 null mutants. Results indicate that lack of methionine synthase induces both complex metabolic and transcriptomic perturbations


290. Phylogenomic analysis of fungal nonribosomal peptide synthetases reveals modular duplication, loss, and shuffling as innovative forces. Scott Kroken1, B. Gillian Turgeon2. 1Division of Plant Pathology and Microbiology, Dept. of Plant Pathology and Microbiology. University of Arizona. kroken@ag.arizona.edu 2Dept. of Plant Pathology, Cornell University. bgt1@cornell.edu


The majority of secondary metabolism in euascomycete fungi is based on a diversity of various and variously modified nonribosomal peptides, polyketides, and hybrids of these two backbone molecules. The evolutionary history of nonribosomal peptide synthetases (NPSs) is similar to that reported for polyketide synthetases (Kroken et al 2003): fungi and bacteria have separate clades of NPSs (with some notable exceptions of horizontal transfer), rampant duplication and divergence, discontinuous distributions of orthologous NPSs, and a rapid drop-off of shared orthologs with increasing phylogenetic distance among fungal species. In addition, the multi-modular construction of NPSs adds another dimension to their evolution. Orthologous NPSs, which are often found in orthologous gene clusters, frequently vary in the presence/absence of certain domains. Paralagous NPSs are the result of modular duplication, and subsequent concatenation or rearrangement of those modules. Rearrangement is also found at the domain level, as the adenylation and condensation domains of each module often have different evolutionary histories. Therefore, an innovative factor of NPS evolution is the reordering of adenylation domains that specify different amino acids, and condensation domains that appear to specify how the amino acid is to be covalently bound in the nascent oligopeptide. For example, the intermediate and terminal condensation domain of fungal NPSs form separate clades, suggesting that the terminal condensation domain also functions as a peptide release factor. Fungal-type NPSs do not have, or apparently need, a thioesterase domain as found in bacterial-type NPSs (which include the ACV synthetases used for penicillin synthesis).


Gene Regulation


291. The Protein Kinase Tor1 Links Nutrient Sensing and Filamentous Growth in Candida albicans. Robert J. Bastidas, Maria Cardenas-Corona and Joseph Heitman. Department of Molecular Genetics and Microbiology. Duke University. rjb10@duke.edu


The life cycle of Candida albicans consists of two phases, a yeast phase and a filamentous phase. The transition between yeast and filamentous growth is controlled by a variety of signaling networks. In eukaryotic cells, the protein kinase Tor (Target of rapamycin) functions in a signaling pathway implicated in regulating cellular responses to nutrients that is conserved from yeast to humans. In this work, we have begun to characterize the function of C.albicans Tor1. Using rapamycin, a pharmacological inhibitor of Tor, we find that CaTor1 is necessary for the ability of C.albicans cells to switch between yeast and filamentous states. First, exposure to rapamycin under non- filamentation inducing conditions elicits differential expression of genes involved in filamentation, as detected by microarray analysis. Second, northern analysis revealed that CaTor1 is required for the repression of hyphal specific gene induction. Third, inhibition of CaTor1 enhances germ tube formation during growth on filamentation inducing conditions. Finally, a screen of transcription factor mutants identified mutations in the filamentous growth repressors CaNrg1 and CaTup1 that result in rapamycin hypersensitivity. Our results support a model in which CaTor1 couples nutrient sensing signaling cascades and filamentous growth possibly by governing the activity of CaTup1 and CaNrg1.


292. Light responses in Mucor circinelloides: a regulatory network involving three white collar-1 genes and a RING-finger protein. Santiago Torres-Martínez, Fátima Silva, and Victoriano Garre. Department of Genetics and Microbiology, Faculty of Biology. University of Murcia. Murcia. Spain


Light regulates many developmental and physiological processes in a large number of organisms, including fungi. The best- known light response in the fungus Mucor circinelloides is the biosynthesis of beta-carotene, although we have recently demonstrated that its sporangiophores also respond to light, exhibiting a positive phototropism. Analysis of both responses to different light wavelengths within the visible spectrum demonstrated that phototropism is induced by green and blue light, whereas carotenogenesis is only induced by blue light. We have identified in M. circinelloides three white collar-1 genes (mcwc-1a, mcwc-1b and mcwc-1c) coding for proteins showing similarity with the WC-1 photoreceptor of Neurospora crassa. All three contain a LOV (light, oxygen or voltage) domain, similar to that present in fungal and plant blue-light receptors. Knockout mutants for each mcwc-1 gene were generated to characterize gene functions. Only mcwc-1c mutants were defective in light induction of carotene biosynthesis, indicating that mcwc-1c is involved in the light transduction pathway that control carotenogenesis. We have also shown that positive phototropism is controlled by the mcwc-1a gene. It seems therefore that mcwc-1a and mcwc-1c genes control different light transduction pathways. Cross-talk between both pathways probably exists because lack of mcwc-1a function clearly affects light regulation of mcwc-1c expression. Studies of interactions between mcwc-1 genes and crgA gene, a RING-finger repressor of photocarotenogenesis in M. circinelloides, revealed that crgA function in carotenogenesis is mediated by mcwc-1b gene, since mutations in this gene suppresses the constitutive phenotype for carotenogenesis showed by the crgA mutants. These results confirm the complex network of pathways controlling the light responses in M. circinelloides.


293. Enhanced Responsiveness and Sensitivity to Blue Light by blr-2 Overexpression in Trichoderma atroviride. Ulises Esquivel-Naranjo and Alfredo Herrera-Estrella Departamento de Ingeniería Genética y Laboratorio Nacional de Genómica para la Biodiversidad, Cinvestav Campus Guanajuato, Apartado postal 629, Irapuato 36500, México. Email: aherrera@ira.cinvestav.mx


Light is an environmental factor that regulates pivotal processes in living organisms, such as growth, development, reproduction and metabolism. Blue light activates asexual reproduction in T. atroviride through the transcription factors BLR-1 and BLR-2 that regulate the light responsive genes. BLR proteins contain PAS and GATA-type DNA binding domains and both proteins are essential for the regulation of blue light responsive genes and photoconidiation. In this work, we show that BLR-2 is a limiting factor for photo-perception. Overexpression of blr-2 resulted in increased photoconidiation and a stronger expression of light induced genes. The characterization of light responses in blr-2 overexpressers allowed us to identify a crosstalk between blue and red light input systems and an interaction of the BLR proteins with other(s) light perception system(s) in T. atroviride. blr-2 overexpression caused a marked reduction of growth when the fungus was grown under defined photoperiods, including a period of strong sensitivity to light, followed by a period of insensitivity. Long periods of incubation under this condition permitted recovery of a rhythmic growth similar to that of the wild type. A novel light dependent role for the BLR proteins was found in the positive posttranscriptional regulation of blr-2 expression, in addition to a transcriptional role, which occurred even in the dark. Finally, we demonstrated that blr-2 overexpression caused higher sensitivity to blue light and therefore propose that the pre-formation of BLR-1/BLR-2 complexes is key for adequate light perception in T. atroviride.


294. Molecular Characterization of Ligth Responses in the Celullolitic Fungus Trichoderma reesei. F.X. Castellanos-Juarez, A. Sánchez, M. Schmoll, C. Kubicek, and A. Herrera-Estrella. Laboratorio Nacional de Genómica para la Biodiversidad, Cinvestav Campus Guanajuato, AP 629, Irapuato, Guanajuato, México 36500 aherrera@ira.cinvestav.mx

Sunlight is a key environmental factor that determines the behaviour of living organisms. Among the diversity of responses triggered by light, in fungi important efforts have been directed to studying photomorphogenesis, phototropism, pigmentation and circadian rhythms. In Trichoderma atroviride, conidiation is activated by blue light. Two genes (blr1/blr2) homologues of the Neurospora crassa wc1/wc2 are essential for photoconidiation and regulation of blue light responsive genes. Recently, envoy, a gene homologous to the N. crassa vivid has been identified in T. reesei, vivid encodes a secondary blue-light receptor, necessary for adaptation to constant illumination and to detect changes in light intensity. Although envoy was unable to complement the wild type phenotype of a vivid mutant, this gene is necessary for light tolerance. In the present work, we analyzed the role of Envoy in photoadaptation, to detect changes in light intensity and its regulation through BLR perception system. Under constant illumination we found Envoy is required to turn off the expression of blue light responsive genes similar to VIVID. This result is consistent with its contribution to the light tolerance during mycelial growth. On the other hand, we observed that an envoy mutant was unable to detect changes in light intensity. In order to scrutinize the participation of BLR proteins in the envoy regulation, we generated mutants in both genes and their characterization showed a lack of photoconidiation and regulation of the blue light responsive genes including envoy.


295. Transcriptional controls of carbon source utilization in Aspergillus nidulans. Michael Hynes. Department of Genetics, University of Melbourne, Parkville, Australia.


In Aspergillus nidulans, acetate utilisation involves acetyl-CoA formation in the cytoplasm and metabolism via the glyoxalate cycle in peroxisomes and via the TCA cycle in mitochondria. A cytoplasmic carnitine-acetyl transferase allows acetyl-CoA to enter the organelles as acetyl-carnitine which is converted back to acetyl-CoA by a second carnitine-acetyl transferase encoded by acuJ, a gene required for growth on both acetate and fatty acids which are converted to acetyl-CoA by peroxisomal beta-oxidation. A peroxisomal targetting mutation results in loss of growth on fatty acids indicating that AcuJ is necessary for transport of acetyl-CoA from peroxisomes to the mitochondria. Growth on acetate is not affected, indicating that acetyl-CoA can be metabolised via the glyoxalate cycle in the cytoplasm. This is supported by the ability of various peroxisome mutants to grow on acetate but not on fatty acids. There are three classes of genes - those induced by acetate only; those induced by both acetate and fatty acids including acuJ and genes for the glyoxalate cycle and a very large number induced only by fatty acids including genes encoding beta-oxidation enzymes and peroxins. The transcription factor FacB, specific for acetate induction, is regulated by glucose repression and induction by acetyl-carnitine. FarA, FarB (orthologs of the highly conserved cutinase transcription factors) and ScfA, are required for fatty acid induction. FarA orthologs are also found in the hemi-ascomycetes, Candida albicans, Debaryomyces hansenii and Yarrowia lipolytica. Carbon sources metabolised via the TCA cycle, like acetate and fatty acids, require gluconeogenesis. Two transcription factors are required for induction of relevant enzymes in response to either malate or oxaloacetate.


296. Carbon sensing regulation of G protein signal pathway in Neurospora crassa. Liande Li and Katherine Borkovich. University of California, Riverside.


N. crassa is an excellent model to study heterotrimeric G protein signaling pathways, as N. crassa can grow in diverse environments, and one of the major systems used by N. crassa to sense extracellular changes involves heterotrimeric G proteins. This study has focused on examining the carbon sensing pathways involving GPR-4, a predicted G protein coupled receptor (GPCR) of N. crassa, and regulated G proteins. Since GPR-4 related GPCRs are found in the genomes of many fungal pathogens, study of GPR-4 may shed light on the functions of these proteins during pathogenesis, growth and development. Our results support the hypothesis that GPR-4 is coupled to GNA-1 in a cAMP signaling pathway that regulates the response to carbon sources in N. crassa. delta gpr-4 mutants have severe defects on glycerol medium suggested that the GPR-4 signaling pathway may be involved in glycerol utilization, but Northern analyses showed that deletion of gpr-4 does not greatly influence expression of many genes implicated in glycerol utilization. delta gpr-4 strains also lack the transient increase in cAMP levels observed in wild type after addition of glucose to glycerol-grown cultures. In order to identify downstream signaling pathways controlled by GPR-4 during carbon sensing, microarray analyses are being performed using samples from wild type and gpr-4 and gna-1 mutants cultured with different carbon sources. The regulated genes will yield information about G protein regulation of carbon sensing in filamentous fungi.


297. Gene silencing in Mucor circinelloides by expression of hairpin RNAs: Role of the dcl-2 and rdrp genes. Rosa M. Ruiz-Vazquez, Juan P. de Haro, Silvia Calo and Santiago Torres-Martinez. Department of Genetics and Micro, Faculty of Biology, University of Murcia, Murcia 30071, Spain.


We have previously demonstrated the existence of a gene silencing mechanism triggered by self-replicative transgenes in the zygomycete Mucor circinelloides. Gene silencing is associated with two size classes of siRNA, 25-nt and 21-nt long, which are differentially accumulated through the vegetative growth of the silenced strains. These two classes of siRNAs are preferentially produced from the 3'-end of the endogenous gene and both are associated with amplification and spreading of the silencing signal. To increase the silencing frequency and the stability of the silencing effects during vegetative growth, we have developed hairpin expressing plasmids designed to silence an endogenous gene. Hairpin constructs containing an intron are highly efficient silencing triggers in M. circinelloides, the expression of hairpin RNA being associated with the accumulation of the two size classes of siRNA. We have isolated two dicer-like genes of M. circinelloides. Phenotypic analysis of null dcl-1 mutants demonstrated that the dcl-1 gene is not essential for sense transgene or dsRNA-induced gene silencing, since dcl-1 mutants are able to silence gene expression and to produce the two classes of siRNA. However, null dcl-2 mutants are severely impaired in gene silencing, revealing an essential role of this gene in RNA silencing and suggesting that a single Dicer enzyme is responsible for the production of the two size classes of siRNAs. We have also isolated a M. circinelloides rdrp gene, which codes for a RNA-dependent RNA polymerase. The role of the rdrp gene in gene silencing triggered by sense transgenes or dsRNA, as deduced from the phenotype of null rdrp mutants, will be presented.


298. RNA expression in Cryptococcus neoformans during pulmonary cryptococcosis. Guanggan Hu, A Sham and JW Kronstad (Michael Smith Laboratories, The University of British Columbia, Vancouver, BC, Canada)

C. neoformans is an encapsulated fungus that causes meningoencephalitis in immunocompromised individuals, and that is believed to be a facultative intracellular pathogen during early stages of pulmonary infection. Alveolar macrophages are the major phagocytic cells in the murine lung and the percentage of Cryptococcus cells inside alveolar macrophages is known to reach a maximum at 8 h after infection, while most of the yeast cells are extracellular by 24 h. Although the course of infection is relatively clear, information on C. neoformans gene expression during infection is quite limited. The goal of the present study was to determine the early transcriptome changes that occur in C. neoformans during pulmonary cryptococcosis. To achieve this goal, we performed long SAGE (serial analysis of gene expression) analysis on C. neoformans cells recovered from infected lungs of mice at 8 h and 24 h after infection. We compared the data with SAGE libraries from cells grown in iron-limiting medium and from cells harvested from the central nervous system of infected rabbits. The analysis revealed elevated expression of a set of functions at 8 h versus 24 h, including multiple transporters (sugar, iron, copper, amino acids and phosphate), as well as proteins involved in secretion, stress response, cellular respiration, protein and carbohydrate catabolism, and virulence. Interestingly, long SAGE analysis also revealed elevated expression of genes for lipid metabolism and for utilization of two-carbon compounds in cells grown in vivo compared with those grown in culture. In contrast, very few of transcripts were elevated at 24 h versus 8 h suggesting that dramatic transcriptional remodeling occurs during the early stages of infection. Real time PCR is being used to confirm the expression of selected transcripts. In addition, a subset of the genes identified by SAGE have been disrupted to determine whether the in vivo gene expression patterns indicate important functional roles during pulmonary infection. Taken together, these findings provide the first insight into the transcriptional response of C. neoformans during the pulmonary infection.


299. Candida albicans CHT4 encodes a sporulation specific gene. Alexander Duenkler1 and Jürgen Wendland1,2. 1 Department of Microbiology, Friedrich-Schiller-University, Jena and Junior Research Group: Fungal Pathogens, Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knöll Institute, Jena; Beutenbergstr 11a; D-07745, Jena, Germany. 2Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10; DK-2500 Valby, Copenhagen, Denmark


The C. albicans genome encodes four chitinases, CHT1, CHT2, CHT3, and CHT4. In a recent analysis we have shown that Cht3 encodes the functional homolog of the Saccharomyces cerevisiae chitinase Cts1 that is involved in mother-daughter cell separation. All four chitinase encoding genes are non-essential. They belong to two groups in which CHT1, CHT2, and CHT3 are more similar to CTS1, while CHT4 is more similar to CTS2. ScCTS2 was described as a "sporulation specific gene".

However, published experimental evidence does not exist. The diploid cts2/cts2 strain sporulates well and the spores do not exhibit germination defects or increased sensitivity against zymolyase. In the filamentous fungus Ashbya gossypii a CTS2 homolog (ACL166w) was identified. The AgCts2 is 490aa in length and shows 42.3% overall identity to ScCts2 (511aa) and 33.2% identity to CaCht4 (388aa). The catalytic centers of the three proteins are highly conserved and shows clear differences to members of the Cts1-chitinase family. We deleted the AgCTS2 gene and carried out phenotypic characterizations. The Agcts2 mutants showed no growth retardation or morphogenetic defects. However, Agcts2 mutants revealed a spore shape defect indicating defects in the assembly of the spore wall. This defect did not lead to increased sensitivity against zymolyase or heat shock and did not affect the germination pattern of A. gossypii spores. Expression of AgCTS2 was analyzed using a lacZ reporter gene. AgCTS2 is expressed in the center of a mycelium that corresponds to the sporogenous mycelium.


300. Molecular Response to Putative Pheromone in Histoplasma capsulatum. Meggan Bubnick1 and George Smulian1,2. 1University of Cincinnati, 2Cincinnati VA Medical Center.


Histoplasma capsulatum (Hc) is an ascomycetous human fungal pathogen found worldwide. Hc is a heterothallic organism with two described mating types, (+) and (-). There is a predominance of (-) mating type strains among clinical isolates; however, little is known about mating on a molecular level, and this must be resolved to address possible virulence differences between (+) and (-) mating type organisms. Predicted mating (MAT) locus idiomorphs, MAT1-1 and MAT1-2, pheromone receptors STE2 and STE3 and a predicted pheromone gene, PPGA, were identified in the Hc genome through homology and syntenic analysis. The predicted pheromone was synthesized, and used to stimulate two Hc clinical strains of opposite mating type, RE1 and VA1. Cultures were also stimulated with a and alpha pheromone extracts isolated from a co-culture of (-) and (+) mating type organisms. RNA was extracted, and quantitative RT-PCR was performed. Expression levels of pheromone receptor, STE2 and the MAT locus transcription factors, MAT1-1-1 and MAT1-2-1, were analyzed. In RE1, expression of MAT1-1-1 and STE2 increased with synthesized pheromone treatment, while in VA1, STE2 and MAT1-2-1 expression showed little change. MAT1-1-1 expression levels in RE1 increased in response to alpha pheromone extract, while MAT1-2-1 expression levels in VA1 increased in response to a pheromone extract. The differential responses of MAT1-1-1 and MAT1-2-1 gene expression to pheromone stimulation confirm their role in regulation of mating type expression. Further studies are required to determine differential gene expression between organisms of opposite mating type and their role in virulence in Hc.


301. Analysis of four putative beta–oxidation genes in Aspergillus nidulans. Kathrin Reiser, Meryl A. Davis and Michael J. Hynes. Department of Genetics, University of Melbourne


Filamentous fungi are able to use fatty acids as sole carbon sources via beta-oxidation. The enzymes required are present in both peroxisomes and mitochondria (Maggio-Hall and Keller 2004 Mol. Microbiol. 54:1173–1185). Two putative fatty acyl-CoA dehydrogenases, AcdA and AcdB, and two putative fatty acyl-CoA oxidases, AoxA and AoxB, were identified in the genome of A. nidulans. Because of their homology to Fox1p of S. cerevisiae and a peroxisomal dehydrogenase of N. crassa, they are thought to be involved in the first step of beta-oxidation. Homologues for each were found in other fungal species. Three of the proteins have a clear PTS1 (peroxisomal targeting sequence), while AcdA has a PTS1-like sequence. Hence, all four proteins are predicted to be peroxisomal. GFP fusion proteins have been constructed to investigate this hypothesis. In the 5’ promoter region (1kb) of each gene, a 6bp sequence (CCGAGG/ CCTCGG) was found at least once. This sequence is predicted to be the core of a fatty acid depending regulation site (Hynes et al. 2006 Eukaryot. Cell. 5:794- 805) suggesting positive regulation by fatty acids. Promoter-lacZ fusions have shown that at least two genes are induced by short- and long-chain fatty acids and induction is altered in the absence of the predicted regulatory proteins. Deletions of all four genes do not give a severe fatty acid growth phenotype. Only the aoxA-deletion phenotype is clearly visible. This implies a great redundancy amongst peroxisomal proteins involved in the first step of beta- oxidation.


302. Role of chromatin modifiers in secondary metabolite production in Aspergillus nidulans. Jin Woo Bok and Nancy P. Keller. Dept of Plant pathology, University of Wisconsin, Madison, U. S. A.


In eukaryotic cells, the DNA is packaged into chromatin by histone proteins. Post-transcriptional modifications of the histones result in epigenetic regulation of gene expression. Epigenetic processes are essential for development and differentiation in eukaryotic cells including fungi. Fungal secondary metabolites are also developmentally regulated. The connection between the epigenetic processes and secondary metabolite production is poorly understood. To unveil epigenetic regulation in secondary metabolite production, a well-known model fungus Aspergillus nidulans was used to introduce mutation in chromatin modifiers. Many chromatin modifiers are conserved from yeast to human including COMPASS (a histone 3 K4 methylase complex), Paf1 complex (a connecting complex between COMPASS and RNA polymerase II), Dot1 (a histone3 K79 methylase) and Lsd (a demethylase of histone3 K4). Here, we present results of secondary metabolite production modification in deletion mutants of those conserved chromatin modifiers. In addition, we examined a role of laeA (a global regulator of secondary metabolites) in these mutants.


303. Activation of the RNAi components as part of the double-stranded RNA induced host defense response in a filamentous fungus. Swati Choudhary1,4, Heng-Chi Lee1,4, Mekhala Maiti1,4, Qun He1,5, Ping Cheng1,6, Qinghua Liu2, and Yi Liu1,3. 1Department of Physiology and 2Department of Biochemistry University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390 USA 3To whom correspondence should be addressed: E-mail: Yi.Liu@UTSouthwestern.edu 4These authors contributed equally in this study. 5Current address: State Key Laboratory for Agro-Biotechnology, College of Biological Sciences, Chinese Agricultural University, Beijing, 100094, China. 6Current address: Merck Research Laboratories, 126 E. Lincoln Ave. Rahway, NJ 07065, USA


Double-stranded RNA (dsRNA) produced in eukaryotic cells can be recognized by the RNA interference (RNAi) pathway to result in posttranscriptional gene silencing. In addition, dsRNA can trigger interferon response as part of the immune response in vertebrates. In this study, we show that dsRNA, but not siRNA, can induce the expression of qde-2 (an argonaute gene) and dcl-2 (a dicer gene), two central RNAi components of the filamentous fungus Neurospora crassa. The induction of QDE-2 associates with enhanced efficiency of gene silencing, suggesting a regulatory mechanism that allows for the optimal function of the RNAi pathway. In addition, we show that DCLs regulate QDE-2 posttranscriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. Finally, genome-wide identification of dsRNA activated genes in Neurospora show that homologs of antiviral and interferon stimulated genes (ISGs), including Mx proteins, 6-16 family proteins, and a RNA helicase are highly induced by dsRNA. Together, our results suggest that the activation of the RNAi components is part of a broader ancient host defense response against viral and transposon infections.


304. QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands. Mekhala Maiti, Heng-Chi Lee, and Yi Liu. Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390 USA


Single-stranded siRNA guides the cleavage of homologous mRNA by Argonaute proteins, the catalytic core of the RNA- induced silencing complex (RISC), in the conserved RNA interference (RNAi) pathway. The separation of the siRNA duplex into single strands is essential for the activation of RISC. Previous biochemical studies have suggested that Argonaute proteins cleave and remove the passenger strand of siRNA duplex from RISC, but the in vivo importance of this process and the mechanism for the removal of the nicked passenger strand are not known. Here, we show that in the filamentous fungus Neurospora, the Argonaute homolog QDE-2 and its slicer function are required for the generation of single-stranded siRNA and gene silencing in vivo. Biochemical purification of QDE- 2 led to the identification of QIP, a QDE-2-interacting protein, with an exonuclease domain. The disruption of qip in Neurospora impaired gene silencing and siRNA accumulated, mostly in duplex form. Furthermore, our results suggest that QIP acts as an exonuclease that cleaves and removes the nicked passenger strand from siRNA duplex in a QDE-2 dependent manner. Together, these results suggest that both the cleavage and removal of the passenger strand from the siRNA duplex are important steps in RNAi pathways.


305. NmrA levels regulate the activity of AreA, the major nitrogen regulator, in Aspergillus nidulans. Koon Ho Wong, Richard B. Todd, Michael J. Hynes and Meryl A. Davis. Department of Genetics, The University of Melbourne, Parkville, 3010, Australia

The GATA transcription factor AreA is the major nitrogen regulatory protein in Aspergillus nidulans. AreA activates the expression of enzymes and permeases required for the breakdown of secondary nitrogen sources. The activator function of AreA is inhibited in the presence of primary nitrogen sources such as ammonium by interaction with the negatively acting protein NmrA. We have investigated the effects of overexpression of AreA and NmrA using the highly inducible xylP promoter. Overexpression of AreA results in partial derepression of catabolic gene expression whereas overexpression of NmrA prevents AreA activity even in the absence of repressing metabolites. The levels of areA mRNA and AreA protein are low in the presence of ammonium and elevated during nitrogen limitation. We have shown that nmrA has the opposite pattern of expression with high levels of NmrA produced on ammonium. To localise sequences required for the regulated expression of nmrA, we have created promoter deletions in the nmrA gene and introduced these constructs into an nmrA deletion strain. Functional studies, together with RT-PCR and Western blot analyses, revealed that promoter sequences between -800 and -512 are required for full expression of NmrA and inhibition of AreA activity. Sequence analysis has revealed that several potential bZIP transcription factor binding sites are located within this region of the nmrA promoter.


306. Coordinated regulation of the melanin biosynthesis gene cluster in Cochliobolus heterostrophus. Sophie Lev, Noa Eliahu1, Aeid Igbaria1, Mark S. Rose2 and Benjamin A. Horwitz1. 1Department of Biology, Technion - Israel Institute of Technology, Haifa, Israel, and 2Syngenta Biotechnology Inc., North Carolina, USA. §email: sophia@tx.technion.ac.il


The maize pathogen Cochliobolus heterostrophus requires two MAP kinases, Chk1 and Mps1, to produce normal pigmentation. Young colonies of mps1 and chk1 deletion mutants have a white and autolytic appearance, which was partially rescued by a hyperosmotic environment. We isolated the transcription factor Cmr1, an ortholog of Colletotrichum lagenarium Cmr1 and Magnaporthe grisea Pig1, which regulates melanin biosynthesis in C. heterostrophus. Deletion of CMR1 in C. heterostrophus resulted in mutants that lacked dark pigmentation, and acquired an orange-pink color. In cmr1-deletion strains the expression of putative scytalone dehydratase (SCD1) and hydroxynaphthalene reductase (BRN1 and BRN2) genes involved in melanin biosynthesis was undetectable, whereas expression of PKS18, encoding a polyketide synthase, was only moderately reduced. In chk1 and mps1 mutants expression of PKS18, SCD1, BRN1, BRN2, and the transcription factor CMR1 itself was very low in young colonies, slightly up-regulated in ageing colonies, and significantly induced in hyperosmotic conditions, as compared to invariable high expression in wild type. These findings indicate that two MAP kinases, Chk1 and Mps1, affect Cmr1 at the transcriptional level, and this influence is partially overridden in stress conditions including ageing culture and hyperosmotic environment. Surprisingly, we found that the CMR1 gene was transcribed in both sense and antisense directions, apparently producing mRNA as well as a long noncoding RNA transcript. Expression of the antisense CMR1 was also Chk1 and Mps1-dependent. Analysis of chromosomal location of the melanin biosynthesis genes in C. heterostrophus resulted in identification of a small gene cluster comprising BRN1, CMR1 and PKS18. Since expression of all three genes depends on Chk1 and Mps1 MAPKs, we suggest their possible epigenetic regulation.


307. The NADH oxidase nadA and its involvement in oxidative stress in Aspergillus flavus. Carrie A. Smith1, Massimo Reverberi2, Niki Robertson1, Gary A. Payne1. 1North Carolina State University, Raleigh, NC, USA, 27606. 2Università "La Sapienza," Rome, Italy 00165.


nadA, which encodes a predicted NADH oxidase, was identified as part of a sugar utilization cluster that lies adjacent to the aflatoxin biosynthetic cluster in several species of Aspergillus. NADH oxidases convert NADH to NAD+, which is a possible coenzyme needed for reactions in the aflatoxin biochemical pathway. In a microarray experiment comparing gene expression between a wild type strain of A. parasiticus and a deletion mutant for the pathway regulatory gene aflR, nadA expression was significantly decreased in the mutant background. Although nadA is transcriptionally controlled by AflR, aflatoxin levels were unaffected in A. flavus nadA deletion strains under several conditions. NADH oxidases can also be a source of reactive oxygen species formation. Previous reports have shown a relationship between oxidative stress and aflatoxin production in Aspergillus sp. The activity of several antioxidant enzymes were examined in nadA deletion strains and peak activity was delayed when compared to wild type. Lower levels of lipoperoxide accumulation were also seen in nadA deletion strains. These data suggest that nadA plays a role in the oxidative stress response in A. flavus. Investigations are underway to characterize additional phenotypes of nadA deletion mutants.


308. Withdrawn


309. Transcriptional activators of Aspergillus nidulans sulfur metabolism. Sebastian Pilsyk, Jerzy Brzywczy and Andrzej Paszewski. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawiñskiego 5A, 02-106 Warsaw, Poland e-mail: Seba@ibb.waw.pl


Aspergillus nidulans metR gene encodes a bZIP transcription factor specific for activation of several sulfur metabolism genes including these encoding the sulfate assimilation pathway enzymes. METR protein level and/or activity is negatively regulated by SCF ubiquitin ligase complex which consists of the proteins encoded by the scon (sulfur controller) genes. Both metR and scon genes constitute a regulatory system known as sulfur metabolite repression (SMR). Loss of function mutations in the metR gene cause methionine auxotrophy while mutations in the scon genes lead to a derepression of the sulfate assimilation pathway enzymes. All metR and scon mutants isolated so far were recessive. Recently, we have isolated three dominant suppressor mutants in a diploid strain homozygotic for metB3 allele. Genetic and molecular analysis showed that Phe48 codon of the metR gene is affected in every of the dominant mutants obtained (MetR20, 21 and 22). The orthologs of the metR gene were also found in other fungal species and Phe48 appears to be one of the most conserved amino acids of the N-terminal domain which suggests that this residue is important for METR protein function and/or stability. MetR20 mutant has elevated acivity of the sulfur metabolism enzymes in comparison to the wild type. Suppressor metR mutants have also an increased level of sulfur compounds in mycelia which suggests that they are resistant to SMR. By searching of the A. nidulans genome sequence, we have found a paralog of the METR protein that encoded protein has similar basic region and leucine zipper sequences. The gene we named metZ is well conserved among other Aspergilli and it has apparently unusual large conserved intron as long as intron in the metR gene.

 

310. The role of MeaB, NMR and AreB as antagonists of AreA in Fusarium fujikuroi. Birgit Schönig, Anne Schmeinck, and Bettina Tudzynski. Universität Münster, Institut für Botanik, Schlossgarten 3, D- 48149 Münster, Germany


In Fusarium fujikuroi, the biosynthesis of gibberellins (GAs) and bikaverin, both nitrogen-free metabolites, is under control of AreA-mediated nitrogen metabolite repression. Glutamine and ammonium strictly repress the expression of AreA target genes. This strong repression can only partially be overruled by rapamycin-mediated inhibition of TOR suggesting that the activity of AreA is controlled by additional negative regulators. In contrast to Aspergillus nidulans, NMR was shown to play only a minor role as inhibitor of AreA activity. In addition to NMR, the bZIP transcription factor MeaB also negatively affects the expression of GA-and bikaverin biosynthesis genes as well as other AreA target genes. In the meaB deletion strain, these genes are up-regulated in a rapamycin-dependent manner. Interestingly, in double areA/meaB mutants, the de-repressing effect of the meaB deletion overrules the down-regulation of the AreA target genes in the single areA mutant. Target genes of AreA and MeaB were identified by an array-based approach. Furthermore, beside AreA, a second GATA-type transcription factor, AreB, is involved in nitrogen regulation. We present a putative model of nitrogen regulation in F. fujikuroi .


311. Identification of genes up- and down-regulated in the nuc-2A mutant strain of Neurospora crassa. Gras, DE1, Silveira, HCS1, Martinez-Rossi, NM1 and Rossi, A2. 1Departamento de Genética, FMRP-USP 2Departamento de Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto, Brazil.


Microorganisms have evolved complex signal transduction networks that enable them to make optimal use of the nutrient sources available. Inorganic phosphate (Pi), an essential nutrient for all organisms, is required for the biosynthesis of nucleic acids, phospholipids and cellular metabolites, as well as for the energy transduction and metabolic signaling responses. The phosphorus acquisition system in Neurospora crassa includes four regulatory genes: nuc- 2+, preg+, pgov+, and nuc-1+. Under limiting Pi, NUC-2 inhibits the functioning of the complex PREG-PGOV through the interaction of ankyrin-like repeats, allowing the activation of the wide domain transcription regulator NUC-1. In an attempt to identify genes involved in metabolic responses to exogenous Pi sensing, we employed suppression subtractive hybridizations (SSH) between RNA isolated from the wild type (St.L.74A) and nuc-2A (FGSC#1996) grown under Pi starvation, pH 5.4. Following SSH, expression of clones was examined using dot-blot macro-arrays. Of the 900 clones arrayed from the SSH cDNA library, approximately 21% were differentially regulated. A total of 66 differentially up-regulated and 124 down- regulated clones were identified and sequenced. Genes encoding proteins involved in signal transduction and regulation were identified among the up-regulated sequences, including translation initiation factor eIF3, ubiquitin and kinases. Among the genes found to be down regulated in nuc-2A, six were involved in gluconeogenesis and oxidative phosphorylation. Virtual and Northern blot analyses of randomly selected genes confirmed the differential gene expression result. Thus, the identification of differentially-regulated genes in nuc-2A is key to the understanding of molecular events involved in phosphorus sensing.


312. Involvement of Ace2 (Activator of Cellulases 2) in the architecture of the xyn2 transcriptosome of Hypocrea jecorina. Astrid R. Stricker*, Peter Trefflinger, Nina Aro, Merja Penttilä, Robert L. Mach *e-mail:stricker@mail.zserv.tuwien.ac.at


Ace2 (Activator of Cellulases 2)-encoding gene was deleted and retransformed in the H. jecorina QM9414 genome. Comparison of xylanase formation and xyn2 transcription of the corresponding strains after cultivation in a batch fermentation or replacement to inducing compounds (xylan, xylobiose) revealed the deletion of ace2 to be responsible for a faster initial inducibility, but final levels of xyn2 transcript as well as xylanase activity formation of the parental strain cannot be reached. Ace2 is not only involved in induction mechanisms but it is also responsible for basal level of xyn2 transcription. Interestingly, Ace2 does not mediate sophorose-specific induction although it plays a role in protein/DNA complex formation under sophorose-inducing conditions. Furthermore, in the present study, identification of a palindrome in the xyn2 promoter consisting of a GGGTAA- and a CCAGCC-element (closely resembling the binding site of Xyr1) is given. Both Xyr1 as well as Ace2 are able to bind the complete motif, the latter also only to one part of it. Phosphorylation as well as dimerization are shown to be a prerequisite for binding of Ace2 to the xyn2 promoter. Finally impact of Ace2 on xyr1 transcription could be demonstrated under inducing conditions. Summarizing, Ace2 can be considered as a factor modulating the overall activation influence of Xyr1 concerning xyn2 expression in the style of Ace1/Xyr1 interaction. This may involve an interplay of both factors on protein/DNA basis as well as direct modulation of xyr1 transcript formation.


313. Differential regulation of xylanase gene expression in Hypocrea jecorina is due to the interplay of the general activator Xyr1 and the fine tuning transcription factors Ace1 and Ace2. Robert L. Mach*, Astrid R. Stricker, Roman Rauscher, Elisabeth Würleitner, Peter Trefflinger * rmach@mail.zserv.tuwien.ac.at


Enzymes of H. jecorina (anamorph Trichoderma reesei) capable of degrading the xylan backbone (encoded by the genes xyn1 and xyn2) have received strong attention because of their application in feed and paper industry. Whereas in Aspergillus the xylanolytic system is strictly co-regulated via the inducer xylose enzymes participating in the respective T. reesei hydrolytic complexes are not. In Trichoderma these more complex induction mechanisms are conferred by modulation of the general regulator of hydrolase formation Xyr1 in its mode of action by additional narrow domain transcription factors such as Ace1 and Ace2. In the case of Ace1/Xyr1-driven xyn1 expression we will shown that Ace1 is a competitor of Xyr1 for only one of the two binding elements. Ace2 is involved in several aspects of xyn2 expression if the inducer is xylobiose or the fungus is cultivated on xylan. Interestingly, Ace2 is not only responsible for the basal transcription of xyn2 but also is important for particular steps of induction. Obtained data indicate that Ace2 acts in a dual role, on the one hand as an antagonist of early induction and on the other hand as an enhancer of a continuous extension of expression of xyn2. Our study revealed a spectrum of different mechanisms modifying the Xyr1-dependent transcriptosomes of xyn1 and xyn2 respectively, including phosphorylation, competition, homo- and heterodimerisation and recruiting of additional regulatory proteins.


314. Expression and transposition of DNA transposon Crawler in Aspergillus oryzae. Hironobu Ogasawara1, Hiroshi Obata2, Yoji Hata2, Saori Takahashi1, and Katsuya Gomi3. 1Akita Research Institute for Food and Brewing, Akita, Japan. 2Research Institute, Gekkeikan Sake Co. Ltd., Kyoto, Japan. 3Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.


An active DNA transposon Crawler has been isolated and characterized from the industrially important fungus Aspergillus oryzae. The transposition events of Crawler were induced by various stress treatments such as CuSO4 or heat shock. The existence of two or more transcripts in different size of Crawler was shown under standard culture conditions. In the present study, we analyzed the transcripts of different size by 3'-RACE analysis. Moreover, relationship between the transposition activity and the proportions of Crawler mRNA molecules was also studied to clarify the control mechanism for transcription of exogenous gene Crawler. The smaller transcribed fragments were resulted from premature polyadenylation and in some cases erroneous intron splicing within the transposase. The erroneous splicing tends to be inhibited by stress treatment of CuSO4, which stimulated the transposition events in conidia allowing the full-length and active transposase to be produced. These results indicate that A. oryzae has a defense system against the exogenous active genes like transposons by mRNA quality control system such as undesirable splicing or polyadenylation resulted in nonstop mRNA decay.


315. Transcription independent chromatin remodelling in Aspergillus nidulans. Harald Berger, Asjad Basheer, Sandra Böck and Joseph Strauss. Fungal Genomics Unit, Austrian Research Centers and BOKU Vienna, Austria.


In Aspergillus nidulans the induction of nitrate assimilating genes (niiA, niaD) is associated with chromatin remodelling processes which depend on the function of a GATA-factor (AreA) and a synergistically acting activator (NirA). Functional dissection of the process revealed that, in addition to nucleosome loss-of-positioning, AreA functions as independent transcriptional activator and also interacts with NirA to stimulate its DNA-binding capacity. Here, we analyzed NirA and AreA-dependent histone acetylation and nucleosomal DNA accessibility at different time points of nitrogen starvation and nitrate induction in the promoter region of niiA and niaD. We find that accumulation of AreA under starvation conditions leads to increased histone H3 acetylation and loss of nucleosome positioning and that this processes are abolished in an areA- strain. High H3 acetylation levels are associated with loss of nucleosome positioning but not with early transcriptional activation. In contrast, transcriptional activation which is strictly dependent on NirA, does not lead to H3 hyperacetylation and only partial nucleosomal loss of positioning is observed under these conditions. Our results indicate that transcriptional activation is not necessarily associated with a drastic chromatin rearrangement in the promoter but AreA-mediated hyperacetylation of H3 is required for maintaining an open chromatin structure facilitating continued transcription from this promoter.


316. Identification of genes up regulated in the palA1 mutant strain of Aspergillus nidulans. Emiliana M. Silva1, Janaina S. Freitas1, Diana Ester Gras2, Juliana Leal2, Fabio M. Squina1, Henrique S.C. Silveira2, Nilce M. Martinez Rossi2, Antonio Rossi Filho1. 1Departamento de Bioquímica e Imunologia, FMRP-USP, Brazil 2Departamento de Genética, FMRP-USP, Brazil e-mail: mimandarano2002@yahoo.com.br


The conserved PacC signal transduction pathway mediates many metabolic events involved in ambient pH sensing in Aspergillus nidulans, and it is widely accepted that it governs the response to neutral-to-alkaline pH. The pacC gene codes for a Zn-finger transcription factor, and the six pal genes (palA, B, C, F, H, and I) are putative members of a signaling cascade, which promotes the proteolytic activation of PacC in alkaline environment. The PalA protein interacts with the YPXL/I motifs in PacC, what is required for the action of PalB, a calpain-like protease. Thus, whatever the growth pH is, palA1 mutation should cause an acidity-mimicking phenotype. This model implies that loss-of-function mutation in any of the six pal genes shall lead to a wild-type acidic growth phenotype regardless on the ambient pH, what was not observed in the palA1 mutant strain. Employing suppression subtractive hybridization, we identified genes up-regulated in the palA1 mutant strain of A. nidulans grown in low-Pi medium, pH 5.0, whose expression should occur only at alkaline pH, as is the case for the expression of the phospholipase C gene. This result shows a derangement in the gene expression profile at pH 5.0, indicating that the palA gene is functional at acidic pH and, therefore, the palA1 mutation did not cause an acidic-mimicking phenotype. Financial support: FAPESP, CAPES, CNPq and FAEPA.


317. Regulated transcript stability in Aspergillus nidulans. Mark X Caddick, Sunthorn Chooluck, Meriel Jones and Igor Morozov. School of Biological Sciences, University of Liverpool, UK. caddick@liv.ac.uk


The level of any transcript is defined by the combined rates of transcription and degradation, both of which are regulated. Nitrogen metabolism in Aspergillus nidulans involves coordinated expression of hundreds of genes, many dependent on the transcription factor AreA, which monitors the nitrogen state of the cell. AreA activity is in part modulated by differential degradation of its transcript in response to intracellular glutamine. We have also determined that glutamine triggers synchronised degradation of a large subset of transcripts involved in nitrogen metabolism. Significantly, we show that two of these transcripts, niaD and niiA, are also stabilised by intracellular nitrate, directly reinforcing their transcriptional regulation. Gln-mediated degradation and the nitrate-dependent stabilisation of the niaD transcript are effected at the level of poly(A) deadenylation and are dependent on the 3’ UTR. To investigate the mechanisms involved in regulated deadenylation we have systematically disrupted a series of genes identified by homology. These include genes encoding three putative deadenylases and five PUF proteins. We have identified and characterised additional components by MS/MS sequencing of proteins specifically associated with the areA 3’ UTR. We will present our current model for the regulated degradation of transcripts in response to nitrogen availability.


318. Identification of genes down regulated in the pregc mutant strain of N. crassa. Fabio M. Squina1, Juliana Leal2, Nilce M. Martinez-Rossi2 and Antonio Rossi1. 1Departamento de Bioquímica e Imunologia and 2Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Brazil.

N. crassa synthesizes nucleases, phosphatases, and Pi transporters in response to signals of Pi starvation, and their regulation includes at least four genes involved in a hierarchical relationship: nuc-2, preg, pgov and nuc- 1. The PREG/PGOV proteins form a cyclin-CDK complex that regulates negatively the translocation of the transcription factor NUC-1 into the nucleus. Nevertheless, cyclins seem to have important participation in other metabolic pathways involved in cell cycle functions by adding specificity to the cyclin-CDK complex. Thus, loss-of- function mutations in the preg gene may alter the expression of other genes rather than those for the Pi- repressible phosphatases. In the present study we showed, by employing differential display reverse transcription polymerase chain reaction, that the expression of the genes for iso-orotate decarboxylase (IDCase) and ubiquinone metiltransferase is reduced in the pregc mutant strain grown under Pi shortage. The IDCase is a step of the thymidine salvage pathway, which reuses pyrimidines to form nucleotides more advantageously than de novo biosynthesis, and also contributes to maintain the balance in the nucleotide metabolism. It is expected that the pathways for the biosynthesis, catabolism and interconversion of critically important molecules, the nucleotides for instance, are highly integrated and highly regulated. Thus, disruption of the UMP/UDP pool balance by lowering the expression of an enzyme of the thymidine salvage pathway would have a variety of physiological effects in the cell’s life, including both growth and conidiation of the mutant strain. Financial support: FAPESP, CNPq, CAPES and FAEPA


319. Regulation by Blue Light and Heat Shock of Gene Transcription in Phycomyces blakesleeanus: Proteins Required for Photoinduction and Identification of a Novel Mechanism for Adaptation to Light. Julio L. Rodriguez-Romero and Luis M. Corrochano. Departamento de Genetica, Universidad de Sevilla, Spain


The gene hspA for the heat-shock protein HSP100 is induced by blue light and heat shock in the zygomycete fungus Phycomyces. HSP100 proteins are ATPases involved in the tolerance to high temperatures, proteolysis, and protein disaggregation. We have identified the gene products required for hspA photoactivation and found that many of them are also required for mycelial photoresponses, a suggestion for a common signal transduction pathway. The photoactivation of hspA requires the protein MADA, with a Zn finger and a flavin-binding site, the madB gene product, which is also required for all the photoresponses, and a regular supply of beta-carotene. None of the madA alleles that result in mutations in the flavin-binding site or that lacks the Zn finger allow hspA photoactivation suggesting that MADA is a photoreceptor and a transcription factor responsible for gene photoactivation probably through the formation of a complex with a protein similar to Neurospora WC-2. The activation of hspA after blue light-exposure or a heat shock is transient. The adaptation of hspA photoactivation is not the result of photoreceptor desensitization, but the result of a novel mechanism causing a light- dependent blockage of gene transcription. The light-dependent blockage of gene transcription does not prevent the activation of hspA by a heat shock and may be caused by the depletion of the MADA transcription factor from the hspA promoter. Our results have allowed us to propose a model for the regulation of hspA transcription by blue light and heat shock.


320. Mutations in a Gene Repressor or a Glucose Transporter Result in Sustained Gene Photoactivation in Neurospora. Maria Olmedo, Laura Navarro-Sampedro and Luis M. Corrochano. Departamento de Genetica, Universidad de Sevilla, Spain mariaolmedo@us.es


The gene con-10 of Neurospora is expressed during conidiation and after illumination of vegetative mycelia. Photoactivation of con-10 is transient and disappears after two hours of light. The rco mutants were isolated by the abundant expression of con-10 in vegetative mycelia. The gene rco-1, encoding a putative gene repressor, and the gene rco-3, encoding a putative glucose sensor, are required for the repression of con- 10 in vegetative mycelia. We have observed that rco-1 and rco-3 mutants have an enhanced and sustained photoactivation of con-10 and con-6, a phenotype they share with vivid mutants. The abundant photactivation of con-10 and con-6 in rco and vivid strains is best observed after five hours of light. The rco and vivid mutations do not alter the stability of the con-10 and con-6 mRNAs, suggesting that the sustained photoactivation is due to a high transcriptional rate that is not subjected to adaptation to light. The threshold of con-10 and con-6 photoactivation is significantly lowered in the rco-1 mutant, but remains unchanged in the rco-3 and vivid mutants. The photophosphorylation of WC-1 in the rco-1, and rco-3 strains seem to be similar to that of the wild type in contrast to the mutation in vivid that results in a sustained WC-1 photophosphorylation. The rco-1 mutant contains a point mutation at nt 1986 resulting in a change of Ser by Phe in the conserved WD domain. We propose that VVD, RCO-1, and RCO-3 seem to participate directly or indirectly in the mechanism of adaptation to light.


321. A genome approach to understanding nonself recognition, heterokaryon incompatibility and programmed cell death. Elizabeth A. Hutchison, Karine Dementhon, and N. Louise Glass. Plant and Microbial Biology department, UC Berkeley, California, 94720


Neurospora crassa can undergo hyphal fusion during vegetative growth to form a heterokaryon. Heterokaryons are only viable between genetically identical individuals; differences at any of 11 heterokaryon (het) loci result in growth inhibition and death. het-c/pin-c mediated nonself recognition is a model to study heterokaryon incompatibility (HI) and the cell death response. The het-c locus encodes a plasma membrane protein while pin-c encodes a HET domain protein. The HET domain is a conserved protein motif that is present in almost all characterized HI interactions in N. crassa and Podospora anserina. Mutations at the vib-1 locus, which encodes a putative transcription factor, suppress HI. Based on Q-RT-PCR data, vib-1 was required for the expression of 3 of the 55 HET domain genes, including pin-c, tol and het-6. Comparative genome hybridization was used to identify additional polymorphic HET domain genes; 6 of 8 additional HET domain genes showed significantly decreased expression in the absence of vib-1. vib-1 is also required for protease production during nitrogen and carbon starvation. Mutants in two vib-1 paralogs, NCU04729 and NCU09915, will be examined for protease production deficiencies during nutrient starvation. Finally, the effects of both the vib-1 regulated HET domain genes and the vib-1 paralogs on heterokaryon incompatibility will be investigated.


322. mat1-2 gene and cryptic fertility in Aspergillus fumigatus. Wioletta Pyrzak, Bruce L. Miller, Karen Miller. Department of Microbiology Molecular Biology and Biochemistry, University of Idaho, Moscow, ID, U.S.A


The lack of sexual cycle in Aspergillus fumigatus (Af) is a major limitation in the study of its pathogenesis. Recent genomic studies revealed evidence for potential sexuality in A. fumigatus. The mat1-2 gene encodes a homologue of the MatA HMG-box mating transcriptional factor that regulates sexual development in fertile Aspergillus nidulans (An). A non functional mat1-2 gene or HMG-box protein could be one reason for loss of sexuality in A. fumigatus. Gene complementation and ORF swap experiments were performed to determine a potential functionality of Af mat1-2 gene in the control of sexual cycle. Results showed that Af mat1-2 did not complement an A. nidulans matA deletion strain. No sexual cycle was observed. An ORF swap gene replacement demonstrated that Mat1-2 protein functionally replaced A. nidulans MatA protein during sexual development. Sexual development was delayed 1-2 days and cleistothecia were 20 x larger relative to wild type. Normal number and viability of ascospores per cleistothecium were observed. Incomplete asci formation and ascospores maturation were observed after 12 days of development. This data suggest that A. fumigatus Mat1-2 protein is functional and can drive sexual reproduction in A. nidulans. Results presented in this study provide evidence for potential cryptic sexual activity in Aspergillus fumigatus.


323. Identification of carbonic anhydrases in Aspergillus fumigatus and A. nidulans. Kap-Hoon Han1, Hyoun-Young Kim1, Jong Hwa Kim1, and Yong-Sun Bahn2. 1Dept. of Pharmaceutical Engineering, Woosuk University, Wanju, 565-701, Korea. 2Dept. of Bioinformatics & Life Science, Soongsil University, Seoul, 156-743, Korea.


Like vertebrates, carbon dioxide (CO2) plays an important role in respiration of various microorganisms including fungi. Furthermore, differential CO2 concentration can act as a signaling cue. Since mammals maintain high CO2 concentration (~5%) compared to natural environment (~0.036%), animal fungal pathogens must adapt to high CO2 levels for their infection. Although CO2 sensing and metabolism in filamentous fungi have been poorly understood, it has been well known that the reversible interconversion between gaseous CO2 and bicarbonate ions (HCO3-) by carbonic anhydrase (CA) is key for maintaining cellular homeostasis. Since Aspergillus fumigatus is a major fungal pathogen causing invasive aspergillosis in immunocompromised patients, adaptation of different CO2 concentration, probably mediated by CAs, could be important. Genome sequence of A. fumigatus and A. nidulans allowed us to identify three conserved CAs in each organism that are designated as cafA (CA in A. fumigatus), cafB and cafC, and canA (CA in A. nidulans), canB and canC, respectively. Interestingly, plate sealing caused upregulation of cafA and canA expression, suggesting that the limited aeration leads to the shortage of CO2 which promotes upregulation of the CA expression.


324. Cwc1 and Cwc2 are central regulators of blue light photoresponses in Cryptococcus neoformans. Yu-Ling Yeh1, Yu-Sheng Lin, Bei-Jia Su, and Wei-Chiang Shen2. Plant Pathology & Microbiology, National Taiwan Univ. 1 r94633012@ntu.edu.tw ; 2 wcshen@ntu.edu.tw


Light is one of the most important environmental factors regulating developmental and physiological processes in filamentous fungi. Our prior studies have demonstrated that blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in Cryptococcus neoformans, a heterothallic basidiomycetous yeast. To reveal their roles in the regulation of light responses, we generated both CWC2 overexpression strain under the cwc1 mutant background and CWC1 overexpression strain under the cwc2 mutant background. The mating phenotypes of these strains were similar to those of the cwc1 and cwc2 mutant strains. These results suggested that Cwc1 and Cwc2 proteins function interdependently to regulate the light response. Furthermore, to reveal the importance of putative domains identified in the Cwc1 and Cwc2 proteins, we generated partially deleted versions of both proteins under the constitutively expressed GPD1 promoter. Our results confirmed that LOV domain and PAS domains are essential for the function of Cwc1 protein, and PAS domain and zinc finger DNA-binding domain are also crucial for the Cwc2 protein. Additionally, to dissect the photoresponses mediated by Cwc proteins, we used Agrobacterium T-DNA insertional mutagenesis as an approach. A mutant suppressed the CWC1 overexpression phenotype under light condition was identified, and T-DNA was found to integrate at the promoter region of the CWC2 gene. Our results demonstrated that Cwc1 and Cwc2 proteins are two central regulators which form complex to regulate blue light photoresponses in C. neoformans.


325. Isolation and functional analysis of the veA interacting vip genes of Aspergillus nidulans. Hyoun-Young Kim1, Hee-Seo Kim2, Miae Oh2, Kap-Hoon Han1, Keon-Sang Chae2, Jong Hwa Kim1. 1Dept. of Pharmaceutical Engineering, Woosuk University, Jeonbuk 565-701, 2Div. of Life Science, Chonbuk Nat'l University, Jeonbuk, 561-756, Korea.


The vipA, B, and C (VeA-interacting protein) genes were isolated by using yeast two hybrid analysis. The vipA transcript size was 1.3 kb and had an ORF encoding a 334 amino acid polypeptide. Putative vipA protein contains FAR1 domain related to plant-specific phytochrome signaling, suggesting that the VipA may act as a component of far-red light signaling with VeA in A. nidulans. However, vipA-null mutants and over- expressors did not show any remarkable phenotypic difference from a wild type. The vipB and vipC genes were also isolated. The vipB gene had an ORF encoding 332 amino acid polypeptide. The amino acid sequence of VipB showed a 38% identity to that of a methyltransferase. The vipB gene dose not seem to be involved in both of sexual development and asexual development, since the vipB deletion and overexpressoion mutants did not show any phenotypic difference from their control. The vipC negatively regulates sexual development in the presence of light, but not in dark condition. For obtaining further information of the genes, microarray analysis was carried out with RNA samples isolated from mycelia at the vegetative stage of three vip-deletion mutants, and their results were compared.


326. Gene expression analysis under membrane-transfer culture. Motoaki Sano1, Akiko Kobayashi1, Hiromoto Hisada2, Yoji Hata2, Masayuki Machida3, Shinichi Ohashi1. 1Kanazawa Institute of Technology (KIT), Hakusan, Japan 2Res. Inst., Gekkeikan, Kyoto, Japan 3Natl. Inst. of Advanced Ind. Sci. and Technol. (AIST), Tsukuba, Japan

Aspergillus oryzae is one of the most useful microorganisms in many fields of biotechnology; has been used to produce fermented foods such as soy sauce, miso, and sake. In A. oryzae, the gene expression patterns change depending on the culture conditions, and a lot of important enzymes are expressed in solid-culture (SC), but not in submerged culture. However, SC has several disadvantages, such as mass transfer limitation inside the solid substrate and difficulty of control of substrate concentration as well as pH of the medium during cultivation. To overcome these disadvantages of SC, membrane-transfer culture (MTC) was developed. In MTC, A. oryzae are grown onto the surface of the nitrocellulose membrane. The gene expression patterns in MTC were analyzed by using DNA microarray consisting of 11,000 oligonucleotides. The DNA microarray analysis showed that there is global resemblance between MTC and SC in the gene expression. As for a part of SC-strong expression gene, it was confirmed to strongly express in MTC. Some genes which as specifically expressed in MTC were identified, and the promoter analysis of these genes were done.


327. The role of XprG in the response to nutrient limitation in A. nidulans. Margaret E. Katz1, Katharyn S. Sue1, Richard B. Todd2 and Brian F. Cheetham1. 1University of New England, Armidale, NSW, Australia and 2University of Melbourne, VIC, Australia. mkatz@une.edu.au.


We have identified three genes involved in the regulation of extracellular proteases in response to carbon, nitrogen and sulfur limitation. Two of the genes encode atypical hexokinases, HxkC and HxkD (formerly XprF), and the third gene product, XprG, belongs to a newly defined class of DNA-binding proteins. Vib-1, a regulator of genes required for programmed cell death in N. crassa is a homolog of XprG. Genetic evidence indicates that HxkC and HxkD are negative regulators of XprG. We have recently found that unlike HxkD, which is a nuclear protein, HxkC is associated with mitochondria. Mitochondrial hexokinases have been shown to block apoptosis in mammals and programmed cell death in plants. HxkC may play a similar role in filamentous fungi. xprG- mutants are protease- deficient and have pale conidia suggesting the XprG is involved in regulating extracellular protease and conidial pigment production. To investigate whether XprG is required for expression of other genes in response to carbon starvation, we used microarrays provided by the PFGRC. These experiments indicate that expression of many genes is greatly reduced in an xprG- null mutant. Many of the genes with altered expression (e.g. brlA, genes encoding sugar transporters and genes in the sterigmatocystin biosynthetic pathway) are known to be regulated in response to carbon starvation. These results indicate that XprG may play a major role in the response to carbon limitation. Nuclear accumulation of the nitrogen regulator, AreA, is abolished in an xprG- mutant.


328. Transcriptional Control and Protein Specialization have Roles in the Functional Diversification of Two Dicer-like Proteins in Magnaporthe oryzae. Naoki Kadotani, Hitoshi Nakayashiki. Lab of Plant Pathology Kobe University, Kobe Japan


Two Dicer-like genes, Mdl-1 and -2, were identified in the genome of the rice blast fungus Magnaporthe oryzae. Molecular phylogenetic analyses indicated that the paralogs of the Mdl-1 and -2 genes diversified prior to the divergence of the major fungal lineages. Analyses of Mdl-1 and Mdl-2 knockout (KO) mutants revealed that Mdl-2 was solely responsible for the siRNA-mediated gene silencing pathway in the vegetative phase of the fungus where Mdl-2 mRNA was expressed 15 times more highly than was Mdl-1 mRNA. However, overexpression of the full-length Mdl-1 cDNA compensated, albeit partially, for the RNA silencing- deficient phenotype of the Mdl2 KO mutant. The levels of siRNA accumulations in the transformants with Mdl-1 cDNA were significantly lower than in those with Mdl-2 cDNA, even when they were overexpressed at similar levels. Our results indicated that the functional diversification of Mdl-1 and Mdl-2 proteins in RNA-mediated gene silencing pathways was likely to have come about from both transcriptional control and protein specialization.


329. MINUTE, the unusual a-type pheromone of Hypocrea jecorina (Trichoderma reesei) mediates light regulation of cellulase gene expression. M. Schmoll, M. Dorrer, and C. P. Kubicek. Institute of Chemical Engineering, TU Wien, Getreidemarkt 9-1665, A-1060 Vienna, Austria


Light has recently been shown to enhance the induced expression of cellulases in H. jecorina. In a cDNA subtraction assay targeted at genes specific for cellulase induction, we isolated minute (min1) encoding a small (5.1 kDa), alkaline (pI 9.6) α/β-protein, which is composed of three ß-sheets containing the motif (L/I)GC(S/T)VM and two helices. Thus, together with its CAAX-domain at the C-terminus MIN1 shares characteristics of both a- and alpha-type peptide pheromone precursors. It inhibits growth in H. jecorina and enhances perithecia formation H. jecorina CBS999.97. Transcription of min1 is up-regulated upon growth on cellulose in the wild-type strain QM9414 in light. Deletion of min1 results in loss of light-dependent modulation of cellulase gene expression on cellulose. Minute shows light dependent transcription, which is strongly enhanced in a strain in which the H. jecorina orthologue of the N. crassa light modulator VIVID – ENVOY – is non-functional. EMSAs using the cbh2 promoter and cell-free extracts from mycelia grown in light or darkness revealed DNA-protein complexes from the min1-deletion strain do not show the difference between cultivation in light or darkness as found in the wild-type. Thus, the unusual peptide pheromone precursor MINUTE is an important factor in light regulation of cellulase gene expression and after ENVOY it represents the second component of the respective signal transduction pathway in H. jecorina.


330. Transcriptional profiling of Candida albicans late biofilm developmental stage associated with community mobilization. Adnane Sellam1, Peter Suci2, Thamir Al Niemi2, Jean-Sébastein Deneault1 and André Nantel1. 1Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec, Canada 2Center for Biofilm Engineering, Montana State University-Bozeman, Montana

Candida albicans is a major cause of morbidity and mortality in bloodstream infections and is the most common fungal pathogen isolated from immuno-compromised patients. C. albicans is also implicated in many biomaterial- related infections which are typically associated with biofilm formation. One of the main consequences of the biofilm mode of growth is the increased resistance to antimicrobial therapy, which is the main reason why biofilm-associated infections are frequently refractory to conventional antibiotic therapy. Yeast cells of C. albicans have an important role in initiation of biofilm formation and its maintenance by anchoring its basal layer to the substratum. Recent studies suggest that yeast cells have also a key role in biofilm dispersal. These cells may detach from mature biofilm and colonize a new substrate area that is not nutrient deprived, where a new biofilm can develop unhindered. Until now nothing is known concerning the genetic program controlling biofilm cell mobilization. To characterize the detachment stage of yeast cells, we recovered mature biofilm and detached cells from a special flow- bioreactor and performed a detailed transcriptional analysis using a new generation of long oligonucleotide microarray. Four to six biological replicates from these populations were compared to each other and against yeast cells from regular batch cultures. For instance, we identified 249 significantly-modulated transcripts that best distinguish mature biofilm from yeast cells grown in batch culture. In this comparison, we did not identify significant changes in sulfur and amino acid metabolism as previously reported in other bioreactors (García-Sánchez et al. 2004) but we did observe a very significant correlation with the changes in transcriptional profiles that are associated with repression of the Polo-like kinase Cdc5p (Bachewich et al., 2005) suggesting that cell detachment is linked to mechanism regulating cell cycle in C. albicans. Additional results of these studies will be shown, discussed and progresses on functional characterization of candidate genes will also be presented.


331. Withdrawn


332. Effect of Copper on the Expression of the Mco1 Gene from Phanerochaete chrysosporium and its Connection with the ACE1 Transcription Factor. Canessa, Paulo; Alvarez, Jose Miguel; Rivas, Alexis; Bull, Paulina & Vicuna, Rafael. Universidad Catolica de Chile, Fac. de Ciencias Biologicas, Lab. de Bioquimica, Alameda 340, Santiago, Chile. pcanessa@bio.puc.cl


We have previously identified a gene encoding ACE1, the first transcription factor described and functionally characterized in the ligninolytic fungus Phanerochaete chrysosporium (Pc-ACE1). To date, the possible target gene(s) of ACE1 remain elusive. Since mco1 possesses at least two ACE elements in its promoter, we decided to analyze the effect of copper on the expression of this gene. Real-time RT-PCR experiments showed a 4 to 5-fold increase in the mco1 mRNA levels upon copper supplementation of cultures. To further characterize Pc-ACE1, the protein was synthesized in vitro using a commercially available transcription-translation system. By means of electromobility-shift assays (EMSA) and using a DNA probe containing a previously described ACE element from yeast, we showed specific binding of Pc-ACE1 to the probe. In addition, EMSA also revealed that Pc-ACE1 binds specifically to a probe containing one of the putative ACE elements found in the mco1 promoter. These results do not only show that Pc-ACE1 indeed recognizes a canonical ACE element, but also support the hypothesis that mco1 is regulated by the Pc-ACE1 transcription factor. We are now identifying the key nucleotide residues involved in this protein-DNA interaction by site-directed mutagenesis. Financed by grant FONDECYT 1030495 & Millennium Institute (MIFAB)


333. A novel mechanism of regulation of iron-dependent pathways in eukaryotes is mediated by HapX interacting with the CCAAT-binding complex. Martin Eisendle1, Birgit Seeber1, Ernst R. Werner2, and Hubertus Haas1 1Division of Molecular Biology/Biocenter, and 2Division of Biological Chemistry/Biocenter, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria; martin.eisendle@i-med.ac.at


Subtle control systems are required to maintain iron homeostasis as this metal is both essential and potentially toxic. In Aspergillus nidulans iron starvation induces iron acquisition but represses iron dependent pathways. Here we demonstrate that HapX mediates the transcriptional repression of iron-dependent pathways, e.g. heme biosynthesis, via physical interaction with the CCAAT-binding complex (CBC). The transcriptional regulation of iron acquisition, e.g. the siderophore system, is mediated by the GATA-type transcription factor SreA. Mutual transcriptional control of HapX and SreA and synthetic lethality of deletion of both regulators suggests a tight interplay of these two iron homeostatic control systems. Growth phenotypes indicate that the CBC has a general role independent of the iron status, whereas HapX function is confined to iron depletion. In agreement, deficiency in any of the CBC subunits has deleterious consequences not only during iron depleted conditions, as with HapX ablation, but also during iron-replete growth. Consistently, expression of HapX is repressed by iron whereas that of the CBC subunit HapC is constitutive. Moreover, deficiency in HapC but not HapX causes up-regulation of iron-dependent pathways also during iron-replete conditions.


334. G-protein alpha subunit Gpa3 regulates sexual differentiation in Cryptococcus neoformans. Kung-Hung Liu1 and Wei-Chiang Shen2. Department of Plant Pathology and Microbiology, National Taiwan University, Taiwan. 1gloclovlo@yahoo.com.tw 2wcshen@ntu.edu.tw


The conserved G-protein signaling molecules play pivotal roles in transducing the environmental stimuli to trigger appropriate cellular responses. Extensive studies demonstrated that diverse developmental processes and pathogenesis in fungi are intricately regulated by the activities of G-proteins. In Cryptococcus neoformans, an opportunistic human fungal pathogen, three G-protein alpha subunits were identified. Gpa1 was shown to act at the upstream of the cAMP response pathway to regulate sexual reproduction and virulence. Here, we generated and characterized two other G-protein alpha subunit mutants, gpa2 and gpa3 . In the gpa2 mutant, no discernible phenotype was observed under all conditions tested. In contrast, elevated level of sexual filamentation was observed in the gpa3 mutant, especially more pronounced in the monokaryotic fruiting process. Deletion of the heterotrimeric G-beta subunit GPB1 in the gpa3 mutant background blocks mating differentiation as the gpb1 mutant. Our results demonstrated that G-protein alpha subunit Gpa3 and beta subunit Gpb1 are integral components of a heterotrimeric G-protein complex which function upstream of the pheromone response pathway to regulate sexual differentiation in C. neoformans.


335. Establishing new vectors for transformation and gene-silencing in Botryis cinerea. Risha Patel, Gary Foster and Andy Bailey. University of Bristol, Biological Sciences, Bristol, UK.


Botrytis cinerea is a necrotrophic fungus capable of infecting over 300 plant species worldwide. Typically, Botrytis cinerea is referred to as grey mold rot or Botrytis blight. It normally attacks various damaged or cracked plant tissues including leaves, weeds, stems, and seeds. In general, B. cinerea becoming more resistant to current fungicides. As a result, agrochemical companies are seeking new targets capable of minimizing pathogenicity of B. cinerea. The use of gene silencing is a well-established technique in animal, plant and fungal systems. The benefits of down regulating genes are vast, including the potential to identify protein function of targeted genes. An effective gene silencing model must be demonstrated and developed before useful targets may be identified. In our initial investigations, we utilized the widely used vector pLOB1 and derivatives thereof to transform and trigger silencing of laccase and superoxide dismutase in Botrytis. Silencing was successful, with low to intermediate levels of gene silencing observed for both target transcripts, however we also identified unexpected reductions in virulence that did not correlate with the targeted transcript. Investigation shows that these were due to specific sequences within the pLOB1 plasmid and for this reason we strongly recommend that researchers move to a different construct for transformation-based experiments. New transformation and expression vectors have been constructed containing the A. nidulans oliC promoter and trpC terminator. These completely alleviate the difficulties observed with pLOB1. The new vector system displayed increased transformation rates and silencing efficiencies without the detrimental effects or reduction in pathogenicity observed with the pLOB1 system. We have strong evidence that gene silencing is possible within Botrytis cinerea, giving transformants displaying a range of silencing levels and this may be of great benefit when used to assess the amount of a particular gene product necessary for the infection process rather than the all-or-nothing studies achieved through targeted gene disruption.


336. The Fusarium oxysporum fost12 Gene Involved in Virulence Is Upregulated in planta. M. A. García-Sánchez, M. Brisa-Ramos, N. Martín-Rodrígues , J.J. De Vega-Bartol, A. P. Eslava and J. M. Díaz-Mínguez . Centro Hispano-Luso de Investigaciones Agrarias (CIALE) Universidad de Salamanca 37007, Salamanca, Spain Phone Fax: +34 923 294663 chonela@usal.es , josediaz@usal.es


Fusarium oxysporum is an ubiquitous plant pathogen with a broad host range; however the isolates are restricted in their host specificity and may be classified into formae speciales. This feature makes this plant pathogen an atractive model for the study of gene expression patterns involved in patogenicity and virulence. Several studies have demonstrated that the mitogen-activated protein (MAP) kinase signal transduction pathways play a rol in fungal pathogenesis. Previous results have shown that fost12 is involved in virulence. The quantification of fost12 transcript produced in planta by a highly virulent strain, as measured by Real Time PCR, showed little accumulation of fost12 transcript in root and a dramatic increase in stem during the early stages of plant infection. We have also investigated the influence of ambient pH and nutrient starvation in the expression of fost12 when the fungus grows in culture media. No clear correlation between pH and differences in the accumulation of fost12 transcript could be observed, thus indicating that changes in pH alone are not important in the regulation of the gene. On the contrary, starvation conditions (either the lack of a carbon or nitrogen source) affect the accumulation of fost12, as indicated by the Northen analysis results. Interestingly, the fost12 promoter shows motifs involved in the regulation by glucose or nitrogen, but no binding motif for the pH regulator PacC was found. We conclude that the upregulation of fost12 in planta seems to be induced by the scarce availability of nutrients, at least in the early stages of infection.


337. Regulation of Trichoderma virens MRSP1, encoding a novel secreted protein. Ella Pardovitz-Kedmi1, Ada Viterbo2, Yael Mandel-Gutfreund1,Ilan Chet2, Prasun K. Mukherjee3, and Benjamin A. Horwitz1. 1Biology, Technion, Haifa, Israel; 2Biochemistry, Weizmann Institute of Science, Rehovot, Israel, 3Nuclear Agriculture and Biotechnology Division, BARC, Mumbai, India.


MRSP1 (MAP kinase Repressed Secreted Protein 1) is highly overexpressed in the tmkA MAP kinase mutant of the biocontrol fungus Trichoderma virens [1]. Transcript levels of MRSP1 are determined by presence or absence of TmkA, rather than by light or sporulation, making it a molecular marker for the unusual, negative, regulation by TmkA. The predicted protein is 15.9 kDa, has a secretory signal, shows some homology to plant expansins, and contains a four-cysteine pattern C-X29-CP(G)C-X31-C which defines a new fungal cysteine-rich motif [1]. The tight regulation by TmkA, and presence of homologs in other fungal genomes, suggests that MRSP1 may have important functions, but the low expression level in wild type, in axenic culture, raises questions about when the protein acts. As an initial test of whether MRSP1 might be involved in interaction between T. virens and plant roots, we are following its expression during contact of the fungus with cucumber roots and penetration into cortical layers. [1] P.K. Mukherjee, R. Hadar, E. Pardovitz-Kedmi, N. Trushina and B.A. Horwitz, Biochem. Biophys. Res. Commun. 350(2006) 716–722.


338. Characterization of the fumonisin biosynthetic regulatory gene FUM21 and multiple alternative splice forms. Daren W. Brown, Robert A.E. Butchko, Mark Busman and Robert H. Proctor. Mycotoxin Research Unit, U.S. Department of Agriculture-ARS, Peoria, IL 61604. e-mail: browndw@ncaur.usda.gov

Fumonisins are a family of mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. The fumonisin biosynthetic gene cluster contains 17 genes of which one, FUM21, encodes narrow domain transcription factor required for fumonisin biosynthesis. The Fum21p is predicted to include a Zn(II)2Cys6 DNA binding domain and a second domain associated with fungal transcription factors. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs) and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis.


339. Disruption of TUP1 in H99 reveals its role in capsule formation and virulence in Cryptococcus neoformans. Hyeseung Lee, Yun C. Chang, and K.J. Kwon-Chung. LCID, NIAID, NIH, Bethesda, MD 20892, USA.


Cryptococcus neoformans, the etiologic agent of cryptococcosis is divided into two species C. neoformans (serotype A and D) and C. gattii (serotype B and C). Serotype A strains are world-wide in distribution while the other serotypes are geographically more restricted. Although H99 (serotype A) and JEC21(serotype D), the representative strains of serotype A and D, share 95% identity in genome sequence, various serotype-specific pathobiological differences have been observed. Previously, we showed that TUP1 disruption resulted in mating type-specific phenotypes in JEC21 derived strains. Subsequent study revealed a quorum sensing-like phenomenon in tup1Δ of both mating types in serotype D strains. In this study, we have deleted TUP1 from the H99 strain to study the scope of its regulatory role in a serotype A strain. Mating efficiency of H99 tup1Δ was severely reduced as seen in serotype D tup1Δ strains. Unlike serotype D strains, however, H99 tup1Δ showed no quorum sensing-like phenotype and showed growth retardation not limited to 25°C and 30°C. Interestingly, in contrast to the serotype D tup1Δ, H99 tup1Δ produced much larger capsule than the wild type regardless of media. Transcriptional profiling revealed that expression of many genes related to iron homeostasis was affected by TUP1 deletion in H99, possibly explaining the hypercapsular phenotype in H99 tup1Δ. In spite of the hypercapsule in vitro, H99 tup1Δ strain had attenuated virulence in mice compared to wild type strain. Capsule size of the yeast cells in brain smears from mice infected with wild type was similar to those infected with the deletant. Taken together, the global regulator TUP1 plays both conserved and distinct roles in serotype A and D strains.


340. Applying PARAFAC models to detect new regulatory mechanisms in Fusaria. Jens A. Andersson1, Claus A. Andersson2, Rasmus Frandsen1. 1Institute of Ecology, Genetics and Microbiology, University of Copenhagen 2Spectroscopy and Chemometrics group, University of Copenhagen


High performance liquid chromatography (HPLC) analysis offers an easy, cost-efficient and reliable method for detection of fungal metabolites. However, subsequent data-analysis is often time consuming, narrow and dependent on experienced personnel to recognise characteristic UV-spectra. Here a mathematically based method for detecting metabolites and classification of a number of Fusaria-strains belonging to the species F. graminearum, F. pseudograminearum and F. culmorum based on multiway analysis of HPLC-data is presented. By applying a PARAFAC-model in the analysis of 3-dimensional HPLC data it is possible to detect characteristic metabolite patterns within and between species. Characteristic metabolite profiles can serve as an efficient tool for chemotaxonomic classification of Fusaria species. Analysis of correlation between synthesized compounds within and between species of Fusaria may disclose new constituents in the regulatory networks that are known to control the synthesis of secondary metabolites. As expected, the models clearly indicate that the diversity of synthesized metabolites differ between strains, species and according to growth media composition. By linking fungal metabolite profiles with expression patterns of known polyketide synthase (PKS) and non ribosomal peptide synthase (NPS)-genes it is here postulated, that a 3-way analysis may serve as an efficient tool to link genotype to fenotype.


341. Functional characterization of a MAPK cascade in Neurospora crassa. G. Park and K.A. Borkovich. Department of Plant Pathology, University of California, Riverside, CA 92521


Mitogen-Activated Protein (MAP) kinase cascades are composed of MAP kinase kinase kinases (MAPKKK), MAP kinase kinases (MAPKK) and MAP kinases (MAPK). These systems are conserved in filamentous fungi. Three components of a MAPK cascade (mik-1, MAPKKK; mek-1, MAPKK; mak-1, MAPK), homologous to that controlling cell wall integrity in Saccharomyces cerevisiae were annotated in the Neurospora crassa genome sequence. Targeted gene replacement of these three kinases (mik-1, mek- 1, and mkc-1) was performed by the Neurospora genome project. In this study, we characterize the function of this MAPK cascade using these mutants. mik-1, mek-1, and mak-1 knockout mutants exhibited significant reduction in apical extension of basal hyphae on solid medium. All three mutants formed short aerial hyphae and arthroconidia formation was observed in all three mutants.. Macroconidiation was almost abolished in both mek-1 and mkc-1 knockout mutants, whereas the mik-1 knockout strain produced reduced levels of macroconidia in comparison to wild type. All three mutants were unable to form protoperithecia or perithecia when used as females in a sexual cross. We are currently analyzing levels of MAK-1 phosphorylation and effects on gene expression in the three mutants during growth and development in N. crassa.


342. Transcription Factors and Chromatin Remodeling in the Neurospora Clock. William Belden, Jennifer Loros, Jay Dunla.p Department of Genetics Dartmouth Medical School Hanover 03755, USA


Positive and negative transcriptional/translation feedback loops involving the frequency gene are central to circadian rhythms in Neurospora crassa. In this model, the transcription factors White Collar-1 (WC-1) and White Collar- 2 (WC-2) activate frq expression in a circadian and light dependent manner; they are found together and have been assumed to act exclusively as a heterodimeric complex. Surprisingly however, chromatin immunoprecipitations (ChIP) indicate that the WC proteins do not act solely as an obligate complex: in vivo binding of WC-2 to the frq promoter occurs in a rhythmic fashion with the peak binding occurring coincident with the peak in frq transcription, whereas WC-1 is bound continuously with only slight increases observed when frq is being transcribed. Nuclease accessibility experiments show chromatin rearrangement at the frq promoter when the gene is expressed. To identify the enzymes mediating the rearrangement, all 19 Neurospora genes with homology to the swi2/ snf2 family of ATP-dependent chromatin-remodeling enzymes were deleted by gene replacement. Examination of these strains identified a gene, designated clockswitch (csw-1), required for normal clock function. The data suggest a role where CSW-1 is required for disassembly of active WCC at frq and is needed for the sharp transition from the transcriptionally active phase to the repressed state.


343. Identification of genes regulated during asexual development of Neurospora crassa. C. Greenwald, T. Kasuga, L. Glass, B. D. Shaw, D. J. Ebbole and H. H. Wilkinson. Texas A&M University, Plant Pathology and Microbiology, College Station, TX and University of California, Berkeley.


We are interested in identifying gene networks associated with asexual development across the filamentous fungi. As a first step toward this goal we have used the full genome microarray available for Neurospora crassa to profile the mRNAs produced during 1) a developmental time course (0, 2, 4, 8, 10, 12, 14, 18, 24 hours), and 2) a comparison of mycelial vs. aerial tissue at 12 hours. Analysis of variance (ANOVA) was used to discern differences among time points. It yielded >3300 significantly regulated genes, representing a third of the >10,000 N. crassa genes on the microarray. Analysis to establish the major categories of expression patterns present among these significantly regulated genes is ongoing. For example, a set of genes known to be expressed during conidiation (con-10, con-6, eas) were regulated as expected (induced specifically in aerial tissue). A total of 91 genes with similarity (0.95 Pearson's coefficient) to at least one of several of these known conidiation-specific genes have been discovered with these experiments. Further experiments to evaluate the expression of these candidate genes in N. crassa developmental mutants (e.g. fl) are underway.


344. A gene with homology to the Cytochrome P450 conserved functional domain is down-regulated during sexual development in Schizophylum commune. Charles McEntee1, Lucas Bernacki1, and Kirk Bartholomew1*. 1Department of Biology, Sacred Heart University, Fairfield, CT USA; bartholomewk@sacredheart.edu *Corresponding Author


The well-characterized A and B mating-type loci of Schizophylum commune control activation of sexual development when haploid mycelia of non-self mating-type interact. The protein products of both the A and B loci are proposed to initiate the events of sexual development by controlling the expression of target genes in compatible mates eventually leading to the formation of the dikaryotic mycelia capable of progressing through sexual reproduction. A previous study using the reverse transcriptase differential display polymerase chain reaction method to isolate genes expressed differentially during the early stages of sexual development resulted in the cloning and sequencing of ddPCR04_05 10b, a partial cDNA with significant homology to the cytochrome p450 family of proteins (pfam00067). We have confirmed the down-regulation of ddPCR04_05 10b at the 48 hour time-point post-mating by quantitative reverse transcriptase PCR. Current progress in cloning, sequencing, and functional analysis is reported.


345. A novel Zn2Cys6 transcription activator, AtrR, regulates ABC transporter genes conferring azole drug resistance in Aspergilli. Daisuke Miura, Katsuya Gomi. Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.


We have already found that the expression of three genes that each encodes ABC transporter (AoatrA, AoatrF, AoatrG) was highly upregulated in a spontaneous mutant of Aspergillus oryzae showing resistance to azole fungicides. Since the expression of several ABC transporter genes was upregulated simultaneously in the mutant, we assumed that azole resistance is caused by mutation of a common transcription factor that controls these gene expressions. Promoter deletion analyses of the AoatrA suggested that a conserved sequence homologous to the PDR1/PDR3-response element (PDRE) is required for gene expression of ABC transporters. A BLAST search of A. oryzae genome database revealed the existence of several genes with a relatively high similarity to Zn2Cys6 motif of PDR1/PDR3. Overexpression of one of these genes under the control of the amyB promoter resulted in increased drug resistance and also affected the expression level of ABC transporter genes in A. oryzae. In addition, both strains with deletion of this regulatory gene in A. oryzae and with its ortholog deleted in A. nidulans were hypersensitive to azoles. These results indicate that the transcription factor, designated AtrR, regulates gene expression of ABC transporters that would function as drug efflux pumps in Aspergilli.


346. Transgene-induced silencing of the zoosporogenesis-specific PiNIFC gene cluster in the oomycete Phytophthora infestans involves chromatin alterations. Howard S. Judelson and Shuji Tani, University of California, Riverside, California, USA


Transformation-based methods for silencing genes in oomycetes are still in their infancy and the underlying cellular mechanisms are poorly understood. Transformants of the phytopathogen Phytophthora infestans in which the PiNIF family of transcriptional regulators were silenced were therefore generated and analyzed to better understand the process. PiNIFC1, PiNIFC2, and PiNIFC3 are zoosporogenesis-induced and clustered within 4 kb, and 20 kb away resides a sporulation-induced form, PiNIFS. Hairpin constructs of PiNIFC1 or PiNIFC2 triggered silencing of the cognate gene in about one-third of transformants and frequently all three PiNIFC genes became silenced coordinately. This resulted in a defect in the germination of zoospore cysts. PiNIFS was not silenced by the PiNIFC transgenes, even though all genes are closely related in DNA sequence. Silencing of the PiNIFC cluster was transcriptional based on nuclear run-on assays, and associated with tighter chromatin packing based on nuclease accessibility experiments involving DNA blotting and qPCR. The chromatin alterations extended a few hundred nucleotides beyond the boundaries of the transcribed region of the PiNIFC cluster, and were not associated with increased DNA methylation. Silencing of the different members of the PiNIFC family by a spreading heterochromatin domain, as opposed to a diffusible signal, may explain why PiNIFS escaped silencing.


347. Involvement of OS-2 MAP kinase pathway in regulation of fungal-specific catalases in Neurospora crassa. Kazuhiro Yamashita1, Azusa Shiozawa1, Shinpei Banno2, Fumiyasu Fukumori1, and Makoto Fujimura1. 1Life Sciences, Toyo University, Itakura, Gunma, Japan. 2 Plant Function Research Center, Toyo University, Itakura, Gunma, Japan.

Neurospora crassa has three fungal specific catalases, CAT-1 (conidia-specific), CAT-2 (peroxisomal catalase), and CAT-3 (hypha-specific). Transcription of the cat-2 and cat-3 genes was stimulated by heat shock and oxidative stress, respectively. In contrast, expression of the cat-1 gene was significantly stimulated by osmotic stress and fludioxonil in the wild-type strain, but not os-2 MAP kinase mutant. Active staining of catalase after native electrophoresis demonstrated that conidia of the os-2 mutant contained low level of CAT-1 protein. These results indicated cat-1 gene expression was regulated by OS-2 MAP kinase. Additionally, the os-2 conidia were sensitive to oxidative stress than those of the wild-type stain, probably due to the low mount of CAT-1 catalase. Fludioxonil, an activator of OS-2 MAP kinase, induced the conidia specific CAT-1 activity in hyphae of the wild-type strain. Interestingly, this fungicide drastically decreased hypha-specific CAT-3 activity, although impaired activation of CAT-1 and inactivation of CAT-3 was not observed in the hyphae treated by osmotic stress. These results suggest that OS-2 MAP kinase may play an important rule for asexual differentiation through the post-transcriptional regulation of fungal-specific catalase expression.


348. Aspergillus oryzae atfA Encodes a Transcription Factor, which is Required for Vigorous Growth in the Solid-State Fermentation. K. Sakamoto1, O. Yamada1, Y. Okita1, K. Iwashita1, O. Akita2, K. Gomi3, S. Mikami1. 1National Research Institute of Brewing, 3-7-1 Kagamiyama Higashi-Hiroshima, Hiroshima, Japan. 2Jissen Women's University, 4-1-1 Osakaue, Hino, Tokyo, Japan. 3Tohoku University, 1-1 Tsutsumidori-Amamiyatyou Aoba Sendai, Miyagi, Japan


In the solid-state culture, Aspergillus oryzae exhibits phenotypes such as the high production of enzymes and conidiophore development. Though these characteristics should involve various gene expressions, the only a few regulatory systems have been understood. From the EST database of A.oryzae, we found a gene encoding transcription factors that show high homology to atf1 of Shizosaccharomyces pombe and named it atfA. We constructed atfA-deletion strain (DelA51) to analyze the function. The germination ratio of DelA51 conidia was reduced to 13.7%, while that of wild type (wt) conidia was over 90 %. Furthermore the DelA51 conidia were more sensitive to stress than wt. Although DelA51 grew as fast as wt on the agar plate or in the submerged liquid culture, the growth of DelA51 was delayed in solid-state fermentation or on the hyper-osmotic agar plate. Furthermore high humidity of atmosphere restored the growth of DelA51. Thus we concluded that atfA is necessary for the vigorous growth on the low water activity substrate. To determine the genes regulated by atfA, gene expression of DelA51 was compared with that of wt using microarrays under osmotic stress condition and we identified 34 genes down-regulated in DelA51.


349. The expression of the novel sexual developmental activator, nsdC, in Aspergillus nidulans is regulated by complex transcriptional and post-transcriptional control. Hye-Ryun Kim, Yong-Woo Shin and Dong-Min Han Dept. of Life Science, Wonkwang University, Korea 570-749


The nsdC gene predicted to encode a putative transcription factor carrying a novel type of zinc fingers consisting of two C2H2 and a C2HC motifs plays as a positive regulator of sexual development in A. nidulans. Two distinct transcripts, 3.2 and 2.8 kb in size, are synthesized and the smaller one differentially accumulates in various stages of growth and development as well as under the varying cultural conditions. It increases in 10 min after the exposure of hypoxically cultured or carbon starved mycelia to air or glucose, respectively, and is restored to normal level in following 10 min. The smaller mRNA is maintained to increased level in creA204 mutant strain. The results suggest that the alternative transcription of nsdC mRNA is closely related with carbon metabolism. 5' RACE and Northern blot analysis revealed the presence of more than two alternative transcription initiation sites which might be responsible for the generation of two kinds of transcript in different size. Two alternative transcription termination sites were also identified through 3' RACE and Northern blot analysis of the nsdC deletion mutant. The gene carries two relatively long introns in its 5'UTR, one of which is spliced alternatively. Taken together, our studies suggest that the expression of nsdC is controlled by complex transcriptional and post transcriptional regulations in response to the state of carbon metabolism.


350. Withdrawn


351. Modulation of the entrained phase of the circadian clock of Neurospora by large and small isoforms of FREQUENCY. Axel Diernfellner1, Hildur V. Colot2, Orfeas Dintsis1, Jennifer J. Loros2, Jay C. Dunlap2 and Michael Brunner1. 1University of Heidelberg Biochemistry Center, INF 328, 69120 Heidelberg, Germany; 2Departments of Genetics and Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA


The large (l) and small (s) isoforms of the circadian clock protein Frequency (FRQ) of Neurospora are parts of a negative feedback loop repressing transcription of their common gene. The ratio of expression of l-FRQ vs. s-FRQ is regulated by temperature dependent splicing. Strains that express only l-frq support display temperature-compensated circadian rhythms whose periods are shorter than those of strains expressing only s-frq. The free-running period of wt decreases slightly with increasing temperature, reflecting the expression ratio of l- vs. s-FRQ. s-FRQ appears to be a significantly better repressor than l-FRQ. In 24 h LD cycles l-frq entrains to an earlier and s-frq to a later phase than wt. The entrained phases depend on day length and temperature. In contrast, the entrained phase of wt is essentially independent of both parameters.


352. Characterisation of the os signal transduction pathway in Botrytis cinerea. Weiwei Liu, Muriel Viaud, Pierre Leroux and Sabine Fillinger. UMR BIOGER, INRA Versailles, Route de Saint-Cyr, 78026 Versailles cedex, France

Botrytis cinerea, a necrotrophic fungal pathogen, has to defence itself against high oxidative and osmotic condition in planta. In fungi, the best studied case of signal transduction is the HOG pathway of Saccharomycese cerevisiae which is required to respond to high osmolarity and oxidative stress. In addition, the homologous os pathway in Neurospora crassa was demonstrated to be involved also in resistance to fungicides. Interested to understand the function of the os pathway in adaptation to adverse environmental conditions, pathogenesis and certain fungicides, we have studied this pathway in the phytopathogenic fungus B. cinerea via the characterization of the principal components, the histidine kinase Bos1 and the MAP kinase Sak1. BOS1 inactivation had pleiotropic effects in B. cinerea: abnormal morphology, loss of macroconidiation, osmosensitivity, sensitivity to some oxidative stress conditions, resistance to three classes of fungicides and loss of virulence. On the other hand, the Δsak1 mutant showed the same phenotypes as the Δbos1 mutant with a single exception: no resistance to the fungicides was observed in the Δsak1 mutant, suggesting that another pathway independent from MAP kinase Sak1 via histidine kinase Bos1 could be required for fungicide resistance in B. cinerea. The Δbos1 Δsak1 double mutant showed resistance to the fungicides as the Δbos1 single mutant, confirming that the MAP kinase Sak1 is not directly involved in resistance to these fungicides. Another interesting feature of the os signal-transduction pathway is that the regulation of Sak1 operates at different levels, on the mRNA level and through phosphorylation.


353. Survey of Genome Wide Occurrence of Alternative Splice Forms in Fusarium verticillioides. Daren W. Brown1, Robert A. E. Butchko1, and Li-Jun Ma2. 1Mycotoxin Research Unit, U.S. Department of Agriculture-ARS, Peoria, IL 61604 2The Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, 02141


Fusarium verticillioides is a pathogen of maize and synthesizes the fumonisin family of mycotoxins. Contamination of infected kernels with fumonisins contribute significantly to economic losses to the maize grain industry worldwide. Ingested fumonisins cause a variety of animal diseases and are correlated to human cancers and neural tube defects. Understanding basic fundamental strategies regulating fungal processes will aid our efforts to limit F. verticillioides maize diseases as well as fumonisin contamination. Alternative splicing of transcripts in many eukaryotes play a critical role in expanding gene function by increasing protein diversity. The few examples of alternative splice forms (ASFs) described in fungi predominantly involve intron retention. Analysis of large-scale expressed sequence (EST) collections coupled with genomic DNA from a number of fungi indicate that ASFs are more widely prevalent than previously thought. Analysis of over 87,000 F. verticillioides ESTs provided evidence of ASFs for over 300 genes which represent almost 3.1% of the genes detected. Although a majority of ASFs retain introns, we present evidence for a variety of alternative splice mechanisms. These results clearly show that alternative splicing in fungi is more common than previously thought and likely play a significant role modifying gene function by increasing the transcript diversity.


354. L-arginine induces structural and functional changes of the mRNA in Aspergillus nidulans. Borsuk P1, Przykorska A2, Blachnio K2, Koper M1, Pekala M1 and Weglenski P1. 1Institute of Genetics and Biotechnology, University of Warsaw 2Institute of Biochemistry and Biophysics, Polish Academy of Sciences


Expression of arginase gene (agaA) in Aspergillus nidulans depends on growth conditions, especially on the presence or absence of arginine and various nitrogen and carbon sources in the medium. 5’UTR of arginase mRNA contain arginine binding motifs, short open reading frame and intron with two putative 3’ splice site. We present evidence that: - L-arginine binds in vitro directly and stereospecifically to the arginase 5’UTR - L-arginine binds to the 5’UTR at more then one site - only L-arginine (not several other L-amino acids or D-arginine) generates drastic changes in the secondary structure of the 5’UTR - putrescine (poliamine) has no influence on the secondary structure of the 5’UTR - L-arginine and poliamines forces the selection of one of the two 3’ splice sites in the intron present in the 5’UTR We postulate that expression of the agaA gene is regulated at both transcriptional and posttranscriptional levels. Posttranscriptional regulation depends on mRNA stability affected by the riboswitch-mediated alternative splicing in the 5’UTR. The role of poliamine in control of agaA expression is also discussed.


355. Control of Translation and mRNA Stability by the Fungal Arginine Attenuator Peptide. Christina C. Spevak, Heather M. Hood, Jaime McCollum, and Matthew S. Sachs, Oregon Health & Science University, 20000 NW Walker Road, Beaverton, OR 97006-8921


Fungal mRNAs specifying the small subunit of Arg-specific carbamoyl phosphate synthetase contain an upstream open reading frame (uORF) specifying the evolutionarily conserved arginine attenuator peptide (AAP). The synthesis and amino acid sequence of the AAP are critical for cis-acting Arg-specific negative regulation of translation. In cell-free translation systems, AAP-mediated regulation depends on the AAP's ability to stall ribosomes at the uORF termination codon in response to arginine. Certain single missense mutations eliminate ribosome stalling in vitro and Arg-specific regulation in vivo and in vitro. The S. cerevisiae CPA1 and N. crassa AAPs are specified by uORFs whose start codons are in relatively poor translation initiation contexts, resulting in much leaky-scanning past the uORF when Arg levels are low, with no stalling of ribosomes on the uORF when it is translated. When Arg is high, AAP-mediated ribosome stalling blocks ribosomes that scanned past the uORF initiation codon from initiating translation at the downstream start codon. The AAP sequences required for stalling were determined by Ala- and Pro-scanning and by analyzing the minimal domain required to induce stalling. The minimal domain contains the most highly conserved region among yeasts, ascomycetes and basidiomycetes. In addition to translational regulation, CPA1 expression is naturally regulated at the level of mRNA stability via a mechanism involving nonsense-mediated mRNA decay (NMD). Arg-regulated stalling at the CPA1 uORF stop codon triggers NMD and the data suggest a simple model that controlling the density of ribosomes at the uORF termination codon modulates NMD. Analyses of N. crassa arg 2 in isogenic NMD+ and nmd- strains indicate that its levels are also controlled by NMD. Thus, these studies provide a basis for a general understanding of how ribosome occupancy of a uORF termination codon controls both translation and mRNA stability in eukaryotes.


356. Cloning and Characterization of Circadian Output Pathway mutant COP1-4. Vitalini M.W. and D. Bell-Pedersen. Texas A& M University.

Decades of research on the Neurospora crassa circadian clock have resulted in a detailed understanding of core components of the central FRQ/WCC oscillator. However, in Neurospora, as well as in the other model systems, little is known about the pathways by which temporal information is relayed from a circadian oscillator to the rest of the cell to control downstream rhythmic processes. We have set up a genetic selection for clock mutations in order to identify novel components involved in output from the Neurospora circadian clock. This selection is based on the differential expression from the promoters of clock-controlled genes (ccg) fused to a selectable marker (mtr) in response to the presence or absence of clock gene product FRQ. After screening hundreds of mutant colonies we have narrowed our focus to a few carefully chosen mutations that disrupt the rhythmic expression of more than one ccg. We found that two genes, when mutated, affect circadian output and are involved in the ability of cells to respond to hyper- osmotic conditions. We will present the cloning and further characterization of one of these mutations, cop1-4, and discuss the link between circadian output and stress-response pathways.


357. Withdrawn


Developmental Biology


358. Role of putative apoptosis-inducing factor (AIF)-homologs in lifespan control of the filamentous ascomycete Podospora anserina. Diana Brust, Andrea Hamann, Heinz D. Osiewacz Institute of Molecular Biosciences, J.W. Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany E-mail: brust@bio.uni-frankfurt.de


Wild-type strains of the ascomycete Podospora anserina are characterized by a limited life span. After about 30 days of linear growth, the growth rate of the wild type strain s declines and the morphology and physiology of cultures changes dramatically, ending up with the death of the hyphal tips. This process has a clear mitochondrial etiology. The role of mitochondria in aging of cultures raises the question whether or not apoptosis-like processes are involved in the final stage of development of this fungus. An in-silico analysis identified several putative apoptosis factors. Especially the AIF (apoptosis-inducing factor) and AMID (AIF homologous mitochondrion-associated inducer of death) homologs are of great interest since the mammalian proteins are known to be involved in a mitochondrial pathway of apoptosis. The PaAIF1/GFP fusion protein was found to be localized in the cytoplasm, while the PaAMID2/GFP fusion protein localizes to mitochondria suggesting a role in a mitochondria-dependent and caspase-independent signalling pathway. The localization of PaAMID1 is under investigation. Deletion of PaAmid1 results in a moderate lifespan extension on complete medium (59 %), while the PaAif1::ble strain is characterized by a mean lifespan of 150 days (340 % increase) on this medium. Interestingly, the double deletion strain PaAmid1::ble, PaAif1::ble is strongly impaired in fertility. Currently, we investigate the age-dependent translocation of the P. anserina AIF and AMID homologs and the mitochondrial release after an apoptotic stimulus achieved by chemical inductors.


359. The NADPH oxidases NOX-1, NOX-2 and their regulatory subunit NOR-1 regulate growth and differentiation in Neurospora crassa. Nallely Cano; Karen Álvarez; Wilhelm Hansberg and Jesús Aguirre. Instituto de Fisiología Celular,UNAM. Mexico. ncano@ifc.unam.mx


We have proposed that reactive oxygen species(ROS) play essential roles for cell differentiation in microbial eukaryotes. The NADPH oxidases (NOX) are enzymes that catalyze the production of the superoxide. In phagocytic cells, the NOX consists of the membrane-associated catalytic core gp91phox and p22phox subunits (cytochrome b558). The assembly of the cytosolic regulatory proteins p47phox, p67phox, p40phox and Rac1/Rac2 with the cytochrome b558 results in NOX activation. NoxA from A. nidulans, the gp91phox microbial orthologue, was found to produce ROS and to be essential for differentiation of sexual fruiting bodies (Lara-Ortiz et al., 2003). p67phox homologues have been reported in Dictyostelium discoideum (Lardy et al., 2005) and Epichloë festucae (Takemoto et al., 2006). In E. festucae, this subunit (NoxR) regulates Nox1 during symbiosis. Here we show that inactivation of NOX-1 in N.crassa results in decreased asexual development, a slight reduction of radial growth and complete female sterility. Mutation of NOX-2 did not affect any of these processes but led to the production of ascospores that failed to germinate even in the presence of hydrogen peroxide or furfural. Mutation of the gp67phox homologue NOR-1 resulted in reduction of asexual development and radial growth, female sterility and lack of ascospore germination. These results suggest that NOR-1 regulates both, NOX-1 and NOX-2, at different developmental stages.


360. Effect of Ras signaling on fruitbody formation in the fungus Schizophyllum commune. Nicole Knabe, Daniela Schubert & Erika Kothe. Friedrich-Schiller-University Jena, Dept. of Microbiology, Neugasse 25, 07743 Jena, Germany, Nicole.Knabe@uni-jena.de


The involvement of small monomeric G-proteins like Ras has been shown in different fungi for MAPK cascade signaling and/or cAMP-dependent pathways. In addition, cAMP signaling has been implicated in fruitbody development. The involvement of Ras in sexual development and cell morphogenesis was investigated by characterization of ras1 and gap1 genes in S. commune. Disruption of gap1 encoding a Ras-dependent GAP (GTPase activating protein) leads to accumulation of Ras in its activated, GTP-bound state and to constitutive Ras signaling. The phenotype of gap1 deletion strains shows disorientated growth pattern. Dikaryons are not able to produce normal clamp connections because the hook cells failed to fuse with the peg beside them. Instead, the dikaryotic character of the hyphae was rescued by fusion of the hooks with nearby developing branches. Deltagap1/Deltagap1 dikaryons formed increased numbers of fruitbody primordia, whereas the amount of fruitbodies was not raised. Mature fruitbodies formed no or abnormal gills. No production of spores could be observed. To further analyse the function of Ras, amino acid mutations in the GTP-binding region and GTPase domains were introduced in vitro. The phenotypes of the resulting transformants carrying the dominant negative allele RasG15N and the constitutive alleles RasG12V and RasQ61L were examined. Similar phenotypes of the RasGap deletion strain and the constitutive Ras mutant could be observed. Both shows a reduced


361. Transcriptional and post-transcriptional levels of control lead to a fruiting body-specific accumulation of the APP protein in Sordaria macrospora and Neurospora crassa. Minou Nowrousian1, Markus Piotrowski2, Ulrich Kück1. 1Lehrstuhl für Allgemeine und Molekulare Botanik and 2Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, 44780 Bochum, Germany, email: minou.nowrousian@ruhr-uni-bochum.de


During fungal fruiting body development, specialized cell types differentiate from vegetative mycelium. We have isolated a protein from the ascomycete Sordaria macrospora that is not present during vegetative growth but accumulates in perithecia. The protein was sequenced by mass spectrometry and the corresponding gene was termed app (abundant perithecial protein). app transcript occurs only after the onset of sexual development; however, the formation of ascospores is not a prerequisite for APP accumulation. Fruiting body-specific accumulation of an APP-EGFP fusion protein is dependent on regulatory regions upstream and downstream of the app open reading frame. The transcript of the N. crassa ortholog is present prior to fertilization, but the protein accumulates only after fertilization. In crosses of N. crassa delta-app strains with the wild type, APP accumulates when the wild type serves as female parent, but not in the reciprocal cross; thus, the presence of a functional female app allele is necessary and sufficient for APP accumulation. These findings highlight multiple layers of temporal and spatial control of gene expression during fungal development (Fungal Genet Biol, 2006, in press).


362. The novel ER membrane protein PRO41 is essential for sexual development in Sordaria macrospora. Minou Nowrousian1, Sandra Frank1, Sandra Koers1, Peter Strauch1, Thomas Weitner1, Carol Ringelberg2, Jay C. Dunlap2, Jennifer J. Loros2, and Ulrich Kück1. 1Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, 44801 Bochum, Germany, and 2Departments of Genetics and Biochemistry, Dartmouth Medical School, NH 03755, USA


The filamentous fungus Sordaria macrospora develops complex fruiting bodies to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant is complemented by a small open reading frame encoding the predicted PRO41 protein; the open reading frame and adjacent regions are deleted in mutant pro41. pro41 transcript levels are upregulated during sexual development. This increase in transcript cannot be observed in the sterile mutant pro1 that lacks a transcription factor, and microarray analysis of gene expression in the mutants pro1, pro41, and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. PRO41 is predicted to be an ER membrane protein by in silico analyses, and a PRO41- EGFP fusion protein co-localizes with ER-targeted DsRED. Furthermore, Western blot analysis showed that the PRO41- EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted into the medium, indicating that the signal sequence is functional. These data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi.


363. Characterization of the Aspergillus nidulans septin AspA/Cdc11. Rebecca Lindsey, Susan Cowden and Michelle Momany*. Department of Plant Biology, University of Georgia, Athens, Georgia 30602 USA lindsey@plantbio.uga.edu


The septin proteins form filamentous rings at the mother-bud neck in yeast. Septins are found in microsporidia, fungi, and animals and are absent from plants. In addition to their original role in cell division, septins have been shown to have roles in cytoskeletal organization, coordination of nuclear division and trafficking across membranes. In Aspergillus nidulans there are five septins aspA/cdc11, aspB/cdc3, aspC/cdc12, aspD/cdc10, and aspE. We have conducted studies on the A. nidulans septin aspA/cdc11. The null mutant of aspA/cdc11 is viable and shows uncoordinated germ tubes, hooked hyphae, split tips, hyperbranching, disorganized conidiophores and hypersensitivity to the actin depolymerizer Cytocholasin A. Localization of AspA/Cdc11-GFP is dynamic. AspA/Cdc11- GFP is visible as a spot on conidia, throughout the cortex in swelling conidia, at the polarizing surface when germ tubes emerge, at septa in germ tubes and branches, and as filaments at tips and in early branches. In delta aspA/cdc11 SepA-GFP and BimG-GFP localization are lost, but TubA-GFP localization is normal. Our results suggest that AspA/Cdc11 interacts with actin as well as the formin, SepA and the protein phosphatase BimG, possibly in the growing hyphal tip.


364. Characterization of the Aspergillus nidulans septin mutant ΔaspB/cdc3. Yainitza Rodriguez and Michelle Momany. Department of Plant Biology, University of Georgia, Athens, Georgia 30602 USA yrodriguez@plantbio.uga.edu


Septins are filament forming P-loop GTPases found in microsporidia, fungi and animals. Septins play important roles in a variety of processes such as cellular and nuclear division, membrane trafficking and organization of the cytoskeleton. This family of proteins was first discovered in a screen for temperature sensitive cell cycle mutants in Saccharomyces cerevisiae. In yeast, septins form filamentous rings at the mother-bud neck which are necessary for completion of cytokinesis and normal morphogenesis. In Aspergillus nidulans there are five septins aspA/cdc11, aspB/cdc3, aspC/cdc12, aspD/cdc10, and aspE. The A. nidulans septin aspB/cdc3 was previously shown to localize at septation and branching sites and at interface layers of the conidiophore. This gene was reported to be essential. We have now found that deletion of aspB/cdc3 is not lethal, but causes severe defects in asexual reproduction and aberrant morphology in several developmental stages. The mutant shows emergence of multiple germ tubes, hyperbranching and hooked branches and disorganized conidiophores. ΔaspB/cdc3 also shows sensitivity to calcoflour, but not benomyl or cytochalasin A, which suggests that the mutant phenotype is not directly associated with the actin or microtubule cytoskeleton, but perhaps with cell wall stability.


365. Transcriptional changes during the isotropic to polar growth switch in Aspergillus nidulans. Andrew Breakspear and Michelle Momany. Department of Plant biology, University of Georgia, Athens, Georgia 30602 USA abreakspear@plantbio.uga.edu


Isotropic and polar modes of growth are common to both yeast and filamentous fungi and are fundamental in defining cell shape. Following the breaking of dormancy A. nidulans conidia grow isotropically as cell wall components are added uniformly in all directions. After the first nuclear division a switch occurs to polar growth that results in the emergence of a nascent germ tube. The new A. nidulans microarray chips produced by the Pathogen Functional Genomics Resource Center (PFGRC) have facilitated the identification of transcriptional changes associated with the switch in growth modes. Microarray analysis was used to compare gene expression before and after germ tube emergence. Following emergence of the primary germ tube 181 genes were down-regulated by at least 2-fold and 401 genes were up- regulated by at least 2-fold. Among up-regulated genes 53 showed a 5-fold or more increase in transcript levels. The results demonstrate that many genes show significant expression changes during early growth in A. nidulans.


366. Growth and developmental control in Aspergillus fumigatus. Jae-Hyung Mah and Jae-Hyuk Yu. Department of Bacteriology, University of Wisconsin, Madison, WI 53706


The opportunistic human pathogen Aspergillus fumigatus reproduces by forming a large number of asexual spores. We studied the mechanisms regulating asexual development in A. fumigatus via examining functions of four key controllers, GpaA (G alpha), AfFlbA (RGS), AfFluG and AfBrlA. Expression analyses of gpaA, AfflbA, AffluG, AfbrlA and AfwetA revealed that, whereas transcripts of AfflbA and AffluG accumulate constantly, AfbrlA and AfwetA are specifically expressed during conidiation. Both loss of function AfflbA and dominant activating GpaAQ204L mutations resulted in reduced conidiation coupled with increased hyphal mass, indicating that GpaA mediates signaling that activates vegetative growth while inhibiting conidiation. As GpaA is the principal target for AfFlbA, the dominant interfering GpaAG203R mutation suppressed the phenotype resulting from loss of AfflbA function. These results corroborate the idea that parimary roles of G proteins and RGSs are conserved in aspergilli. Functions of the two major developmental activators AfFluG and AfBrlA are then examined. While deletion of AfbrlA eliminated conidiation completely, deletion of AffluG did not cause severe defects in A. fumigatus sporulation in air-exposed culture, implying that, whereas the two Aspergillus species may have a common key downstream developmental activator, upstream mechanisms activating brlA may be distinct. Finally, both AffluG and AfflbA mutants showed reduced conidiation and delayed accumulation of AfbrlA mRNA in developmental induction, indicating that these upstream regulators are associated with the proper progression of conidiation.


367. Pdc1, a Ustilago maydis 14-3-3 Homologue, Regulates Cell Growth and Separation. Cau D. Pham, Zhanyang Yu, and Michael H. Perlin. University of Louisville, Department of Biology, Program on Disease Evolution, Louisville, Kentucky, USA


14-3-3 proteins are a group of conserved eukaryotic proteins that participate in the regulation of many important cellular processes, e.g., signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. Most organisms have multiple forms of these proteins to provide redundancy or with each isoform fulfilling specific roles. Inspection of the recently-available U. maydis genome sequence reveals that this organism may be ideal for the study of 14-3-3 proteins since, in contrast to other organisms, U. maydis has only one predicted 14-3-3 protein. We named the U. maydis 14-3-3 protein, Pdc1, for its expected role as a phosphorylation-domain coupling protein. Pdc1 appears required for normal cell growth. Haploid strains do no tolerate pdc1 gene disruption, while Pdc1 knock-down leads to 10-fold reduction of cell density in liquid culture, as well as filamentation and elongation of cells. Calcofluor and DAPI stains show that such elongated or filamentous cells contain multiple septa and nuclei, possibly due to failure of mother and daughter cells to separate. Finally, yeast-2-hybrid data indicate that the Pdc1 protein interacts with a number of proteins (Actin, FPR1 and FBR2, AFG3). Interestingly, Pdc1 interacts with the Rho1 homologue, Uro1, another protein required for cell growth and division, as well as the formin, Bni1, whose interaction with Rho1 in S. cerevisiae is required to recruit Ste5 to its G protein. Not unexpectedly, Pdc1 interacts with itself in yeast 2-hybrid experiments. Pdc1 protein can suppress bmh1bmh2 deletion defects in S. cerevisiae, suggesting its functional homology with the 14-3-3 proteins, Bmh1 and Bmh2. Site-directed mutagenesis of the putative protein kinase A target residue in Pdc1 does not prevent dimer formation, but does interfere with the ability of the corresponding mutant Pdc1 proteins to complement bmh1bmh2 S. cerevisiae mutant growth defects.


368. Identifying cis- and trans-factors involved in spore-specific gene expression in the oomycete Phytophthora infestans. Qijun Xiang, Audrey Ah Fong, Kyoung Su Kim, Howard S. Judelson. Department of Plant Pathology, University of California, Riverside, CA 92521


Using microarray analysis, we have identified a large number of genes induced during sporulation and zoospore formation in Phytophthora infestans, the cause of the potato late blight disease. The promoters of several of these genes are being analyzed to define the sequences that confer sporulation or zoospore-specific patterns of gene expression, using a GUS reporter system to test deleted or mutagenized versions of the promoters in P. infestans transformants. This approach has been used to identify a 7-nt "cold box" in the promoters of several genes induced during zoospore formation, and to define the binding sites of development-associated transcription factors in the promoters of other genes such as those encoding a sporulation-specific serine-threonine kinase and a protein phosphatase. DNA fragments from the promoters are being used to purify the relevant transcription factors using DNA affinity approaches. EMSA (electrophoretic mobility shift assay) is being used to examine the DNA binding activities of the proteins.


369. A pheromone receptor is involved in mound expression in Schizophyllum commune. Goutami Banerjee and Dr. Thomas J Leonard. Clark University, Worcester, MA.


Restriction enzyme-mediated DNA integration (REMI) was used to induce mutations in a homokaryotic strain of Schizophyllum commune, Hk28, which forms prolific numbers of large, abnormal multicellular growths called mounds. Mutant transformants were screened for their inability to form mounds. One such moundless transformant was isolated, analyzed and found to have a single disrupted gene caused by plasmid integration. When the disrupted gene was isolated and sequenced, it proved to be an unreported pheromone receptor in Schizophyllum. This transformant was analyzed for its ability to express the hydrophobin genes Sc1, Sc3 and Sc4. Hydrophobin Sc3 is essential for the formation of aerial hyphae in homokaryons and dikaryons, whereas Sc1 and Sc4 are dikaryon specific and are associated with the formation of fruiting bodies. Using Real time PCR, the parent strain HK28 was found to show normal levels of Sc3 gene expression, barely detectable levels of Sc1, and very high levels of Sc4. The moundless transformant was found to express normal levels of Sc3, but showed a sharp decrease and eventual non-expression of hydrophobin Sc4 gene expression during the time period in which mounds are normally produced. These results suggest homokaryotic mound formation and Sc4 gene expression are regulated by a pheromone receptor. When homokaryotic fruiting body tissue was examined for hydrophobin expression, Sc4 was not detected, suggesting the nature of hyphal cohesion involved in homokaryotic fruiting and mound development utilize different hydrophobins for hyphal cohesion.


370. Secondary metabolism and sporulation signalling in Aspergillus nidulans. Olivia Márquez1, Angel Trigos1, J. Luis Ramos2, Gustavo Viniegra3, Holger Deising4, Nallely Cano2 and Jesús Aguirre2*. 1Universidad Veracruzana, México. 2Universidad Nacional Autónoma de México, México. 3Universidad Autónoma Metropolitana, México. 4Martin-Luther University Halle-Wittenberg, Germany. *E-mail: jaguirre@ifc.unam.mx

We characterized Aspergillus nidulans strains carrying conditional (cfwA2) and null (DcfwA) mutant alleles of the cfwA/npgA gene, encoding an essential phosphopantetheinyl transferase (PPTase). We identified the polyketides shamixanthone, emericellin and dehydroaustinol, as well as the sterols ergosterol, peroxiergosterol and cerevisterol in extracts from developmental cultures. The PPTase CfwA/NpgA was required for production of polyketide compounds, but dispensable for fatty acid biosynthesis. The asexual sporulation defects of cfwA, DfluG and DtmpA mutants were not rescued by the cfwA-dependent compounds identified here. However, cfwA2 mutation drastically enhanced the sporulation defects of both DtmpA and DfluG single mutants, suggesting that unidentified CfwA-dependent PKS and/or NRPS are involved in production of unknown compounds, required for sporulation. In addition, we show that tmpA and tmpB genes define two new fluG-independent sporulation pathways.


371. Withdrawn

 

372. Deletion of the gene encoding the metacaspase homolog PaMCA1 results in lifespan extension in P. anserina demonstrating a connection between apoptosis and lifespan control. Andrea Hamann, Diana Brust, Heinz D. Osiewacz Institute of Molecular Biosciences, J. W. Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany Phone: + 49 (0)6979829279 Fax: + 49 (0)6979829363 E-mail: a.hamann@bio.uni-frankfurt.de


The wild-type strain s of the filamentous ascomycete Podospora anserina is characterized by a mean lifespan of 25 days. The well-characterised senescence syndrome of wild-type strains is accompanied by gross rearrangements of the mitochondrial DNA due to the accumulation of a small circular element, the so-called plDNA finally leading to mitochondrial deficiency. Since mitochondria also play a key role in controlling apoptotic processes, the involvement of an apoptosis machinery in the mechanism by which senescent cultures finally die, was analysed. The genome of P. anserina encodes several important apoptosis factors, e.g. two metacaspases and several AIF (apoptosis-inducing factor) and AMID (AIF-homologous mitochondrial inducer of death) homologs. Their putative role in lifespan control was investigated by deleting some of the corresponding genes. Especially the deletion of PaMca1 results in a 2.5 fold increase in lifespan demonstrating that apoptosis factors are involved in lifespan control. It appears that the strongly elevated level of hydrogen peroxide in senescent mycelium activates a metacaspase-dependent cell death program. This hypothesis is confirmed by the observed enhancement of caspase activity after hydrogen peroxide treatment. Accordingly, during aging the sensitivity of wild-type protoplasts to hydrogen peroxide treatment increases, while it remains stable in a PaMca1::ble strain explaining the extended lifespan of this strain. The results obtained in P. anserina point to a connection between conserved mechanisms of apoptosis-like processes and organismic aging.


373. The novel regulator of spore maturation VosA controls development and trehalose synthesis in Aspergillus nidulans. Min Ni and Jae-Hyuk Yu, Departments of Genetics and Bacteriology, 1925 Willow Drive, University of Wisconsin, Madison, WI 53706, USA


The asexual spore (conidia) is the primary agent for dispersal, survival and propagation in fungi. Aspergillus nidulans has served as an excellent model system for studying the mechanism of asexual development. In this report, we identified and characterized the novel developmental regulator VosA that represses asexual/sexual development when present in multiple copies. The VosA (viability of spores) protein is highly conserved in many filamentous fungi, and is similar to VeA, an important regulator of balanced development. As VosA negatively controls conidiation, deletion of vosA resulted in conidiophore development in liquid submerged culture and over-expression of vosA blocked conidiation completely. The regulatory role of VosA in conidiation is primarily via repressing the expression of brlA, a key activator conidiation in Aspergillus. Importantly, the conidia and sexual spores (ascospores) of the vosA deletion mutant lose cytoplasmic fluids upon prolonged incubation, which results in extremely low (2%) viability of the spores. Chemical analyses revealed that trehalose, a crucial disaccharide serving as a stress protectant, is absent in the conidia of the deletion vosA mutant. These findings coupled with the observation that vosA is highly expressed only in conidia and ascospores indicate that VosA plays an essential role in controlling spore maturation and proper regulation of development. In addition, yeast one hybrid experiments indicate that the C-terminal half of VosA may contain a transcriptional activation domain.


374. Functional study of SMR1/MAT1-1-2, a mating-type gene which does not control self-non self recognition in Podospora anserina. E. Coppin, S. Arnaise, K. Bouhouche, X. Robellet, D. Zickler and R. Debuchy. Institut de Génétique et Microbiologie, UMR 8621 CNRS-Universite Paris Sud, Orsay, France


The sexual reproduction of P. anserina requires coordinate action of 4 mating-type genes. Three encode transcriptional factors which control the recognition between mat- /MAT1-1 and mat+/MAT1-2 sexually compatible cells during fertilization, and between nuclei after fertilization, at the transition from a plurinucleate to a dikaryotic stage when ascogenous hyphae are formed. The fourth gene, SMR1/MAT1-1-2, is essential for development of dikaryotic hyphae; its inactivation results in barren perithecia. In contrast, it is dispensable for development of abnormal uniparental ascogenous hyphae, observed when the other mat genes contain mutations affecting nuclear pairing [1]. MAT1-1-2 is present in all Sordariomycetes analyzed to date. All MAT1-1-2 proteins contain a 17 aa stretch with conserved and invariant residues forming a HPG domain proposed to define a new DNA- binding domain [2]. However, the cytosolic localization of the SMR1-GFP protein does not support the hypothesis that SMRl could be a transcription factor. To better understand the structure-function relationships of SMR1 we analyzed 35 alleles constructed by site-directed mutagenesis. Deletion of the HPG domain led to a mutant phenotype, confirming its role in SMR1 activity. However, while 12 aa of this domain were replaced with alanine, only the W193A change inactivated SMR1. We identified a second mutant allele (A347D) in the C terminus, indicating that this region, not conserved in orthologs, is functionally important for sexual development of P. anserina. To date the molecular function of SMR1/MAT1-1-2 remains unknown. 1- Arnaise et al., Genetics (2001) 159: 545 2- Debuchy et al., Mol Gen Genet (1993) 241: 667


375. Is RIP abolished at the mating type locus of Podospora anserina? K. Bouhouche, E. Coppin and R. Debuchy. Institut de Génétique et Microbiologie, UMR 8621 CNRS-Université Paris Sud, Orsay, France

A mild version of the Repeat Induced Point mutation (RIP), a homology-dependent gene-silencing mechanism, was shown to affect both cis- and trans-duplicated sequences in P. anserina (1 and unpublished data). Surprizingly, silencing was never observed for duplications of the SMR2/MAT1-1-3 mating type gene created at the resident mat- /MAT1-1 locus by gene replacement, while the same duplication introduced ectopically was silenced by RIP mutations. These data were obtained using a powerful genetic system which permits screening of mutations inactivating SMR2, by restoration of sporulation. To extend these results, we introduced a duplication of the hph gene conferring hygromycin resistance at the same site of the resident mat- idiomorph. As a control, the mat- sequence with the duplicated hph insertion has been integrated at different ectopic sites. By loss-comparison of hph expression in the progeny of crosses involving the different transgenic strains we will be able to determine whether the mat- idiomorph is really protected against RIP. This situation, if confirmed, would open a new field of investigation in understanding why and how RIP is specifically abolished at the mat- locus (and perhaps at the mat+ locus). In addition, a genetic system based on mat genes expression is already available to seek mutations able to restore RIP at the mat- locus after mutagenesis. 1- Graïa et al, Mol. Microbiol. (2001) 40: 586


376. Role of PaTrx1, Patrx2 and PaTrx3, three cytosolic thioredoxins, in the life cycle of the filamentous ascomycete Podospora anserina. Fabienne Malagnac1,2, Benjamin Klapholz1 and Philippe Silar1,2. 1Institut de Génétique et Microbiologie, UMR CNRS - Université de Paris 11, UPS Bât. 400, 91405 Orsay cedex, France 2UFR de Biochimie, Université de Paris 7 - Denis Diderot, case 7006, 2 place Jussieu, 75005, Paris, France


Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2 and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2 and PaTrx3 showed that PaTrx1 is the major thioredoxin involved in methionine biosynthesis. Deletions also show that either PaTrx1 or PaTrx3 is necessary for sexual reproduction and for the development of the Crippled Growth cell degeneration, processes that are also controlled by the PaMpk1 MAP kinase pathway. Analysis of the phosphorylation and the nuclear localization of PaMpk1 shows that thioredoxins do not participate in the activation of this MAP kinase and thus acts through another pathway.


377. The gene for the ubiquitin ligase regulator Cand1 is putatively split into two genes in the filamentous fungus Aspergillus nidulans. Elke U. Schwier, Martin Christmann, Krystyna Nahlik, Silke Busch and Gerhard H. Braus. Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, 37077 Göttingen, Germany; e-mail: eschwie@gwdg.de


Ubiquitin dependent proteolysis plays an important role in many cellular processes in eukaryotes. Cullin containing ubiquitin ligases like the SCF (Skp1-Cullin-F-box protein) complex mark proteins for degradation by ubiquitinylation. It has been shown that the protein Cand1 binds to cullins. The Cand1 C-terminus blocks the SKP1 binding site of the SCF complex component Cul1 and affects thereby the assembly/disassembly of the ubiquitin ligase. The N-terminus of Cand1 buries the neddylation site on Cul1, which prevents its modification by Nedd8, an ubiquitin-like protein and alters the activity of the complex. In A. nidulans the gene encoding the putative homolog of human Cand1 seems to be split in two, both independent genes having about 20% identity to the human protein. Deletion of the A. nidulans gene coding for the protein Cand1_C similar to the c-terminal part of human Cand1 leads to a red hyphae phenotype. Cand1_C is expressed during vegetative growth but not during sexual development. It localizes to the nucleus and interacts with CulA, CulD and Cand1_N in the yeast two hybrid system. Currently we are investigating the function of the smaller protein Cand_N. The split cand1 gene makes the fungus A. nidulans an attractive model organism for studying the putative different functions of the two parts of the Cand1 protein.


378. B-regulated development is influenced by sts1, a putative sugar transporter in the basidiomycete Schizophyllum commune. Dionna Kasper, Michael Gallagher, Thomas Hickernell, and Stephen Horton. Department of Biological Sciences, Union College, Schenectady, NY 12308 USA


Sexual development in the basidiomycete mushroom Schizophyllum commune is dependent upon activation of signaling pathways controlled by the A and B mating-type genes. This activation normally occurs as a result of a mating interaction between two compatible haploid individuals, yielding a dikaryon capable of mushroom development. Activation of the B, but not A-regulated pathway results in a characteristic phenotype called flat. The gene sts1 encodes a putative sugar transporter from S. commune, and has been previously implicated in mushroom development. sts1 null mutant strains were created by homologous integration of a knockout construct in haploid transformation recipients. Dikaryons homozygous for the sts1 null mutation displayed a severely attenuated ability to fruit. Unexpectedly, when haploid sts1 null mutants were outcrossed, a proportion of the null progeny displayed the flat phenotype characteristic of B pathway activation. The same phenomenon was observed in regenerated protoplasts of haploid sts1 null strains. In both cases, the flat individuals were found to donate, but not accept nuclei in test matings, a genetic behavior consistent with the idea that B-regulated development was indeed activated in these haploid strains. From these data, we hypothesize that sts1 plays a role in both B-regulated development and fruiting body production in S. commune.


379. Cis-acting elements regulating transcription initiation and sporulation-specific expression of the PiCdc14 gene of Phytophthora infestans. Audrey M. V. Ah Fong and Howard S. Judelson. Department of Plant Pathology, University of California, Riverside, California, USA.


Asexual spores are central to the disease cycle of Phytophthora infestans, the cause of tomato and potato late blight. To understand transcriptional processes involved in sporulation, functional regions were identified within the promoter of PiCdc14, a gene normally expressed only during asexual spore development. These were defined by testing truncated promoters or site-directed mutants in transformants of P. infestans, using the GUS reporter. Two functional classes of motifs were identified, one directing sporulation-specific expression and others regulating the position of transcription initiation. Two apparently redundant binding sites for transcription factors directing expression during sporulation were localized just upstream of nt -66 (relative to transcription start site), within a region showing high conservation in four Phytophthora spp. The site of transcription initiation was controlled both by sequences flanking the normal major start site and the motif CTCAAC, which was present four times between -74 and -138. Promoters in which either the normal start site or all four CTCAAC motifs were eliminated still exhibited sporulation- specific expression, but initiation commonly occurred at nt -109. Deletions of single CTCAAC motifs had intermediate effects. Although the region containing the major start site of PiCdc14 lacks the Inr-like consensus described previously for many other Phytophthora genes, it does not appear to represent a sporulation-related determinant since exchanging it with the Inr-like region of the PiExo1 gene did not impair PiCdc14 expression. Experiments to better define the biochemical and genetic interactions between these sites and their cognate transcription factors are underway.


380. GzPP2A, encoding a putative protein phosphatase 2A, is involved in sexual development and pathogenesis in Gibberella zeae. Hee-Kyoung Kim1, Yin-Won Lee2, and Sung-Hwan Yun1. 1Department of Biological Resources and Technology, Soonchunhyang University, Asan 336-745; 2School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea.


Gibberella zeae is an important pathogen of maize, wheat and rice. We used REMI to identify a self-sterile mutant of G. zeae, designated R2629. The REMI mutant showed pleiotropic phenotypes other than no perithecial formation, including abnormal conidia morphology and reduction in mycelial growth and virulence on host plants. Outcrossing analysis confirmed that all of the phenotypic changes in R2629 were tagged with the hygromycin B resistance marker. The vector insertion appears to cause a complicated genomic rearrangement in R2629. The genomic region flanking 5' of vector insertion site was identified as the KpnI site 713 bp upstream of the ORF annotated as FG01141.1 located in the contig 1.56 of the G. zeae genome databases, which showed a similarity to delta-1- pyrroline-5-carboxylate dehydrogenase, whereas the 3' flanking region was matched to a KpnI site within the ORF designated GzPP2A (FG05894.1 located in the contig 1.236) highly similar to B56-delta regulatory subunit of protein phosphatase 2A. Subsequent targeted gene deletion and complementation analyses confirmed that GzPP2A was responsible for the pleiotropic phenotypic changes in the original REMI mutant. The GFP gene under the control of the GzPP2A promoter was strongly expressed in both mycelia and conidia, but not in ascospores, indicating that GzPP2A is down-regulated during the sexual reproduction mode. Taken together, it is conclusive that GzPP2A regulates important cellular processes in G. zeae, including fungal development (during both asexual and sexual stages) and pathogenicity on host plants.


381. Identification of proteins interacting with the Sordaria macrospora WD40 repeat protein PRO11 by a yeast two-hybrid screen. Yasmine Bernhards and Stefanie Pöggeler. Georg-August-University of Göttingen, Institute of Microbiology and Genetics, Department Genetics of eukaryotic microorganisms, Göttingen, Germany.


Fruiting-body development in fungi is a complex cellular differentiation process that is controlled by more than 100 developmental genes. Mutants of the filamentous ascomycete Sordaria macrospora showing defects in fruiting- body formation are pertinent sources for the identification of components of this multicellular differentiation process. Recently, we have shown that the sterile mutant pro11 carries a defect in the pro11 gene encoding a multimodular WD40 repeat protein. PRO11 shows significant homology to several vertebrate WD40 proteins, such as striatin and zinedin, which are supposed to function as scaffolding proteins linking signaling and eukaryotic endocytosis. In this study, a yeast two-hybrid screen of a S. macrospora cDNA library with the pro11 cDNA has been used to identify interaction partners of PRO11. With PRO11 as a bait, amongst others we discovered phocein, a putative homologue of the mammalian phocein as putative interaction partner. In mammals, phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Phocein bears a few homologies with the sigma-subunits of clathrin adaptor proteins. Using phocein as a bait in a yeast two-hybrid screen, we identified novel interacting proteins. Amongst the proteins of interest are subunits of the COPI vesicle coat and subunits of the 20S proteasome.


382. Networks of Sordaria macrospora transcription factors: interactions between the MADS box protein MCM1, mating type proteins and the homeodomain transcription factor STE12. Nicole Nolting* and Stefanie Pöggeler. Department of Genetics of Eukaryotic Microorganisms - Institute of Microbiology and Genetics –Georg-August University Göttingen, Germany


The MADS box protein MCM1 controls diverse developmental processes and is essential for fruiting body formation in the homothallic ascomycete Sordaria macrospora. In S. macrospora, deletion of mcm1 results in a pleiotropic phenotype. S. macrospora deltamcm1 strains display a reduced biomass, increased hyphal branching and reduced hyphal compartment length during vegetative growth. Furthermore, they are unable to produce fruiting bodies during sexual development. Little is known about interaction partners of MADS box proteins in filamentous fungi. In S. macrospora, MCM1 interacts with the mating-type protein SMTA-1. In order to identify more interaction partners, we have screened a S. macrospora cDNA library. From this screen, the S. macrospora ste12 gene, a homologue of Saccharomyces cerevisiae STE12, was identified. In S. cerevisiae, STE12 is responsible for activating genes in response to MAP kinase cascades controlling mating and filamentous growth. The ste12 gene of S. macrospora encodes a protein containing both, a homeodomain and a zinc finger region. Deletion of the ste12 gene in S. macrospora neither affects vegetative growth nor fruiting body formation. However, ascus and ascospore development are highly impaired by the deltaste12 mutation. Our data provide an example for the functional divergence of STE12 homologues in filamentous ascomycetes.


383. The Aspergillus nidulans F-box Protein Project Özgür Bayram1, Heike S. Rupprecht1, Marc Dumkow1, Marcia R. v. Z. Kress1, Thomas Linger2, Özlem Sarikaya Bayram1, Gustavo H. Goldman3, Gerhard H. Braus1. 1Institute of Microbiology & Genetics, Georg-August-University Goettingen, Grisebachstr. 8, 37077 Goettingen, Germany; e-mail: obayram@gwdg.de; hruppre@gwdg.de 2Institute of Microbiology & Genetics, Dept. of Bioinformatics, Georg-August-University Goettingen, Goldschmidtstr. 1, 37077 Goettingen, Germany 3Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, CEP 14040-903, Ribeirão Preto, São Paulo, Brazil


F-box proteins are part of E3 SCF (Skp1,Cullin,F-box) ubiquitin ligases which are the specificity factors of the ubiquitin dependent protein degradation machinery. They contain the F-box as conserved N-terminal domain which acts as interface to the Skp1 protein. In addition they have a substrate binding domain for the protein to be ubiquitinated. There are at least 70 F-box-like proteins encoded by the Aspergillus nidulans genome. To address the cellular functions of F-box proteins in A. nidulans, we have started to systematically disrupt F-box encoding genes via homologous gene replacement method. The characterization of the F-box mutants, which has been carried out so far, will be presented.


384. Mutations in mating-type genes greatly decrease repeat-induced point mutation in the fungus Podospora anserina. Sylvie Arnaise, Denise Zickler, Anne Bourdais and Robert Debuchy. Institut de Genetique et Microbiologie, Bat 400, Univ Paris-Sud, UMR 8621, Orsay, F-91405.


RIP (Repeat-Induced Point mutation) and PR (Premeiotic Recombination) are two developmentally regulated silencing processes in filamentous ascomycetes. RIP detects and mutates duplicated sequences, while PR results in deletion of the interstitial sequence between cis-duplicated sequences. These processes take place in the fruiting bodies, between fertilization and premeiotic replication. Fertilization is controlled by the mating types, which have been demonstrated to control also subsequent developmental events preceding meiosis in Podospora anserina and Neurospora crassa. We have investigated the relationship between mating type and silencing processes in P. anserina. We thus tested several mutations in the mating-type genes which affect fruiting-body development, for their effects on RIP and PR. These mutations strongly decrease the frequency of the silencing processes, a clear indication that the two processes are connected. Possible models for the relation between the mating-type genes and the silencing processes will be presented.


385. A mutation in a GDP mannose pyrophosphorylase encoding gene leads to aberrant hyphal growth. *Brian D. Shaw, Gustavo Rebello, Soo Chan Lee, Srijana Upadhyay and Melissa Long. Program for the Biology of Filamentous Fungi, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, 77803, USA.


The temperature sensitive swoN1 mutant results in an aberrant hyphal growth pattern that differs significantly from wild type. Growth from conidia results in germlings with pronounced swollen sub-apical cell compartments as much as 20 micron in diameter. Growth and development at hyphal apices appears to proceed similarly to wild type. The swoN1 mutant was complemented using a genomic library, in which two genes were identified with potential to restore growth of the mutant to wild type levels. These genes are AN1911.3 and AN5586.3 as designated by the Broad Institute A. nidulans genomic database; each encode proteins with predicted similarity to GDP mannose pyrophosphorylase. We designate AN1911.3 swoN since it contains a point mutation at position 1240 bp after start of genomic sequence and 1093 bp after the start codon of coding sequence. This point mutation results in a predicted residue change of serine to a phenylalanine at amino acid 365 of the protein. The swoN1 mutant also exhibits an altered staining pattern of the mannoprotein stain Alcian Blue, relative to wild type. This gene along with swoM, manA and swoA represents the fourth identified in A. nidulans that is likely to participate in protein mannosylation. Each of these mutations results in swollen cells with aberrant hyphal growth, suggesting that cell wall associated mannoproteins are important for maintaining hyphal shape.


386. The role of ADP-ribosylation factors in cell morphogenesis of Aspergillus nidulans. Soo Chan Lee and Brian D. Shaw. Program for the Biology of Filamentous Fungi/Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, 77840


ADP-ribosylation factors (ARFs) are small GTPase proteins with several biological activities including vesicle formation and trafficking and, in yeast, bud site selection. In filamentous fungi, numerous vesicles are found at the growing tips and in the Spitzenkorper where they are thought to be active in secretion of cellular components, endocytosis, and maintenance of tip growth. The exact roles of the ARFs in filamentous fungi have not been established. ArfA::GFP localizes to cellular compartment which may be Golgi. ArfA::GFP was not co-localized with endocytosis machineries indicating ArfA is involved in exocytosis. ArfB::GFP localized to septa, a new cell wall synthesis site. Disruption of ArfB by transposon insertion resulted in loss of polarity during germ tube emergence and hyphal growth. A compromised Spitzenkorper was observed in the mutant. In addition, the arfB::Tn strain displayed delayed endocytosis. The Arf proteins have a conserved N-myristoylation motif. In swoF1 (N-myristoyl transferase) mutant cells, ArfA::GFP and ArfB::GFP showed non-specific localization. In wild type cells, ArfAG2A::GFP and ArfBG2A::GFP, each with a G2A amino acid substitution that disrupts myristoylation, mislocalized. Interestingly overexpression of ArfA protein partially rescues the polarity defect of the swoF1 mutant. These observations suggest that both endocytosis and exocytosis by the Arf proteins play a critical role in hyphal polarized growth in filamentous fungi and N-myristoylation determines subcellular localizations for ArfA and ArfB.


387. A link between N-myristoylation and proteasome activity. Soo Chan Lee and Brian D. Shaw. Program for the Biology of Filamentous Fungi/Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, 77840


Ubiquitin dependent proteolysis is a fundamental biological process regulating the half-life of proteins. The interaction between the 19S and 20S proteasome particles is essential for this activity. A subunit of the 19S particle, RptA in Aspergillus nidulans, has a conserved N-myristoylation motif. Orthologs of RptA are known to control substrate entry and gate the channel of 20S particle, but less is known about the role of N-myristoylation of the protein on its function. In our analyses of genetic suppressors of an A. nidulans N-myristoyl transferase mutant (swoF1), we found a mutation in a 20S proteasome alpha subunit that partially bypasses phenotypic defects of the swoF1 mutant. To investigate the mode of suppression, we used monoclonal anti-ubiquitin antibody to measure the amount of the ubiquitinated proteins. The swoF1 mutant accumulates fewer ubiquitinated proteins than does wild-type. The suppressor mutant, however, accumulated more. In addition, the abnormal hyphal growth phenotypes of the swoF1 mutant were partially bypassed in the presence of MG132, a proteasome inhibitor. These data suggest that N- myristoylation negatively regulates the proteasome activity. We will discuss our ongoing investigation of the role of myristoylation of RptA protein on i) function of the proteasome, ii) the interaction between 19S and 20S proteasome and iii) the localization of 19S proteasome.


388. PRM-1 is involved in germling fusion and is essential for male and female fertility in Neurospora crassa. Andre Fleissner, Spencer Diamond and N. Louise Glass. Department of Plant and Microbial Biology, University of California, Berkeley, USA


Germ tubes of N. crassa conidia show mutual attraction and fusion resulting in the connection of many individuals to one functional structure. In Saccharomyces cerevisiae Prm1p is essential for efficient plasma membrane fusion between mating cells. The N. crassa genome contains a PRM-1 homolog. To study the potential role of the respective protein in germling fusion, we constructed a prm-1 knock out mutant in N. crassa. Germ tubes of germinating prm-1 conidia show normal attraction and make contact similar to the wild type. Quantitative fusion analysis showed that more than 50% of mutant germling pairs remain unfused, suggesting a role of PRM-1 in membrane fusion. Additionally prm-1 mutants exhibit a dominant phenotype during sexual development. The mutant shows normal protoperithecia development but is sterile both as the male or female mating partner in crosses with wild type. Perithecia show initial normal development after fertilization, such as enlargement and darkening, suggesting that fusion between the mating partners is normal. However, further development is arrested and perithecia are devoid of progeny. These data indicate a novel essential role of PRM-1 in post-fertilization events.


389. Characterization of genes expressed during sexual reproduction in Phytophthora. Xiaofan Niu, Waraporn Prakob, and Howard S. Judelson. Department of Plant Pathology, University of California, Riverside CA 92521 USA.


Sexual reproduction serves an important role in the life and disease cycles of Phytophthora since the sexual spores (oospores) result in new genotypes and are an important means of long-term survival. In order to understand the mechanism of sexual development in the genus, P. infestans, the causal agent of potato and tomato late blight, is being used as a model in our study. Microarrays representing much of the P. infestans transcriptome were used to profile gene expression during mating. Approximately 87 genes induced more than ten-fold were identified using these arrays and validated by semi-quantitative RT-PCR. A majority of these genes are induced exclusively during mating, but some are also up-regulated during asexual spore development. Most are expressed during oosporogenesis in the homothallic species P. phaseoli. To understand the temporal and spatial patterns of expression of the genes, the activity of ten genes transcribed only during mating are being tested in P. infestans transformants by fusing their promoters with the GUS reporter gene. Deletions, site-directed mutagenesis, and electrophoresis mobility gel shift assays will be used to define the cis-elements regulating the genes. In addition, the function of selected mating-induced proteins are being tested, such as a RNA-binding protein belonging to the Puf family. Affinity strategies are identifying mRNAs bound and presumably regulated by Puf.


390. Investigations on protein interactions between HET-C and PIN-C during heterokaryon incompatibility in Neurospora crassa. Jianping Sun, Julie Welch and N. Louise Glass. Plant and Microbial Biology Department, University of California, Berkeley, California, 94720


Self/nonself discrimination is a ubiquitous and essential function in both multicellular and microbial species. In filamentous fungi, such as Neurospora crassa, nonself recognition is important during both sexual reproduction and vegetative growth. During vegetative growth, hyphal fusion between individuals that differ in specificity at het loci results in rejection of heterokaryon formation and programmed cell death. Heterokaryon incompatibility (HI) has been shown to reduce the risk of transmission of infectious cytoplasmic elements. In N. crassa, het-c HI is mediated by nonallelic interactions between the closely linked loci, het-c and pin-c, suggesting that physical interaction between HET-C and PIN-C is required for nonself recognition. In order to investigate the possible physical interaction between HET-C and PIN-C, localization and affinity purification (LAP) will be used. The het- c locus encodes a glycine-rich plasma membrane protein, while pin-c encodes a HET domain protein. Population analyses of het-c/ pin-c showed that alleles at het-c and pin-c show severe linkage disequilibrium; pin-c alleles are extremely polymorphic and show only ~50% DNA sequence identity. Analysis of the transcriptional start site of pin-c alleles by 5' RACE showed alternative start sites. These experiments will provide further information towards understanding the mechanism and evolution nonself recognition in filamentous fungi.


391. Classic sexual reproduction and monokaryotic fruiting are differentially regulated by the mediator protein Ssn8 in Cryptococcus neoformans. Yu-Sheng Lin and Wei-Chiang Shen. Department of Plant Pathology and Microbiology, National Taiwan University, Taiwan. wcshen@ntu.edu.tw


Cryptococcus neoformans is an important human fungal pathogen with defined life cycle. The well developed molecular and genomic resources have established it as an excellent model for studying fungal virulence and physiology. Our previous study demonstrated that C. neoformans can perceive blue light and blue light represses sexual filamentation via the conserved white collar proteins, Cwc1 and Cwc2. To further understand how blue light negatively regulates mating filamentation, we conducted a suppressor screening using Agrobacterium-mediated transformation under CWC1 overexpression strain background. After screening 4132 nourseothricin-resistant transformans, we obtained 64 strains with different levels of filamentation phenotypes. In this presentation, we report the characterization of an Agrobacterium transformant AY18 in which T-DNA integration affected the activity of a gene homologous to the Saccharomyces cerevisiae SSN8. To reveal the roles of C. neoformans SSN8 homologue, we created the ssn8 deletion mutants under different genetic backgrounds. SSN8 gene deletion in the CWC1 overexpression strain displayed similar filamentation as the original AY18 T-DNA integrant. Furthermore, the wild type MATalphaƒnand MATa ssn8 mutants were obtained, and both mutants displayed the reduction level of filamentation in classic alpha-a cells mating. More interestingly, dramatic production of monokaryotic filamentation, a rare sexual differentiation involved the same sex of mating cells, was observed in the MATalphaƒnand even in the MATa ssn8 mutants. Our data suggest that C. neoformans SSN8 homologue plays critical but divergent roles in two sexual differentiation processes of C. neoformans. This is the first demonstration of mediator component involved in the differentiation of C. neoformans. Its role related to the Cwc complex is under investigation.


392. The smtA Gene Encoding a Putative SAM Methyl Transferase is Necessary for Progression of both Sexual and Asexual Development of Aspergillus nidulans. Hyo-Jung Kim, Jee-Hyun Kim*, Keon-Sang Chae1, Kwang-Yeop Jahng1 and Dong-Min Han. Institute of Basic Natural Science, Wonkwang University, 1 Div. Biological Science, Chonbuk National University

The smtA(S-adenoslymethionin Methyl Transferase) gene was identified as a multi-copy suppressor of various mutations such as sndE80, silC188, silD6, silE181 and silF174, which overcame the inhibitory effect of stresses on sexual development. The gene is predicted to encode a putative methyl transferase carrying an S-adenosylmethionin binding domain. The smtA ORF consists of 353 amino acids and is disrupted by 9 introns. The null mutant showed defects both in sexual and asexual developments. Conidiation and cleistothecia maturation were delayed and the amounts were reduced. The smtA gene was expressed in high level during vegetative growth and maintained only upto the early stages of asexual and sexual development. The smtA mRNA level was largely reduced in veA or fluG null mutant indicating that the smtA gene expression was positively controlled by those genes. The veA null mutation was epistatic to smtA mull mutation. These results together suggest that SmtA play some role in progression of both sexual and asexual development downstream of VeA and FluG.


393. Analysis of pceA, a regulatory gene for early stage of conidiation in Aspergillus oryzae. Kenichiro Matsushima1, Osamu Hatamoto2, Genryou Umitsuki1 and Yasuji Koyama1. 1Noda Institute for Scientific Research, 2Product Development Division, Kikkoman Corporation. Japan.


The pceA gene of A. oryzae, encoding a putative transcription factor with a GAL4-like Zn(II)2Cys6 motif, is an ortholog of the A. nidulans nosA gene, which is involved in regulation of sexual development. KN16-10, a pceA overexpressing strain of A. oryzae, exhibited an earlier and increased conidiation compared to the wild-type strain. In addition, KN16-10 produced conidiospores in liquid medium, whereas the wild-type strain did not produce them under the same conditions. The expression of conidiation-related gene homologs, such as brlA and flbA, and that of the pceB, a paralog of pceA, were investigated in KN16-10 and the wild-type strain. The Real-Time PCR analysis showed that expression of brlA, a regulatory gene for the early stage of conidiation in A. nidulans, increased in KN16-10 both in liquid and solid medium. At the same time, the expression of pceB increased in parallel with the expression of pceA. These findings suggest that pceA plays an important regulatory role in the early stage of conidiation in A. oryzae by upregulating the expression of brlA. Finally, considering that the expression of pceB seems to be also upregulated by pceA, pceB might be involved in conidiation as well, although its exact function remains unclear.


394. Two G protein alpha subunits share redundant roles in pheromone sensing in Cryptococcus neoformans. Yen-Ping Hsueh, Chaoyang Xue and Joseph Heitman. Department of Molecular Genetics and Microbiology, Duke University, Durham, NC


A vast majority of the cell-environment communication is dependent on the G protein-coupled receptors (GPCR) and their cognate G proteins that act as molecular switches for signaling cascades that respond to different cues. In fungi, one such signaling cascade is the well characterized pheromone response pathway triggered by pheromone/pheromone receptor recognition. Unlike Sacchromyces cerevisiae which expresses two G alpha subunits (one coupled to the pheromone receptor and the other to the nutrient sensor Gpr1), most filamentous ascomycetes and basidiomycetes have three G alpha subunits. Studies in many fungi have successfully identified and characterized the G alpha subunit that acts upstream of the cAMP-PKA pathway, while it has been unclear which of the G alpha subunits is coupled to the pheromone receptor and pheromone response pathway. Here we report that in Cryptococcus neoformans , a pathogenic basidiomycetous yeast, two G protein alpha subunits (Gpa2 and Gpa3) are both involved in pheromone sensing and mating. Genetic analysis reveals that gpa2 gpa3double mutants, but not either the gpa2 or gpa3 single mutant, are sterile in bilateral cross, indicative of a shared role in the mating process. Deletion of GPA3 causes constitutively active pheromone response and hyperfilamentation in haploid cells. Furthermore, the expression of GPA2 and GPA3 is differentially regulated. GPA3 expression is induced in the nutrient-limiting condition while GPA2 is specifically induced during mating. Overexpression of a dominant active GPA2 allele enhances pheromone expression while overexpression of a dominant active GPA3 allele results in mating inhibition. This suggests that Gpa2 and Gpa3 have both redundant and divergent signaling activities governing the sexual development. The incorporation of an additional G protein alpha subunit into the regulatory network enables increased signaling complexity and may facilitate cell fate decisions involving the choice between filamentous sexual and asexual development.


395. Photoregulation of apothecial morphogenesis and patterning in Sclerotinia sclerotiorum. Jeffrey A. Rollins and Selvakumar Veluchamy. Department of Plant Pathology, University of Florida, Gainesville, FL E-mail: rollinsj@ufl.edu


We have undertaken spectral characterization studies to determine the wavelengths of light that positively influence apothecial photomorphogenesis and phototropisms in Sclerotinia sclerotiorum . Our results indicate that wavelengths between 320 and 400 nm (UV-A) are sufficient and necessary for apothecial morphogenesis and positive phototropism. Wavelengths in the blue to green (425-520 nm) range of the spectrum are negatively phototropic at low fluence rates and can de-etiolate stipes at high fluence rates. Insight into the spatial coordination of apothecial development was also investigated by light deprivation experiments. The effect of removing light after the initiation of photomorphogenesis resulted in the abortion of the existent disc development and stage-specific redevelopment of new stipes or discs. To identify the primary photoreceptor(s) governing these photoresponses, we have begun functional characterization of putative photoreceptor-encoding genes identified from the S. sclerotiorum genome sequencing project (https://www.broad.mit.edu/annotation/fgi). Based on Northern hybridization analyses, Ss- cry1 which encodes a member of the CRY-DASH photolyase sub-family, is the first candidate gene targeted for functional analysis. Ss- cry1 transcripts do not occur in dark germinated stipes, but accumulate by 15 min. and peak around 6 h. in stipes exposed to UV-A (3.5µmoles/m 2 /s). No wavelengths in the visible spectrum were found to stimulate significant Ss- cry1 transcript accumulation. We have isolated Ss- cry1 gene replacement mutants and are working to genetically purify and phenotypically characterize these mutants.


396. The roles of fimbrin, fimA, and alpha-actinin, acnA, in hyphal growth. Srijana Upadhyay1, Aleksandra Virag 2, Soo Chan Lee 1, Steven D. Harris2, and Brian D. Shaw 1. 1Dept of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, 77803, USA. 2Plant Science Initiative, University of Nebraska-Lincoln, N234 Beadle Center, 1901 Vine Street, Lincoln, NE 68588, USA.

We investigated the roles of actin (ActA) binding proteins fimbrin (FimA) and alpha-actinin (AcnA) in hyphal growth in A. nidulans. We have used live cell imaging to examine the distribution of ActA::GFP, FimA::GFP and AcnA::GFP. In actively growing hyphae cortical ActA::GFP and FimA::GFP patches are highly mobile and are concentrated near the hyphal apex, but a patch depeleted zone occupies the hyphal apex. FimA::GFP localizes transiently to septa. Localization of both ActA::GFP and FimA::GFP was disrupted after cytochalasin treatment. AcnA::GFP localizes to septal rings and has not been visualized at hyphal apices. A transposon insertional strategy was used to disrupt fimA resulting in germinating conidia with an extended isotropic growth phase followed by simultaneous emergence of multiple germ tubes. Colonies of the fimA disruptants are compact and conidiate poorly. Deletion of acnA, results in a severe hyphal growth defect leading to compact colonies that not sporulate. Endocytosis was severly impaired in the fimA disruption strain but was unaffected in the acnA deletion strain. ActA::GFP distribution in the fimA disruption strain, results in abnormal ActA::GFP distribution. A model for the roles of these proteins in hyphal growth is proposed.


397. Involvement of G-alpha subunits of heterotrimeric G proteins and adenylyl cyclase in fruiting body development of Sordaria macrospora. Jens Kamerewerd, Malin Jansson, Minou Nowrousian, Stefanie Pöggeler and Ulrich Kück. Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Universitätsstr. 150, 44780 Bochum, Germany, jens.kamerewerd@rub.de


Heterotrimeric G proteins play an important part in signal transduction and regulation of fungal differentiation and growth. The relative contribution of Sordaria macrospora G-alpha subunits GSA1, GSA2 and GSA3 in vegetative and sexual development has been shown through analysis of strains deleted for various combinations of the corresponding genes. All mutants lacking one of the G-alpha subunits are restricted in vegetative growth. Strains lacking the gsa1 gene in addition to one of the other subunits are completely sterile. Defects caused by the deletion of gsa2 are observed only in combination with loss of gsa1, suggesting that this G-alpha subunit acts complementary to the other subunits in S. macrospora. GSA3 seems to be involved in the germination or viability of ascospores. To characterize the possible involvement of adenylyl cyclase as downstream effector, we generated a mutant lacking the cr1 gene. Besides restricted mycelial growth, the perithecia of the deletion strain accumulate mainly under the surface of solid media. The mutant is fertile, but germination of ascospores is reduced. Analysis of double knockout mutants with G-alpha subunits and adenylyl cyclase suggests a major role of GSA1 and CR1 for perithecial development as the double mutant develops no mature perithecia. The sum of our results indicates that G-alpha subunits and adenylyl cyclase are not essential for viability of this filamentous fungus but are important factors for sexual propagation.


398. The Woronin body-associated PRO40 protein is required for fruiting body development in Sordaria macrospora. Ines Engh 1, Christian Würtz2, Konstanze Witzel-Schlömp 1, Hai Yu Zhang 1, Birgit Hoff 1, Minou Nowrousian 1, Hanspeter Rottensteiner 2 & Ulrich Kück1. Lehrstuhl für Allgemeine und Molekulare Botanik 1 und Institut für Physiologische Chemie, Abteilung für Systembiochemie, 2 Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, Germany, ines.engh@rub.de


Fruiting body formation in ascomycetes is a highly complex process that is under polygenic control. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant of the homothallic ascomycete Sordaria macrospora is defective in a 120 kDa WW domain protein that plays a pivotal role in fruiting body maturation. Complementation analysis with different pro40 mutant strains using full-sized or truncated versions of the wild-type pro40 gene revealed that the C-terminus of PRO40 is crucial for restoring the fertile phenotype. Microscopic investigations using DsRed or GFP polypeptides showed a co-localization of PRO40 with HEX-1, a Woronin body specific protein. However, fluorescence microscopy, sedimentation and immunoblotting analyses as well as growth tests with the S. macrospora pro40 mutant and Neurospora crassa mutants of the pro40 homolog so revealed that Woronin body integrity is not affected in mutant strains. We discuss the function of PRO40 in fruiting body formation.


399. Pheromone functions of Gibberella zeae. Jungkwan Lee 1, Robert L. Bowden 2, and John F. Leslie 1. 1 Department of Plant Pathology, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, KS66506-5502, 2 USDA-ARS Plant Science and Entomology Research Unit, Throckmorton Plant Sciences Center, Kansas State University, Manhattan, KS66505-5502.


In heterothallic Ascomycete fungi, mating specificity for sexual reproduction is controlled by two idiomorphs (MAT- 1 and MAT-2). Two sex pheromone/receptor pairs function in recognition and attraction of opposite mating types. In the homothallic fungus Gibberella zeae (Fusarium graminearum), the MAT locus has been rearranged such that both of the idiomorphic alleles are next to one another on the same chromosome. Our objective was to characterize pheromone precursor genes (ppg1 and ppg2) and pheromone receptor genes (pre1 and pre2) from this fungus. ppg1 is expressed in germinating conidia and mature ascospores of G. zeae, but ppg2 expression was not detected in any cells. pre2 was expressed in all cells including mycelial cells induced on carrot agar, whereas pre1 was weakly expressed only in mature ascospores. Deletion of ppg1 or pre2 (Δppg1 or Δpre2) reduced the number of perithecia but Δppg2 and Δpre1 mutants had no discernable change in morphological phenotype or sexual fertility. A Δppg1/Δppg2 double mutant has a phenotype similar to that of the Δppg1 single mutant, suggesting that neither pheromone is essential for either male or female fertility. Δppg1 or Δppg2 in combination with a partially disabled MAT allele did not result in any other changes in either male or female fertility beyond those expected for the Δppg1 or Δppg2 mutations alone. We conclude that one of the pheromone/receptor pairs (ppg1/pre2) found in many Ascomycetes has a role in, but is not essential for, selfing and outcrossing in G. zeae, whereas the other pheromone/receptor pair (ppg2/pre1) no longer has a function in sexual reproduction.


400. Catalases during the sexual cycle of the filamentous fungus Podospora anserina. Eric Espagne, Anne Bourdais, Denise Zickler and Veronique Berteaux-Lecellier. Institut de Genetique et Microbiologie, Universite Paris-Sud-11, UMR8621,F-91405 Orsay, France.

Reactive oxygen species are not only toxic molecules but they are also signaling molecules that control various processes such as pathogen defense, programmed cell death and cell differentiation. Our previous studies of the pex2 peroxisome assembly mutants, which dikaryotic cells never engage into karyogamy and meiosis but remain in a proliferative state, suggested a role of hydrogen peroxide in P. anserina differentiation. An increase in hydrogen peroxide level might be responsible for the failure in the meiotic engagement and/or for the anomalous maintenance of the mitotic state. To test this hypothesis and more generally to study the role of hydrogen peroxide during P. anserina life cycle, we proposed to increase or reduce the amount of hydrogen peroxide by deregulating genes involved in its production or degradation. In a first attempt, we focused on catalase genes which encode enzymes that convert hydrogen peroxide to oxygen and water. We have identified 4 monofunctional catalases and one bifunctional catalase- peroxidase in the P. anserina genome. Expression of the 5 catalases has been followed during the sexual development and 3 of them have been deregulated. Characterization of the strains is under investigation. The results as a whole will be discussed in the frame of the role of hydrogen peroxide in the fungus life cycle.


401. Peroxisomal ABC transporters and beta-oxidation in the filamentous fungus Podospora anserina. Stephanie Boisnard, Eric Espagne, Denise Zickler, Anne-Laure Riquet, Anne Bourdais and Veronique Berteaux-Lecellier. Institut de Genetique et Microbiologie, Universite Paris-Sud, UMR8621, F-91405 Orsay, France. The first two authors contributed equally to the work.


The two P. anserina peroxisomal ABC transporters pABC1 and pABC2 display respectively 39% and 45% identity with the human ALDP peroxisomal ABC transporter (adrenoleucodystrophy). Although ALDP's function remains unclear, various data suggest that it could be involved in the transport of acyl-CoAs across the peroxisomal membrane where they will be oxidized. We have shown that overproduction of human PMP70 and pABC1 transporters in P. anserina, act as partial suppressors of the differentiation defect of the pex2 peroxisomal assembly mutant (Zellweger syndrome in humans). To determine more precisely the involvement of peroxisomal ABC transporters in both acyl-CoA transport across the peroxisomal membrane and their possible role in differentiation, we knocked out both pABC1 and pABC2 genes. Contrary to their counterpart in budding yeast, the single and double deleted strains do not display growth deficiency on oleic acid as sole carbon source. The further discovery of a mitochondrial beta-oxidation in filamentous fungi suggested that this pathway might compensate for the lack of peroxisomal oleic acid oxidation in the delpABC1 delpABC2 double mutant and lead us to characterize mutants affected in both mitochondrial and peroxisomal beta-oxidation pathways.


402. Identification of two genes in Gibberella zeae necessary for spore discharge. Heather Hallen1, Brad Cavinder1, and Frances Trail1,2. 1Department of Plant Biology and 2Department of Plant Pathology, Michigan State University, East Lansing, MI 48824-1312 hallenhe@msu.edu


We have identified two genes, encoding a calcium channel and a transcription factor, which are necessary for ascospore discharge in Gibberella zeae. Mutants of both genes are incapable of forcible spore discharge, but produce phenotypically normal perithecia and viable ascospores. Expression analysis of the mutants revealed that they do not regulate each other. Affymetrix GeneChip analysis yields hundreds of genes with 4-fold or greater expression difference between each mutant and wild-type (p < 0.003), suggesting that they play a significant role in both gene activation and repression in G. zeae. Interestingly, expression of many of the genes is affected in both mutants. These genes will allow us to begin to unravel ascus structure and function.


403. Defects in light regulation of sexual and asexual development in Coprinopsis cinerea. Wassana Chaisaena, Monica Navarro-Gonzalez, and Ursula Kües. Institute of Forest Botany, Georg-August-University, Göttingen, Germany


Fruiting body development in Coprinopsis cinerea is adapted to the light and dark phases of the normal day-night rhythm. If young developing structures (day 1 to day 4 of development) are transferred into constant darkness, normal cap and stipe differentiation arrests. Instead, the stipe base proliferates to give an elongated structure known as “etiolated stipe” or “dark stipe”. Recessive mutations in genes dst3 and dst4 cause such phenotype in the normal day- night regime. These genes are distinctive from dst1 and dst2 identified before in Japan. From morphological analysis of the cap tissues of the etiolated stipes, dst3 and dst4 appear to act later in development (at day 3 and day 4 of development, respectively) than dst1 and dst2 (defects at day 2 of development). Unlike dst3 and dst4, dst1 and dst2 also act in light-control of asexual sporulation. Only the dst2 mutant is totally blind and forms etiolated stipes also under constant light incubation. Normally in constant light, fruiting body development is totally repressed. WC and MNG acknowledge scholarships from the Rajamangala University of Technology (Thailand) and CONACYT (Mexico), respectively. The Deutsche Bundesstiftung Umwelt financially supported the laboratory.


404. A constitutively activated Ras-protein has multiple effects on vegetative and sexual development in Coprinopsis cinerea. Prayook Srivilai, Monica Navarro-Gonzalez, and Ursula Kües. Institute of Forest Botany, Georg-August-University, Göttingen, Germany


A mutation in codon 19 of a cloned ras gene of Coprinopsis cinerea altering a glycine into a valine codon (rasval19) gives rise to a constitutively activated GTPase. The rasval19 mutant allele has been transformed into different monokaryons of C. cinerea and found to affect the mycelium of monokaryons and, after mating, also of dikaryons. Growth rates were reduced, hyphae lost growth orientation, many abnormal short side-branches were formed and mycelial growth was invasive. rasval19 dikaryons had many abnormal unfused clamps at hyphal septa whose hyphal tips tended to further elongate, thereby passing the subapical pegs formed for clamp cell fusion. In fruiting body development, tissue formation within the primordia was altered. Gills appeared to be oversized compared to the upper stipe tissues and the plectenchyma in the pileus. Fully developed fruiting bodies were minute (1-1.5 cm) compared to normal 4-5 cm sized fruiting bodies. Basidiospore production was affected in quantity and quality. Many basidiospore were abnormal small. PS and MNG acknowledge scholarships from the Mahasarakham University (Thailand) and CONACYT (Mexico), respectively. The Deutsche Bundesstiftung Umwelt financially supported the laboratory.


405. Microarray-based gene expression profiling during fruitbody initiation in Agaricus bisporus. B. Herman, D.C. Eastwood, S. Sreenivasaprasad, A. Dobrovin-Pennington, R. Noble, K.S. Burton. Warwick HRI, University of Warwick, Wellesbourne, CV35 9EF, UK.

Homobasidiomycete Agaricus bisporus goes through a phase change from vegetative mycelium to reproductive growth to form fully differentiated mushroom fruitbodies. The molecular mechanisms controlling this process are poorly understood. In the early stages of the development, defined as mushroom initiation, a series of environmental parameters play key roles. These include a reduction in temperature and CO2 concentration, heavy watering and biotic factors (e.g. microbial flora influencing the levels of eight-carbon volatiles). Little is known about how each parameter affects differentiation in A. bisporusand how the fungus senses and responds to the changes in the environment. This investigation is aimed at understanding the molecular basis of the phase change and to determine the effect of the environmental triggers on A. bisporus development and gene expression.Stage-specific cDNA suppression subtractive hybridisation procedure was used to identify genes (more than 300 unisequences) differentially expressed during the mushroom initiation process. A database of more than 1000 A. bisporus genes representing the 300 unisequences and other cDNAs and ESTs was established. Bioinformatics was used to analyse these sequences and to design gene-specific probes which were processed through the Agilent Technologies eArray software to create a 60 base oligonucleotide microarray. The custom array is being used for comparative gene expression profiling during the initiation process in A. bisporus under different growth regimes.


406. Molecular biology of recognition between mating partners in Mucor-like fungi. Johannes Wostemeyer, Anke Burmester, Christine Schimek, Kornelia Schultze, Jana Wetzel. Institute of Microbiology, Chair of General Microbiology and Microbe Genetics, FSU-Jena, Germany.


Trisporoids are a general principle of communication between mating partners in mucoralean fungi. Their synthesis starts by cleavage of beta-carotene by a dioxygenase reaction and procedes via several reactions towards trisporic acid. Several of these reactions are specific for one of the mating types, resulting in the necessity for cooperative synthesis of the actice mating signal, trisporic acid. We have identified and cloned three of the genes encoding functions in the pathway from carotene to trisporic acid. Especially the TSP1 gene for dihydromethyltrisporate dehydrogenase has been studied in detail with respect to its regulation. The gene product is active predominantly in the minus mating type in three different species analyzed, Parasitella parasitica, Mucor mucedo and Absidia glauca. Although the gene is controlled in a mating type-specific manner in all three organisms, the levels of its regulation differ considerably in these species between posttranscriptional reguation in the mycoparasite P. parasitica, posttranslational regulation in M. mucedo and a tight temporal regulation in A. glauca. In none of these species regulation of TSP1 took place at the transcriptional level. Data on the regulation of the TSP2 gene for the minus type- specific dihydrotrisporin dehydrogenase and the gene for the carotene dioxygenase will also be presented.


407. Characterisation of FlbB, a putative B-zip type transcription factor needed for early stages of conidiation induction in Aspergillus nidulans. Oier Etxebeste1, Aitor Garzia1, Min Ni2, Reinhard Fischer3, Jae-Hyuk Yu2, Eduardo Espeso4 and Unai Ugalde1. 1Faculty of Chemistry, San Sebastian, Spain 2FRI, Univ. Wisconsin, Madison 3Dept Applied Microbiol.Univ. Karlsruhe, Germany 4CIB, CSIC, Madrid, Spain


Conidiation in Aspergillus nidulans requires the activation of brlA, which in turn induces expression of other genes required for development. Genes expressed during vegetative growth participate in sensing the right conditions for brlA induction. These include genes collectively called flb (their mutants are fluffy with low brlA expression). Whilst one such mutant has been linked with G-protein signaling (flbA), others encode transcription factors with as yet unknown roles. We have cloned flbB as AN 7542.3 encoding a putative 427aa B-zip type transcription factor. The N-terminal B-zip DNA binding domain shows high similarity with that of Pap1, a transcription factor involved in adaptive responses to mild oxidative stress in Schizosaccharomyces pombe. In addition, FlbB also shares similarities with Pap1 at the C-terminal region, where two cysteine residues determine proper cellular localisation in response to mild oxidative stress. The pattern of expression, cellular distribution of FlbB, and relationship with other flb genes will be presented, with preliminary evidence that it may be responding to developmentally significant ROS signals.


408. The bd mutation in Neurospora crassa is a dominant allele of ras-1 implicating ras-signaling in circadian output. Luis F. Larrondo, William J. Belden, Allan C. Froehlich, Mi Shi, Chen-Hui Chen, Jennifer J. Loros, and Jay C. Dunlap. Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA. Luis.F.Larrondo@dartmouth.edu


The band (bd) mutation in Neurospora crassa has been an integral tool in the study of circadian rhythms for the last 40 years by enabling clear visualization of circadianly regulated conidia production (banding). The bd mutation was mapped using single nucleotide polymorphisms (SNPs), cloned, and determined to be a point mutation in the ras-1 gene, changing threonine 79 to isoleucine. There is a defect in light-regulated gene expression in ras-1bd mutants suggesting that the Neurospora photoreceptor, the WHITE COLLAR complex, is a downstream target in RAS signaling. There are also increases in the circadian regulated transcription of both wc-1 and fluffy consistent with the idea that regulators of conidiation are elevated in ras-1 bd . Comparison of bd with dominant active and dominant negative ras-1 mutants and in biochemical assays of RAS function indicates that RAS-1 bd has only minor alterations in GTP/GDP exchange and GTPase activity. In addition to the bd mutation, we found that oxidative stress can also increase conidia production suggesting a link between generation of reactive oxygen species (ROS) and RAS-1 signaling. Altogether, the data indicate that multiple interconnected signaling pathways including RAS signaling and elevated ROS levels can elicit heighten conidiation and regulate the amplitude of circadian and light-regulated gene expression in Neurospora.


409. Evidence for a lectin-mediated defense of mushrooms against predators, parasites and pathogens. Martin Wälti1, Anke Grünler1, Michaela Bednar1, Alex Butschi2, Michael Hengartner2, Markus Aebi1 and Markus Künzler1. 1Institute of Microbiology, ETH Zürich, Switzerland. 2Institute of Zoology, University of Zürich, Switzerland.

The galectins CGL1 and CGL2 as well as the homologous lectin CGL3 of the homobasidiomycete Coprinopsis cinerea are strongly induced during sexual development and highly enriched in the fruiting body. Despite this differential expression pattern in time and space, neither ectopic expression during vegetative growth nor silencing of the respective genes had any obvious effect on fruiting. These results make a role of these lectins in mushroom development unlikely. In search of an alternative function, we found a pronounced toxicity of the galectin CGL2 towards the nematode Caenorhabditis elegans. The phenotype of the toxicity resembles the one of the nematotoxic crystal toxins from Bacillus thuringiensis. We are currently investigating the specificity and the mechanism of this CGL2-mediated toxicity. Moreover, preliminary data show that CGL3, which has a different sugar binding specificity than CGL2 and reveals no toxicity towards C. elegans, is able to specifically agglutinate some bacteria and to inhibit vegetative growth of some fungi. These results suggest that mushrooms and probably also other multicellular fungi contain, analogous to plants, a lectin-mediated defense against predators, parasites and pathogens that might be ecologically important. We are interested in the spectrum of defense molecules (with emphasis on lectins) and the regulation of this defense system in C. cinerea .


410. Role of WetA During Conidiation and Yeast Growth in the Thermo-dimorphic Fungal Pathogen Penicillium marneffei. David Cánovas1, Mariam Ghosn, Luke Pase2 and Alex Andrianopoulos. Department of Genetics, University of Melbourne, Victoria 3010, Australia 1 Current address: Departamento de Biotecnologia Microbiana, Centro Nacional de Biotecnologia - CSIC, Campus UAM - Cantoblanco, Madrid 28049, Spain. 2 Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia.


Penicillium marneffei is a thermally dimorphic fungal pathogen of humans. At 25ºC the saprophitic form is filamentous in growth and composed of hyphal cells which are usually multinucleate and separated by incomplete septa. At 37ºC, the pathogenic form is an elongated unicellular and uninucleated yeast cell, which divides by fission. Besides the dimorphic switch, P. marneffei can also undergo asexual development (conidiation) at 25ºC resulting in the production of specialized structures called conidiophores, which bear the infectious form (conidiospores). The different cell types produced during the developmental programmes are strictly controlled. We cloned the homologue of the Aspergillus nidulans conidiation gene wetA from P. marneffei. Expression of wetA is dependent on the developmental transcriptional activators BrlA and AbaA during conidiation, but it is only AbaA- dependent during yeast growth. The wetA gene is not expressed during vegetative growth at 25ºC. Accordingly, a deletion of the gene showed morphological defects during conidiophore development and yeast growth but not during hyphal growth. A proportion of the conidiophores displayed abnormal morphologies, including cotton-ball shapes, aberrant cell types and multinucleated conidia. Over time conidial viability decreased and conidia displayed inappropriate germination. At 37ºC a proportion of yeast cells were multinucleated. The wetA deletion mutant yeast cells were generally shorter in length than wild type cells, had an increased tendency to lyse, and were more sensitive to SDS. The phenotype of the deletion strain at 37ºC can be explained by defects in cell wall/membrane structure and this is supported by TEM studies.


411. Mechanisms by which the adhesin Flo11 determines morphology in S. cerevisiae . Yang Yang, Sungsu Lee and Anne M. Dranginis. Department of Biological Sciences, St. John’s University, Queens, NY. drangina@stjohns.edu


Flo11, a cell wall adhesion molecule, is an important determinant of cellular morphogenesis and development in S. cerevisiae. In various strains of yeast, Flo11 may be required for pseudohyphae formation, biofilm formation, formation of mats containing specialized hub and spoke structures on semi-solid medium, and unusual colony morphologies. We find, however, that only a subset of these phenotypes are displayed by any given Flo11-expressing strain. In strain Sigma 1278b, for example, Flo11 is required for the formation of the hub and spoke structures but this strain does not flocculate. Strain S. cerevisiae var. diastaticus, on the other hand, requires FLO11 for flocculation but does not form hub and spoke structures. The Flo11-dependent phenotypes of strain SK1 include the development of a lacy colony morphology which is not exhibited by other strains. We are using these differences in strain-specific Flo11 phenotypes to investigate the mechanisms by which this adhesin determines morphology. In one approach, the domains of Flo11 have been separately purified and tested in vitro for adhesion to cells and other substrates. The amino terminal domain has proven to be the adhesion domain, and its cellular receptor is the mannosylated central domain of Flo11 on adjacent cells. Primary sequence differences in these domains correlate with phenotypic differences in some strains; gene- swap experiments are being used to determine whether these sequence differences suffice to confer phenotypic differences. Levels of expression and subcellular distribution of Flo11 in each strain are also being determined.



Animal and Plant Pathogens; Fungal-Host Interactions



412. Modulating the occurrence and distribution of ROS during nitrogen deprivation and early developmental stages of Colletotrichum acutatum infecting strawberry. Sigal Horowitz Brown1,2, Oded Yarden1, Natan Gollop3, Songbi Chen3, Aida Zveibil2, and Stanley Freeman2. 1Dept. of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100; 2Dept. of Plant Pathology, and 3Dept. of Food Science, ARO, The Volcani Center, Bet Dagan 50250, Israel. Freeman@volcani.agri.gov.il


Significant alterations in the abundance of different proteins were found during wildtype Colletotrichum acutatum appressoria formation, under nitrogen limiting conditions or complete nutrient supply, when compared to a nonpathogenic mutant. CuZn superoxide dismutase and glutathione peroxidase were up-regulated at the appressoria formation stage and nitrogen limiting conditions, compared to growth in complete nutrient supply, whereas abundance of bifunctional catalase was up-regulated predominantly at the appressoria formation stage. Fungal ROS (reactive oxygen species) was detected within germinating conidia during strawberry host pre-penetration, penetration and colonization stages. Application of an exogenous antioxidant quenched ROS production and reduced appressoria formation frequency. A general up-regulation in metabolic activity during appressoria formation and under nutrient deficiency conditions was observed. Glyoxylate cycle and lipid metabolism–related proteins (malate dehydrogenase, formate dehydrogenase, and acetyl coA acetyltransferase) showed enhanced levels during the appressoria production phase in contrast to down- regulation of isocitrate dehydrogenase. This study indicates that developmental processes occurring under nutritional deprivation in C. acutatum infecting strawberry are facilitated by metabolic shifts which may be mediated by fungal ROS production.


413. Development of a Molecular Genetic Linkage Map for Colletotrichum lindemuthianum and Segregation Analysis of Two Avirulence Genes. Francisco Luna-Martínez1, Raúl Rodríguez- Guerra2, Mayra Victoria-Campos1, June Simpson1. 1 Department of Genetic Engineering, CINVESTAV, Unidad Irapuato, A.P. 629, Irapuato, Guanajuato, México. 2 Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), A.P. 112; Celaya, Guanajuato, México. jsimpson@ira.cinvestav.mx


A framework genetic map was developed for Colletotrichum lindemuthianum, the causal agent of anthracnose of common bean (Phaseolus vulgaris L.). This is the first genetic map for any species within the family Melanconiaceae and the genus Colletotrichum and provides the first estimate of genome length for C. lindemuthianum. The map was generated using 106 haploid F1 progeny derived from crossing two Mexican isolates differing in two avirulence genes (AvrclMex and AvrclTO). The map comprises 165 AFLP markers covering 1897 cM with an average spacing of 11.49 cM. The markers are distributed over 19 major linkage groups containing between 5 and 25 markers each and the genome length was estimated to be 3241 cM. AvrclMex and AvrclTO segregate in a 1:1 ratio supporting the gene for gene hypothesis for the incompatible reaction between C. lindemuthianum and P. vulgaris, but could not be incorporated into the genetic map. This map forms the basis for the development of a more detailed C. lindemuthianum linkage map which would include more markers and allow the location of genes previously characterized in this species.


414. The Unfolded Protein Response and the transcription factor Hac1p, in Candida albicans. Tithira T. Wimalasena1, Thomas Guillemette1, 3 , Andrew Plumridge1, Brice Enjalbert2, Alistair J. P. Brown2 and David B. Archer1. 1School of Biology, University of Nottingham, University Park, Nottingham, NG7 2RD, UK 2Aberdeen Fungal Group, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK 3Present address: UMR 77 Pathologie Vegetale, Faculte des Sciences, Université d'Angers, France


The Unfolded Protein Response (UPR) is a stress response that regulates the folding of proteins in the endoplasmic reticulum and the delivery of secretory proteins to the cell surface and exterior. Candida albicans is an opportunistic human fungal pathogen: its morphology and the secretion of enzymes and cell surface components are important factors in its virulence. We therefore aimed to describe the UPR in C. albicans and to determine its importance in nitrogen-dependent processes in C. albicans, such as the yeast-mycelial transition. In S. cerevisiae, the UPR induces the dimerization of a ER-resident type I transmembrane protein, Ire1p which activates splicing of HAC1u, to HAC1i mRNA by excising a 252-bp non- conventional intron towards the 3’end of the mRNA, which produces the functional Hac1p. This activates transcription of UPR-sensitive genes via direct binding to the cis-acting UPR element (UPRE) to activate its target genes. In Aspergillus niger, a similar mechanism is observed, but the intron is limited to 20-bp. In addition to this unconventional splicing in A. niger , a 5’-UTR truncation occurs which affects the size of the transcripts. Here we report the characterization of the gene encoding Hac1p in C. albicans. In this study, the UPR was induced by treating C. albicans yeast cells with dithiothreitol (DTT) and tunicamycin. The analysis of cDNA made from HAC1 mRNA shows that there is a difference between the HAC1i and HAC1u towards the 3’ end of the mRNA: HAC1i mRNA lacks a 19-base intron that is present in HAC1u. Furthermore, we deleted both copies of the HAC1 gene in C. albicans to create a null mutant strain. We confirm the role of the UPR in the delivery of cell surface proteins and show also that the UPR has a role in determining the morphology of C. albicans .


415. Aso1 is required for anastomosis and pathogenicity in Alternaria brassicicola. T.K. Mitchell1, K. D. Craven2, C. Yangrae3, C. B. Lawrence3. 1North Carolina State University, Dept. of Plant Pathology, Raleigh, NC 27695; 2Samuel Roberts Noble Foundation, Ardmore, OK; 3Virginia Bioinformatics Institute, Blacksburg, VA.


Vegetative fusion, or anastomosis, has been documented for many fungal species and provides an efficient conduit for the movement of nutrients, proteins, signaling molecules, and even viruses through a colony. Using nitrate-utilization mutants of the necrotrophic plant pathogen Alternaria brassicicola and two related species, we characterized anastomosis capacity and investigated the possibility of an operating vegetative incompatibility system. All eight A. brassicicola isolates tested, as well as Alternaria mimicula and Alternaria japonica, were capable of self- anastomosis, with only two isolates capable of non-self fusion. In no case did we observe cell death or the development of a barrage between incompatible isolates. Interrogation of the genome sequence resulted in the identification of het and so-1 homologs. The het genes are central to the vegetative compatibility system in Podospora anserina. Functional characterization of our deletion mutant of aso-1 (Alternaria so-1) showed a loss of the ability to undergo self-anastomosis. Additionally, the deletion mutants were no longer pathogenic on cabbage leaves. Here we present our analysis of the aso-1 locus, its functional characterization, and a model of how vegetative compatibility is functioning in this important plant pathogen.


416. Interaction with plants stimulates mating of the human fungal pathogen Cryptococcus neoformans. Chaoyang Xue1, Yasuomi Tada2, Xinnian Dong2, and Joseph Heitman1. 1Department of Molecular Genetics and Microbiology, 2Department of Biological Science, Duke University, Durham, NC 27710. Email: xuecy@duke.edu


Cryptococcus species such as C. neoformans and C. gattii are globally distributed human fungal pathogens that primarily afflict immunocompromised individuals. How and why this human fungal pathogen associates with certain plant species, such as Eucalyptus trees, in its environmental niche has been mysterious. Here we established defined Cryptococcus-Arabidopsis and Cryptococcus-Eucalyptus interaction systems. Fungal cell proliferation and mating occurred on plant surfaces. The presence of plants (Arabidopsis or Eucalyptus) stimulated fungal mating even under constant light conditions, indicating that plant materials overcome the known inhibition of mating by light. Mating efficiency of C. gattii strains was markedly enhanced by the presence of plants. Completion of the sexual cycle in nature enables production of basidiospores - the suspected infectious propagule. On the plant side, dwarfing and chlorosis of Arabidopsis were often observed following infection with a fungal mixture of two opposite mating type strains under sterile growth conditions. The plant jasmonate- dependent defense pathway was induced by fungal infection, while the salicylate-dependent defense pathway was partially repressed. These findings reveal that Cryptococcus can parasitically interact with plants, compromising pathogen- induced resistance mechanisms to gain benefit from plants, especially during mating. Plant derived materials including myo-inositol and indole-3-acetic acid (IAA) have been discovered to stimulate mating, which may explain the association in nature between plants and this human fungal pathogen. The study of how fungal cells sense plant signals is underway.


417. Differences in mating of Cryptococcus species on pigeon guano defines their ecological niches. Kirsten Nielsen, Anna Lisa De Obaldia, and Joseph Heitman. Department of Molecular Genetics and Microbiology Duke University Medical Center, Durham, NC


Cryptococcus neoformans is a pathogenic yeast that predominantly infects immunocompromised individuals. C. neoformans exists in two varieties - grubii (serotype A) and neoformans (serotype D), and is commonly isolated from pigeon guano (PG). By contrast, Cryptococcus gattii often infects immuncompetent individuals, and has been traditionally associated with other environments, including eucalyptus trees. C. neoformans and C. gattii diverged ~ 40 million years ago. The ecological niche a species occupies is determined by resource requirements and the physical conditions necessary for survival. The ecological niche to which an organism is most highly adapted is the realized niche, while the complete range of habitats the organism can occupy is considered the fundamental niche. To determine whether C. neoformans and C. gattii occupy different ecological niches, we examined the growth and development of these species on PG. This study shows that PG can support growth of C. neoformans and C. gattii. However, we found that C. neoformans exhibits prolific mating on PG, while mating of C. gattii is dramatically inhibited. The observation that C. neoformans has adapted to complete the lifecycle on pigeon guano suggests that PG represents a realized niche of C. neoformans but not C. gattii.


418. The role of the small GTPase Rheb in the control of the development of the phytopathogenic fungus Botrytis cinerea. Heber Gamboa Meléndez1, Weronika Podlesna1, Michel Droux2 and Géraldine Mey1. 1Laboratoire de Pathogénie des Champignons Nécrotrophes, Université Claude Bernard- Lyon 1, UMR CNRS-INSA- UCBL 5122, 10 rue Dubois, Bât Lwoff, 69622 Villeurbanne Cedex, France. E-mail: hgamboa@univ-lyon1.fr 2 Laboratoire Mixte CNRS/Bayer CropSciece, Centre de Recherche de la Dargoire, Bayer CropScience, 14-20 rue Pierre Baizet, 69263, Lyon, France


The success of the infection process partly relies on the ability of fungal pathogens to adapt their metabolism to the use of the nutrients present in the host. Necrotrophic fungi have expended this skill to the complete colonization of the target plant subsequent to the enzymatic maceration of plant tissues. The nutrients released during the degradation process may both be included in the anabolic reactions required for the parasitic growth and act as signal molecules. The growth and the differentiation of eukaryotic cells is regulated via the TOR (Target of Rapamycin) dependant signalling cascade which seems to be conserved from yeast to humans: in the presence of extracellular nutrients, the small GTPase Rheb (Ras homologue enriched in brain) acts as a molecular switch and activates the kinase TOR. We investigated the role of the TOR pathway in the control of the growth of necrotrophic fungi, using as a model Botrytis cinerea, the causal agent of the gray mold disease on more than 200 plant species. RNA interference was used to decrease the expression of the gene encoding Rheb in B. cinerea. The saprophytic growth of the rheb transformants appeared affected: the growth and the sporulation rates were reduced. The infection assays showed that the rheb mutants were less virulent than the wild type, indicating that the parasitic growth was also altered. Mutation of the gene rhb1 in Saccharomyces cerevisiae (Rheb orthologue) increases the uptake of amino acids. Complementation experiments of the yeast rhb1 mutant revealed that Rheb from B. cinerea restored the down regulation of certain but not all amino acid transport functions. The involvement of Rheb in the control of amino acid uptake in B. cinerea is currently further investigated using HPLC experiments and expression studies of transporter encoding genes by qRT-PCR. Taken together, these results will contribute to a better understanding of the role of Rheb in the control of amino acid metabolism and its involvement in the necrotrophic development of B. cinerea.


419. Mannitol is required for asexual sporulation in Stagonospora nodorum. Waters, Ormonde D.C., Solomon, P.S., Trengove, R.D. and Oliver, R.P. ACNFP, SABC, DHS, Murdoch University, Perth 6150, WA, Australia.


 The physiological role of the mannitol cycle in the wheat pathogen S. nodorum has been investigated by reverse genetics and metabolite profiling. A putative mannitol 2-dehydrogenase gene was cloned and disrupted. The resulting strains lacked all detectable mannitol dehydrogenase activity. The mdh1 strains were unaffected for mannitol production but surprisingly, were still able to utilize mannitol as a sole carbon source suggesting a hitherto unknown mechanism for mannitol catabolism. The mutant strains were not compromised in their ability to cause disease or sporulate. A previously developed mannitol 1-phosphate dehydrogenase disruption construct was introduced into the mdh1 background, resulting in a strain lacking both enzyme activities. The mpd1mdh1 strains were unable to grow on mannitol and produced only trace levels of mannitol. The double mutant strains were unable to sporulate in vitro when grown on minimal medium for extended periods. Deficiency in sporulation was correlated with the depletion of intracellular mannitol pools. Significantly sporulation could be restored with the addition of mannitol. Pathogenicity of the double mutant was not compromised, although like the previously characterised mpd1 mutants, the strains were unable to sporulate in planta. These findings question not only the currently hypothesised pathways of mannitol metabolism, but also identify for the first time that mannitol is required for sporulation of a filamentous fungus.


420 . The role of the fungal extracellular matrix in lesion development on plants. Giovanna Sassi, Natalie Catlett, Charlotte Bronson, and B. Gillian Turgeon. Cornell University, Department of Plant Pathology, gs242@cornell.edu


Polysaccharide capsules of fungi are virulence factors for several fungal-host pathosystems. The extracellular matrix (ECM) of the corn pathogen Cochliobolus heterostrophus surrounds germ tubes, appressoria, and plant-colonizing hyphae. A UV-generated ecm1 mutant shows greatly reduced virulence on corn compared to wild type. Pre- penetration (attachment, spore germination, appressorium formation) and penetration activities are all normal suggesting the mutant is perturbed in plant colonization. The ecm1 mutation has been mapped by AFLP/ RFLP analysis to chromosome 4 (Zhu et al. Genome 1998). Genome sequencing identified two large supercontigs flanking the Ecm1 locus with the closest RFLP markers about 20 cM away. To provide closer markers for cloning by chromosome walking, the hygromycin B and nourseothricin resistance genes (hygB and NAT) were introduced at the proximal supercontig ends. As a second approach to cloning ECM1, the Stagonospora nodorum genome was queried with the proximal ends of the supercontigs flanking ECM1; a single supercontig was identified that spans the two C. heterostrophus contigs. This contig carries 3 candidate ECM1 encoding genes. We hypothesize that ECMs play a role in plant pathogenesis by facilitating both virulence factor delivery and host response evasion.


421. Transcriptional adaptation of Mycosphaerella graminicola to programmed cell death (PCD) of its susceptible wheat host. John Keon1, John Antoniw1, Raffaella Carzaniga2, Siân Deller1, Jane L. Ward3, John M. Baker3, Michael H. Beale3, Kim Hammond-Kosack1 and Jason J. Rudd1. 1Wheat Pathogenesis Programme, Plant-Pathogen Interactions Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK2Centre for Molecular Microbiology and Infection, Department of Infectious Diseases, South Kensington Campus Imperial College London, London. SW7 2AZ, UK3National Centre for Plant and Microbial Metabolomics, Rothamsted Research, West Common, Harpenden, Herts. AL5 2JQ E-mail address: Jason.rudd@bbsrc.ac.uk

Many important fungal pathogens of plants spend long periods (days to weeks) of their infection cycle in symptomless association with living host tissue followed by a sudden transition to necrotrophic feeding as host tissue death occurs. Little is known about either the host responses associated with this sudden transition or the specific adaptations made by the pathogen to invoke and/or tolerate it. Here we describe the host responses of wheat leaves infected with the Septoria Leaf Blotch pathogen, Mycosphaerella graminicola during the development of disease symptoms, and use microarray transcription profiling to identify adaptive responses of the fungus to its changing environment. We show that symptom development on a susceptible host genotype has features reminiscent of the hypersensitive response (HR), a rapid and strictly localized form of host programmed cell death (PCD) more commonly associated with disease resistance mechanisms. The initiation and advancement of this host response is associated with a loss of cell membrane integrity and dramatic increases in apoplastic metabolites and fungal growth rate. Microarray analysis of the fungal genes differentially expressed before and after the onset of host PCD supports a transition to more rapid growth. Specific physiological adaptation of the fungus is also revealed with respect to membrane transport, chemical and oxidative stress mechanisms and metabolism. Our data support the hypothesis that host plant PCD plays an important role in susceptibility towards fungal pathogens with necrotrophic lifestyles.


422. Functional Characterisation of Cellulose Synthases from Phytophthora infestans using Proteomics and RNA interference. Laura J.Grenville-Briggs1, Vicky L. Anderson1, Catherine R. Bruce 1, Anna O. Avrova2, Stephen C. Whisson2, Paul R. J. Birch2, and Pieter van West1. 1 Aberdeen Oomycete Group, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, UK. p.vanwest@abdn.ac.uk 2 Plant-Pathogen Interactions Programme, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK


The appressorial stage of the interaction between the late blight pathogen, Phytophthora infestans, and its hosts is one of the first points in which direct contact between the pathogen and the plant occurs via the formation of specialised infection structures, incl. the appressorium, penetration peg, and the infection vesicle. We have identified proteins that are abundant in appressoria using proteomics, one of which is a cellulose synthase (Pi-Cel1). Three other cellulose synthase genes, Pi-Cel2, Pi-Cel3, and Pi-Cel4 were identified within the P. infestans EST collections. Real time RT-PCR reveals that all four genes are up-regulated in germinating cysts with appressoria. Gene silencing studies of the cellulose synthases perturb the morphology and structure of appressoria. Also culturing germinated cysts in the presence of cellulose synthase inhibitors resulted in identical changes in appressoria structure and morphology. Pathogenicity studies employing P. infestans zoospores in the presence of the cellulose synthase inhibitor resulted in greatly reduced infection rates.


423. Pathogenesis-related developmental processes in the rice blast fungus Magnaporthe grisea. Ane Sesma1, Sara Tucker1, Steve Lenhert2; Bruno Moerschbacher2 and Anne Osbourn3. 1Dept. of Disease and Stress Biology, John Innes Centre NR4 7UH Norwich, UK; 2Westfälische- Wilhelms-Universität Münster, Hindenburgplatz 55, D-48143 Münster, Germany; 3Dept. of Metabolic Biology, John Innes Centre NR4 7UH Norwich, UK; e-mails: sesma@bbsrc.ac.uk; Sara.Tucker@bbsrc.ac.uk; bruno.moerschbacher@uni-muenster.de; anne.osbourn@bbsrc.ac.uk


Host infection and virulence is commonly associated with dimorphism in fungal pathogens. M. grisea develops an invasive hyphal growth to colonise the epidermal cells which contain different morphological features from the fungal filaments produced on the leaf surface. Interestingly, we have found that the dimorphic transition which M. grisea undergoes in order to invade the plant cells can also be induced on smooth hydrophilic polystyrene (PS-PHIL) surfaces. This developmental switch is dependent on PMK1, a mitogen-activated protein kinase required by M. grisea to infect leaves and roots. To date 2,800 insertional transformants have been screened for abnormal growth on PS-PHIL surfaces. Two groups of mutants with different phenotypes have been identified. In the first group the mutants show hyper branched and accelerated growth compared to the wild-type. The other group of mutants lack the ability to undergo the developmental switch on PS-PHIL surfaces. Several mutants from both groups are defective in plant infection. This new system offers an easy and fast method to screen for mutants unable to infect plants.


424. Colonization of maize roots by Colletotrichum graminicola leads to symptomless systemic colonization of above ground plant tissues. Verónica M. García, Brian D. Shaw, Michael R. Thon and Serenella Sukno. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX. 77843 ssukno@tamu.edu


Anthracnose of maize is caused by the filamentous fungus, Colletotrichum graminicola, (Ces.) G. W. Wils. The disease can affect most plant tissues though the stalk rot and seedling blight forms of the disease are the most economically damaging. Little is known about its ability to infect roots. To better understand the process of root colonization, we constructed a GFP-expressing strain of, C. graminicola, and used it to perform a detailed histological study of plant tissue colonization upon root infection. Structures produced by other root pathogenic fungi were observed, including runner hyphae, hyphopodia and microsclerotia. A mosaic pattern of infection resulted from specific epidermal and cortical cells becoming infected by intercellular hyphae while surrounding cells are uninfected. Interestingly, falcate conidia, normally restricted to acervuli, were also found filling epidermal cells and root hairs. We observed the development of several fungal structures that may serve as survival structures that enable the fungus to over- winter. The microsclerotia produced in culture were able to infect plant roots, demonstrating that the structures were viable and could serve as a source of inoculum. Twenty-eight percent of plants challenged with soil-borne inoculum became infected in above ground plant parts (stem and/or leaves) indicating that root infection can lead to asymptomatic systemic colonization of the plants. These observations suggest that root infection may be an important component of the maize anthracnose disease cycle.


425. A mutant of Leptosphaeria maculans unable to produce the toxin sirodesmin PL has reduced pathogenicity on stems of Brassica napus. Candace E. Elliott, Donald M. Gardiner*, Anton Cozijnsen, Georgina Thomas, Angela Van de Wouw and Barbara J. Howlett. The School of Botany, University of Melbourne, VIC 3010 Australia. *Current address: CSIRO Plant Industry, St. Lucia, Qld, 4072 Australia.


Sirodesmin PL is a non host-selective phytotoxin produced by the ascomycete Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This toxin belongs to the epipolythiodioxypiperazine class of fungal secondary metabolites and its biosynthesis involves a cluster of 18 co-regulated genes. Disruption of the two module non-ribosomal peptide synthetase gene (sirP) in this cluster prevents the production of sirodesmin PL. Loss of sirodesmin in the sirP mutant did not affect growth or fertility in vitro, but resulted in an isolate with less antibacterial and antifungal properties than the wild type isolate. While this sirodesmin-deficient sirP mutant was able to infect cotyledons of B. napus as efficiently as wild type, it was less able than wild type to infect stems. The expression of two genes in the cluster, sirP and the ABC transporter (sirA) was studied in planta through the use of isolates containing fusions of the green fluorescent protein (GFP) with their promoters. GFP fluorescence of both isolates was observed during infection of cotyledons and of stems, and was especially strong in pycnidia and in hyphae growing in necrotic stem tissue. This pattern of gene expression was consistent with the distribution of sirodesmin in planta. Reverse phase high performance liquid chromatography and ElectroSpray Ionization – Mass Spectrometry/ Mass Spectrometry showed the presence of sirodesmin PL in infected cotyledons and stems of B. napus. This is the first report of the detection of sirodesmin PL in planta.


426. Identification of differentially expressed proteins of Verticillium longisporum during infection of Brassica napus (Rapeseed). Seema Singh1, Susanna A. Braus-Stromeyer1, Oliver Valerius1, Sven Krappmann1, Gerhard H. Braus1. 1Institute of Microbiology and Genetics, Department of Molecular Microbiology & Genetics, George-August University, Göttingen, Germany. Tel: +49-551-393817, e-mail: ssingh1@gwdg.de


Verticillium longisporum is a devastating soil-borne pathogen on rapeseed crops causing vascular wilt disease. During growth of V. longisporum in the xylem vessels, it is likely that some proteins of V. longisporum, important for fungal growth or pathogenesis, may be differentially expressed on receiving possible defense signals from the host-plant. This project aims at the identification of such differentially expressed proteins. It involved quantitative comparison of proteomes of cell extracts of different culture conditions i.e. the fungus cultured with or without xylem sap from Brassica napus by 2D-PAGE. ESI-MS/MS identification of the digested 2-D spots was employed later. Some differentially expressed protein spots were successfully identified. Seven protein spots are up-regulated and five are down- regulated. These identified proteins will serve as a basis for the cloning of corresponding coding genes. Reporter constructs of identified genes, tagged with GFP and integrated in V. longisporum will be tested on induction by xylem sap and in planta. This will establish a suitable system for the identification of the possible fungus-plant interaction by changed gene expression.


427. Iron source preference and regulation of iron uptake for Cryptococcus neoformans. Won Hee Jung, Tianshun Lian, Anita Sham and James W. Kronstad. Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, B.C., V6T 1Z4, Canada whjung@interchange.ubc.ca


Iron is required by virtually all organisms for their growth and proliferation. The competition for iron between host and pathogen is a critical aspect of many infectious diseases. The availability of iron in mammalian tissues and fluids is maintained at extremely low levels (10-18 M) by iron-binding proteins such as transferrin (Tf). However, pathogenic microbes require 10-6 to 10-7 M to accomplish key metabolic processes. Thus, they must steal iron from extracellular proteins by binding ferrated Tf, by elaborating siderophores, or by degrading hemoglobin or other iron containing proteins. We have been studying iron uptake and regulation in the human pathogen C. neoformans. These studies included global analyses of iron-regulated gene expression using serial analysis of gene expression and microarrays. Functional studies of target genes led to the characterization of Sit1, a siderophore transporter of the non-reductive iron uptake system, and Cir1, a global iron regulator that controls the regulon of genes for iron acquisition. More recently, we have characterized components of the reductive iron uptake system to achieve a more thorough understanding of iron utilization in C. neoformans. Here we present identification and functional characterization of CFT1 (Cryptococcus Fe transporter). Reverse genetic analysis revealed that the gene is responsible for ferric iron uptake and transferrin utilization in vitro. Importantly, a mutant lacking CFT1 displayed significant attenuation of virulence suggesting that C. neoformans utilizes transferrin as the primary iron source in host cells. Regulatory mechanisms for CFT1 and other iron-responsive genes have also been studied in various strains including mutants lacking CIR1 or components of the cAMP pathway. Our results indicate that CFT1 is a direct downstream target of Cir1 and that the cAMP pathway also influences iron utilization in C. neoformans.


428. Mapping of the avirulence gene, AvrLepR3 in the blackleg fungus, Leptosphaeria maculans. Angela Van de Wouw1 and Barbara Howlett1. 1School of Botany, The University of Melbourne, Parkville, VIC, 3010 Australia


The blackleg fungus, Leptosphaeria maculans, is the most serious pathogen of canola (Brassica napus) world wide. Current practices to control this disease include breeding disease resistance genes into canola varieties and management such as rotations with other crops. Several disease resistance genes have been mapped in B. napus and the corresponding avirulence genes in L. maculans identified, and in two cases cloned. Disease resistance from a related Brassica, B. sylvestris conferred by a major gene, LepR3, has been bred into Australian canola varieties. However, within three years of commercial release, the frequency of virulent isolates in fungal populations has changed such that resistance has been overcome with up to 95% yield losses in some regions. We are mapping the avirulence gene, AvrLepR3 in the pathogen corresponding to the LepR3 resistance gene. Crosses have been set up between L. maculans isolates with either avirulent or virulent phenotypes on varieties with LepR3. More than 200 progeny have been generated and they segregate in a 1:1 ratio of avirulence:virulence. Using the raw sequence files from the L. maculans genome project (Genoscope, France), we are developing microsatellite markers and minisatellite markers. These are currently being tested on progeny to identify linked markers to AvrLepR3.


429. Molecular studies of the Saprolegnia-fish interaction. Andrew J. Phillips, Emma J. Robertson, Vicky L. Anderson, Chris J. Secombes and Pieter van West. Aberdeen Oomycete Group, University of Aberdeen, IMS, Foresterhill, Aberdeen, AB25 2ZD, UK. E-mail: p.vanwest@abdn.ac.uk

Oomycetes of the genus Saprolegnia are responsible for devastating infections of fish. The disease (Saprolegniosis) is characterized by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish and is of particular problem to aqua-cultural businesses. We are investigating the molecular mechanisms which enable Saprolegnia to successfully infect fish, the molecular processes that suppress host defenses during infection, and the nature of the pathogen/host interaction. To enable us to study the fish pathogen interaction we have developed an in vitro infection model. In this model system a cultured-monolayer of a primary fish cell-line (RTG-2) is infected with cysts of S. parasitica. This model has enabled us to harvested material from several stages of the interaction between fish and Saprolegnia, allowing us to investigate the kinetics of the infection using a range of molecular, microscopic and biochemical techniques. We are particularly interested in the early time-points of the interaction, and are studying the mechanisms which allow Saprolegnia to establish an infection, and the defensive mechanisms employed by the host. We are currently addressing the latter by conducting microarray studies to detect changes in the host transcriptome in response to infection by S. parasitica. Our latest findings will be presented.


430. Functional characterisation of putative SNARE proteins from the fish pathogen Saprolegnia parasitica. Emma J. Robertson1, Vicky L. Anderson1, Andrew J. Phillips1, Chris J. Secombes2, and Pieter van West1. 1The Aberdeen Oomycete Group, College of Life Sciences and Medicine, University of Aberdeen, Foresterhill, Aberdeen, Scotland, UK. E-mail: p.vanwest@abdn.ac.uk. 2 Scottish Fish Immunology Research Centre, Aberdeen, Scotland,UK.


The oomycete S. parasitica causes Saprolegniosis, a serious fish disease whereby mycelium grows on incubating eggs, or into the fins and body of freshwater fish. Severe infection result in death of the host, due to an imbalance in osmoregulation. Very little is known about the molecular biology of S. parasitica, and pathogenicity is largely undetermined. To gain more information about which genes are expressed at the onset of an infection and during pathogen development, we constructed an cDNA library from the pre-infectious stage of the S. parasitica life cycle, namely cysts. This resulted in the isolation of two EST sequences with homology to syntaxin and synaptobrevin, both members of the SNARE protein superfamily. SNAREs are involved in vesicular transport and membrane trafficking and fusion in a range of organisms, and play a critical role at different stages of the organism’s development. Consequently, it is anticipated that SNARE proteins may be involved in encystment of S. parasitica zoospores, an important stage for the organism’s pathogenicity. Herewe present experiments to identify and characterise syntaxin and synaptobrevin from S. parasitica.


431. Identification of Fusarium oxysporum f. sp. lycopersici pathogenicity genes through Agrobacterium-mediated insertional mutagenesis. Caroline B. Michielse, Ringo van Wijk, Ben J.C. Cornelissen and Martijn Rep. Plant Pathology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands.


Fusarium oxysporum f. sp. lycopersici is a soil-born fungus that causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the plant xylem vessels. In order to identify genes involved in pathogenicity 10,209 random insertion mutants were generated using T-DNA of Agrobacterium tumefaciens as an insertional mutagen. Currently, over 6800 mutants have been assessed in bioassays. So far, 16 non-pathogenic mutants and 65 mutants with reduced pathogenicity have been identified. Using Southern analysis, an estimate of the average T-DNA copy number was obtained for the entire collection, as well as the T-DNA integration pattern for all identified pathogenicity mutants. In addition, TAIL-PCR was used to isolate the genomic regions flanking the T-DNA of the pathogenicity mutants. Several known pathogenicity genes were identified, such as class V chitin synthase and phosphomannose isomerase. Work is in progress to identify, verify and characterize the remaining pathogenicity genes.


432. The impact of Rho-GTPases on polar growth, differentiation and pathogenesis in the grass pathogen Claviceps purpurea. Yvonne Rolke and Paul Tudzynski. Institut für Botanik, Westf.Wilh.Universitaet,Schlossgarten 3, Muenster, Germany


The infection of rye by Claviceps purpurea is an interesting model for directed growth of fungal hyphae in host tissue. C. purpurea infects the stigma and grows in a strictly directed, unbranched modus through the different tissues of the floret (following the pollen tube path), to tap the vascular tissue. We are interested in the signalling mechanisms which guide the pathogen during this early colonisation phase. Since the Rho-GTPases Cdc42 and Rac are known as key players in differentiation and in establishment of polarity in fungi we analysed their impact on the C. purpurea/rye system. Cpcdc42 is involved in this directed growth phase and controls branching and conidiation (Scheffer et al 2005). Deletion of the gene encoding the C. purpurea homologue of Rac resulted in a much stronger phenotype: Δcprac mutants grow in a three dimensional, corral like shape with shortened, hyperbranched hyphae; they are non-sporulating and apathogenic.Thus Cpcdc42 and Cprac have strong and partially opposite effects on differentiation and pathogenicity in C.purpurea. Deletion of the PAK kinase gene cpcla4 yielded a Δrac-like phenotype; since Y2H analyses confirned interaction of Cpcla4 with Cprac, these data indicate that CpCla4 is activated by Cprac. The influence of Cpcdc42, Cprac and Cpcla4 on gene expression and on the phosphorylation status of different MAP-kinases is currently analyzed. Scheffer et al.( 2005) EC 4:1228-1238


433. Agrobacterium-mediated Transformation of gfp into Muscodor albus, an Endophytic, Gas-emitting Fungus. David Ezra and Tami Kroiter. Department of Plant Pathology and Weed Research, ARO, the Volcani center, Bet – Dagan 50250; Israel


M. albus is an endophytic fungus originally isolated from Cinnamomum zeylanicum from Honduras. This fungus produces a mixture of volatile organic compounds (VOCs) which is lethal to a wide variety of fungal and bacterial plant pathogens. M. albus has been the subject of a number of studies which have suggested the use of this fungus as a biological control agent against plant pathogens. Our study examines the possible use of M. albus for the control of systemic diseases of fruit trees. M. albus is completely sterile; no spores or fungal fruiting bodies could be observed under different laboratory conditions. Unlike plant pathogens that induce disease or plant defense responses, when inoculated into the host plant, symbiotic endophytes do not usually cause visible reactions. This may be a problem for the researcher trying to decide whether the plant was successfully infected by the endophyte or not. In order to properly study the establishment, development and progress of the endophyte in the host plant and host-endophyte interactions, methods for the identification and localization of endophytic microorganisms are needed. One approach is to attempt to re-isolate the endophyte from the plant. Another strategy involves generating specific PCR primers for the identification of the endophyte in the plant. A third approach involves the transformation of a reporter gene into the endophyte and the subsequent localization of its expression in the host plant. We have used all three of these methods in our study of M. albus in planta. This report will describe the Agrobacterium-mediated transformation of the reporter gene gfp into Muscodor albus hyphae.


434. Molecular and cellular responses of Alternaria brassicicola to plant defense metabolites. Adnane Sellam1,2, Anita Dongo2, Thomas Guillemette2, Pascal Poupard2, Piétrick Hudhomme3, and Philippe Simoneau2. 1 Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount, Montreal, Quebec, Canada 2 UMR PaVé N°77, Faculté des Sciences, 2Bd Lavoisier, F-49045 Angers, France 3 Chimie, Ingénierie Moléculaire et Matériaux, UMR CNRS 6200 - Université d'Angers, 2 Bd Lavoisier, 49045 Angers, France


Alternaria brassicicola is the causative agent of black spot disease of Brassicaceae belonging to Brassica and Raphanus genus. During host infection, A. brassicicola is exposed to high levels of antimicrobial defense compounds such as indolic phytoalexins and glucosinolates breakdown products. To investigate the transcriptomic response of A. brassicicola when challenged with brassicaceous defense metabolites, suppression subtractive hybridization (SSH) and Differential Display were performed. Following exposure to ITC, A. brassicicola displayed a response similar to that experienced during oxidative stress. Indeed, a substantial subset of differentially expressed genes was related to the glutathione (GSH) cycle and to other mechanisms involved in cell protection against oxidative damages. Generation of Reactive Oxygen Species (ROS) following the exposure of A. brassicicola to ITCs was clearly demonstrated using two different fluorogenic probes. Treatment of germinated condia with the phytoalexin camalexin induced the expression of another functional set of genes. Indeed, this phytoalexin appeared to activate a compensatory mechanism to preserve cell membrane integrity. Thus , among the camalexin-elicited genes, several were involved in sterol and sphingolipid biosynthesis. An uptake assay using the fluorigenic dye SYTOX green confirmed the alteration of A. brassicicola membranes during exposure to camalexin. During interaction, the upregulation of all fungal selected genes was observed during the infection of Raphanus sativus while only a subset was overexpressed during the incompatible interaction with Arabidopsis thaliana ecotype Columbia. The Glutathione S-transferase (GST) gene AbGst1, differentially expressed in response to ITCs, was functionally characterized. The obtained results point to a function of AbGst1p in ITC conjugation and neutralization, enabling A. brassicicola to tolerate ITC during host infection.


435. Towards a comprehensive metabolome of Stagonospora nodorum. Peter S. Solomon1, R.D. Trengove2 and R.P. Oliver1. 1Australian Centre for Necrotrophic Fungal Pathogens and 2Separation Science Laboratory, SABC, DHS, Murdoch University, WA 6150, Australia. Email: psolomon@murdoch.edu.au


Stagonospora nodorum is a potent pathogen of wheat causing yield losses throughout many parts of Australia, the United States and Israel. In an effort to develop strategies to control yield losses, the Australian Centre for Necrotrophic Fungal Pathogens (ACNFP) has taken a multi-discipline approach to understanding the molecular basis of the disease. Metabolomics is rapidly emerging as a fast and powerful complementary technique to genomics and proteomics, particularly for the dissection of previously generated targeted gene knockout strains. Several platforms currently exist to study metabolomics including GC-MS, GC-TOF-MS, NMR, CE-MS and FT-MS. Advantages and disadvantages are evident for each of these techniques. Recently, the Separation Science Laboratory at Murdoch University has acquired a suite of new instruments that will complement the existing metabolomics platforms available. Of particularly interest is a GC × GC-TOF-MS that allows two-dimensional separation prior to time-of-flight analysis. This new instrument provides greater sensitivity and vastly improved separation compared to existing GC-MS platforms. This poster will provide a comparison of the detectable metabolites extracted from S. nodorum using GC-MS and GC × GC-TOF-MS platforms.


436. Functional analysis of the role of a stress-activated MAP kinase in the association between a fungal endophyte and its grass host. Carla Eaton1, Simon Foster2, Jeremy Hyams1, and Barry Scott1. 1 Massey University, New Zealand. 2Sainsbury Laboratory, Norwich, UK.


The association between the fungal endophyte Epichloe festucae and its grass host Lolium perenne is regulated by the multicomponent NADPH oxidase complex. This complex catalyses production of reactive oxygen species (ROS) from molecular oxygen. Previous work has shown that disruption of a number of components of this complex results in an abnormal plant interaction phenotype, in which hyphal growth is unregulated and the host undergoes premature senescence. In Aspergillus nidulans, disruption of the stress-activated MAP kinase SakA has been shown to affect regulation of the NADPH oxidase, suggesting a link between SakA and ROS signalling (Lara-Ortiz et al, 2003). This suggests that SakA may also have a role in controlling the association between Epichloe festucae and its grass host. The sakA gene has been cloned based on conserved microsynteny at sakA locus in a number of filamentous fungi. This gene was then disrupted and the mutant shown to display increased osmosensitivity in culture. The mutant also displays resistance to the phenyl-pyyrole fungicide fludioxonil, as has been seen in other filamentous fungi defective in the stress-activated MAP kinase. The E. festucae sakA mutant also displays an abnormal host interaction phenotype, suggesting a possible role for SakA in regulating the symbiosis.


437. Magnaporthe grisea Mstu1, an APSES protein homolog of Aspergillus nidulans StuA, is required for regulation of appressorial turgor generation. Marie Nishimura, Junji Fukada and Nagao Hayashi. National Institute of Agrobiological Sciences, Tsukuba, Japan.

APSES proteins are transcription factors with highly conserved basic helix-loop-helix (bHLH) DNA binding domains to regulate fungal differentiation during asexual and sexual developments. We have identified a deletion mutant of the APSES protein Mstu1 (Magnaporthe STUA homolog 1) in M. grisea that showed reduced conidiation and mycelial growth, and was defective in plant penetration even though it formed comparable number of melanized appressoria relative to the wild type. M. grisea penetrates plant cuticles using appressorial turgor, which is generated by accumulation of glycerol synthesized from conidial reserves such as glycogen and lipids. In mstu1, delay in transfer of conidial glycogen and lipids to appressoria, and in sequential catabolism of those conidial reserves was observed. Furthermore, appressorial turgor was insufficient for plant penetration in mstu1. A single point mutation introduced at the potential PKA phosphorylation site in Mstu1 (T112A) resulted in low appressorial turgor and a delay in mobilization of lipids, and in catabolism of glycogen and lipids in the engineered mutant. Our results indicate that Mstu1 is a downstream target of the PKA and controls mobilization of lipids, and catabolism of the glycogen and lipids during appressorial turgor generation, and that glycogen mobilization is regulated by Mstu1 in PKA independent manner.


438. A distinct class of RNA binding proteins in Magnaporthe grisea implicated in pathogenicity. Sara L. Tucker1, Anne E. Osbourn2 and Ane Sesma1. 1Department of Disease and Stress Biology, 2Department of Metabolic Biology, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK. (sara.tucker@bbsrc.ac.uk)


A random mutagenesis approach was used to generate a library of insertional mutants in M. grisea strain Guy-11. The mutant bank was screened using a root infection assay, which led to the identification of several candidate strains with a reduced pathogenicity phenotype. One mutant strain, RBP35, is considerably affected in its ability to cause disease on roots while there is a small reduction in virulence on the aerial parts of rice plants. The mutant shows a slight reduction in growth rate and is impaired in conidiation compared to the wild type strain. RBP35 has undergone insertional inactivation of a gene encoding a predicted RNA binding protein. Such proteins are characterised based on the presence of one or more RNA recognition motifs (RRMs). The RRM is a nucleic acid binding domain that can act together and/or in association with other domains which then contribute to protein cooperativity and/or affinity for ssRNA. Characterisation of RRM-containing proteins in other systems has revealed that they can have a wide subcellular distribution and various functions in pre-mRNA processing and splicing, RNA stability, sequestration and degradation, mRNA transport, rRNA metabolism, telomere metabolism and protein recognition. Our initial efforts have focused on defining this class of proteins in M. grisea. We have set out to determine the subcellular location of RBP35 and to identify other proteins with which it may interact.


439. Functional analysis of Nep1-like proteins in Sclerotinia sclerotiorum. Alicia Greenhill1, Rebecca Barnett1, Megan Drew1, & Kim Plummer1. 1La Trobe University, Melbourne, Australia. k.plummer@latrobe.edu.au


Sclerotinia sclerotiorum is an aggressive necrotrophic phytopathogen causing white mould in >400 plant species worldwide. S. sclerotiorum is estimated to cost the US agricultural industry in excess of $200 million pa. Effective control methods of S. sclerotiorum are lacking and no resistant crop cultivars have been identified. We are using the S. sclerotiorum genome sequence (Broad Institute) to investigate necrosis inducing proteins secreted during infection. Two candidates, SS1G_03080.1 (SsNLP1) and SS1G_11912.1 (SsNLP2), have high similarity to the Fusarium oxysporum necrosis and ethylene-inducing peptide 1 (Nep1). Nep1-like proteins (NLPs), ranging from 24-26 kDa, are conserved across a wide range of taxa including bacteria, fungi and oomycetes. Bioinformatic analysis indicated SsNLP1 and SsNLP2 encode putative proteins 246aa and 245aa in length, with predicted secretory signal peptides of 20aa and 19aa, respectively. SsNLP1 and SsNLP2 had significant amino acid sequence identity to NLP family members, and contained conserved cysteine residues. Semi- quantitative RT-PCR analysis indicated that SsNLP1 and SsNLP2 are differentially regulated in vitro and during infection of Brassica napus. Gene silencing and knockout studies are presently underway to determine the role of SsNLP1 and SsNLP2 in pathogenicity of S. sclerotiorum.


440. ROS signalling in the grey mould Botrytis cinerea. Nora Temme, Nadja Segmueller and Paul Tudzynski Institut fuer Botanik, Westf.Wilhelms Universitaet Muenster, Germany


Reactive oxygen species (ROS) play a major role in plant-pathogen interactions. The necrotrophic plant pathogen Botrytis cinerea causes an oxidative burst on its host plants. Its virulence correlates with (and might even depend on) the intensity of this induced plant defence reaction. To analyse ROS signalling in B. cinerea, components of the oxidative stress response system were characterised. The stress-activated MAPK (SAPK) BcSak1, a homologue of Hog1, is phosphorylated under oxidative stress;.its deletion showed that in B.cinerea this MAPK cascade is essential for vegetative differentiation (conidiation) and pathogenicity (penetration). The Botrytis activator protein 1 (Bap1) is a bZIP transcription factor structurally related to Yap1 from yeast. Deletion of bap1 resulted in increased sensitivity to oxidative stress. Bap1 controls expression of typical oxidative stress response genes,e.g. bccat2 (catalase 2), bctrr (thioredoxine reductase) and bcglr (glutathione reductase). Only bccat2 is also under control of Bcsak1, indicating that Bap1 acts mostly independently of the SAPK signalling cascade. In contrast to bcsak1, bap1 is not essential for conidiation or virulence. BcHk1 is a histidine kinase homologous to Mak2/3 which are involved in the oxidative stress response in Sch. pombe. Deletion of bchk1 results in increased sensitivity to oxidative stress and to the dicarboximide iprodione; activation of BcSak1 by oxidative stress caused by low levels of H2O2 is affected by BcHk1. Analysis of the response regulator Bcrr1 (a homologue of Skn7/Prr1) is under way.


441. Characterization of extracellular phospholipase D activity in Phytophthora. Harold J.G. Meijer and Francine Govers. Laboratory of Phytopathology, Plant Sciences Group, Wageningen University, Binnenhaven 5, NL-6709 PD, Wageningen, The Netherlands

Phospholipase D (PLD) catalyzes the hydrolysis of structural phospholipids, such as phosphatidylcholine, leading to the production of phosphatidic acid and a free headgroup. In comparison to other eukaryotes, Phytophthora spp. have a more complex and diverse set of PLD genes. Mammals have only one type of PLD called PXPH-PLD. Plants also have PXPH-PLDs but in addition they have a large family of C2-PLDs. In these proteins, PX, PH and C2 are the lipid- binding domains that precede the catalytic PLD domain. Phytophthora has one PXPH-PLD, lacks C2-PLDs, but has three additional novel PLD sub-families (Meijer and Govers 2006), one of which is a large family of 12 members that all have a signal peptide and are probably secreted. The substrates for these secreted PLDs could be phospholipids that reside in plant membranes, one of the barriers faced by the pathogen when it enters the host. To test the hypothesis that the secreted PLDs play a role in the infection process, we first analyzed PLD activity in the pool of extracellular proteins secreted by Phytophthora. Here we present the first biochemical characterization of PLD activity present in extracellular fluid and of the substrate specificity. Meijer, H.J.G., and Govers, F. 2006. Genomewide analysis of phospholipid signalling genes in Phytophthora spp.: novelties and a missing link. Mol. Plant Microb. Inter. 19:1337-1347.


442. Comparative transcriptional profiling of Blumeria graminis development on host and non-host surfaces. Petro D Spanu, Maike Paramor, Calin Andras. Imperial College London


Blumeria graminis f sp hordei is a fungus that causes powdery mildew on barley. When conidia land on barley epidermis, they germinate and grow following a very tightly controlled series of developmental stages: production of a short primary germ tube then a secondary germ tube that enlarges to form an appressorium, from which a penetration peg breaches the cell wall and grows into a feeding haustorium. Proliferation of the extracellular mycelium, and the associated secondary haustoria, eventually lead to massive production of conidia for further dispersal. On non- host plants, such as wheat, development is arrested early, at penetration or shortly thereafter. On artificial surfaces, e.g. cellophane or glass, development is aborted prior to differentiation of appressoria or secondary germ tubes respectively. In the work reported at this meeting, we have carried out a comparative analysis of the transcriptional profiles of over 2000 genes during early development on barley, wheat, cellophane and glass. We find genes whose regulation is specifically affected by the surfaces on which Blumeria grows. Surprisingly, Blumeria differentiates between barley and wheat surfaces well before differences are visble. We will report here on the genes that appear to characterise growth on host, non host and artificial surfaces. Our ultimate aim is to identify the common regulatory elements of these genes that are likely to be the target of networks controlling development of early stages of infection in  Blumeria.


443. Expression of a dominant active MgRas2 bypassed surface recognition signals for appressorium formation in Magnaporthe grisea. Xinhua Zhao, Chaoyang Xue, Yangseon Kim, and Jin-Rong Xu Department of Botany and Plant Pathology, Purdue University


Ras proteins are low molecular weight GTP-binding proteins that function as a molecular switch in signal transduction. We have identified two Ras homologues in Magnaporthe grisea. The mgras1 deletion mutant had no detectable phenotypes but the efforts to generate a mgras2 mutant failed, indicating that MgRAS2 may be an essential gene. Expression of a dominant active MgRas2G23V allele in the wild-type strain had no effect on vegetative growth but enhanced conidiation. Transformants expressing MgRas2G23V(DA66) efficiently formed appressoria on both hydrophobic and hydrophilic surfaces. Interestingly, DA66 formed melanized appressorium- like structures on the tip of aerial hyphae. We then transformed the MgRas2G23V construct into other appressorium-forming fungi. In the resulting transformants, appressorium-like structures were observed at hyphal tips. When an appressorium-specific reporter was transformed into DA66, GFP signals were observed in these appressorium- like structures. DA66 had an elevated intracellular cAMP level and enhanced phosphorylation of the Pmk1 MAP kinase. However, expressing MgRas2G23V had no effect in the mutants that blocked the Pmk1 pathway. These data indicated that MgRas2 may function upstream from both the cAMP/PKA and Pmk1 MAP kinase pathways. The G23V mutation in MgRas2 enables M.grisea to bypass surface recognition and attachment signals for appressorium formation.


444. The application of laser microdissection to in planta gene expression profiling of the maize anthracnose stalk rot fungus Colletotrichum graminicola. Weihua Tang, Sean Couphlan, Edmund Crane, Mary Beatty, Dong Zhang and Jon Duvick . Shanghai Inst. Plant Phys. Eco.


Laser microdissection (LM) offers a potential means for deep sampling of a fungal plant-pathogen transcriptome during the infection process using whole-genome microarrays. Using a fluorescent protein-expressing fungus can facilitate the identification of fungal structures for LM. However, fixation methods that preserve both tissue histology and protein fluorescence, and that also yield RNA of suitable quality for microarray applications, have not been reported. We developed a microwave-accelerated acetone fixation, paraffin-embedding method that fulfills these requirements. We successfully used LM to isolate individual maize cells associated with Colletotrichum graminicola (AmCyan) hyphae at an early stage of infection. The LM-derived RNA, after two-round linear amplification, was of sufficient quality and quantity for global expression profiling using a fungal microarray. Comparing replicated LM samples representing an early stage of stalk cell infection with samples from in vitro-germinated conidia, we identified 437 and 370 C. graminicola genes showing significant up- or downregulation, respectively. We confirmed the differential expression of several representative transcripts by quantitative RT-PCR and documented extensive overlap of this dataset with a PCR- subtraction library enriched for C. graminicola transcripts in planta. Our results demonstrate that LM is feasible for in planta pathogen expression profiling and can reveal clues about fungal genes involved in pathogenesis.


445. Yeast LHS1 homologue, MoLHS1, plays multiple roles in the vegetative growth, asexual sporulation and pathogenicity of the rice blast fungus, Magnaporthe oryzae. Mihwa Yi and Yong-Hwan Lee. Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea


Heat shock protein 70 (Hsp70) family of genes are highly conserved through out the organisms including plants, animals, and fungi. The functions of any Hsp70 proteins in the rice blast fungus, Magnaporthe oryzae, however, have not been elucidated yet. The one matching to the yeast LHS1 (Lumenal Hsp Seventy) of the homologues in M. oryzae was deleted by targeted fungal transformation via Agrobacterium tumefaciens-mediated transformation. The vegetative growth of the mutants was slightly delayed and the pigmentation on the medium supplemented with manganese was reduced. Germination and appressorium formation of the molhs1 mutants were not different from those of the wild type strain. However, both penetration and invasive growth stages were defective, which results in significant reduction on the pathogenicity of the fungus. Furthermore, the asexual reproduction was largely decreased in the molhs1 compared to the wild type strain. A single ectopic insertion of partial fosmid clone including the full ORF of MoLHS1 recovered the defective phenotypes on the molhs1 mutant. The transcriptional expression of MoLHS1 was specifically induced by endoplasmic reticulum stress conditions such as dithiothreitol or tunicamycin treatment, which shows the possibility that MoLHS1 might function as chaperone under ER stress like LHS1 in yeast. Here we report novel functions of Hsp70 gene in M. oryzae as an important player on the pathogenicity, mycelial growth, and the sporulation development.


446. Degradation of plant cell wall polysaccharides in relation to the infection process of Magnaporthe grisea on cereals. Ronald P. de Vries1, Charissa de Bekker1, Arnaud Lagorce2, Cecile Ribot3, Marc-Henri Lebrun3. 1Microbiology, Utrecht University, Utrecht, The Netherlands; 2Bayer Cropscience, Lyon, France; 3UMR 2847 CNRS-Bayer Cropscience, Lyon, France.


The Magnaporthe grisea species complex attacks a wide range of grasses, including wheat, barley or rice, and is a model organism for studying plant fungal interactions. The early stage of the infection process is characterized by the formation of an appressorium that mediates the penetration of the fungus into host plant tissues through the puncture of cuticle and plant cell wall. Although this penetration process is mainly mechanical, requiring a high turgor in the appressorium, it can not be excluded that cell wall degrading enzymes (CWDE) could play a role in this process and in subsequent fungal spreading into plant tissues Analysis of M. grisea genome revealed a large number of genes encoding plant polysaccharides degrading enzymes. The set of M. grisea CWDE encoding genes was compared to the repertoire of Aspergillus nidulans CWDE, highlighting evolutionary trends specific of M. grisea that suggest an adaptation of M. grisea to its host plants. Analysis of the expression of these genes in rice leaves infected by M. grisea was performed using DNA micro array and real time RT-PCR demonstrating that some of these genes are highly expressed during the infection.


447. Molecular cloning of Avr-Pia, the avirulence gene in Magnaporthe oryzae toward the rice blast resistance gene Pi-a. Teruo Sone1, Shinsuke Miki1, Kotaro Matsui1, Taketo Ashizawa2, Hideki Kito3, Kazuyuki Hirayae2, and Fusao Tomita4. 1 Hokkaido Univ., Sapporo Japan, 2 Nat. Agric. Res. Cen., Niigata Japan 3Nat. Inst. Agric. Sci. Tsukuba Japan, 4Univ. of Air, Sapporo Japan


Avirulence genes in the genome of fungal plant pathogens are the main factor for the determination of their host (cultivar) specificity. We aimed to clone and analyze the avirlulence gene from the Japanese field isolates of M. oryzae. First, a mutant of strain Ina168, named Ina168m95-1, which gained the virulence toward rice cultivar Aichi- asahi with resistance gene Pi-a, was isolated. RAPD primers were screened for polymorphic amplification between the mutant and the parent. One primer, named OPM-1 resulted a DNA (named PM1) fragment absent in the mutant. Three cosmid clones including PM1 flanking region were screened from genomeic DNA library. One of these clones named 46F3 could complement the mutant phenotype, and thus strongly indicated that this clone contains the avirulence gene Avr-pia. The clone 46F3 contained many fragments of transposable elements. We divided 46F3 insert into six fragments, I to VI, that does not contain transposable elements, and individually cloned into hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. The inoculation assay of transformants revealed that fragment V (3.5 kb) contains Avr-pia. This was also confirmed by PCR amplification assay of fragment V from field isolates. From isolates that infect Pi-a, no amplification was detected. Deletion mutants of the fragment V were used for the further focusing of Avr-pia locus. Finally we found an 1199 bp-DNA region responsible for Avr-pia, which includes a 255 bp-ORF with weak homology to bacterial cytochrome-like protein.


448. The MAD1 Adhesin of Metarhizium anisopliae Links Adhesion to Insect Cuticle with Blastospore Production and Virulence; A Second Adhesin (MAD2) Enables Attachment to Plant Surfaces. Chengshu Wang and Raymond J. St. Leger. Department of Entomology, University of Maryland, College Park, MD 20742, USA. stleger@umd.edu.


Metarhizium anisopliae is a fungus of considerable metabolic and ecological versatility, being a potent insect pathogen that unusually can also colonize plant roots. The mechanistic details of these interactions are unresolved. We provide evidence that M. anisopliae adheres to insects and plants using two different proteins, MAD1 and MAD2 that are differentially induced in insect hemolymph and plant root exudates, respectively, and produce regional localization of adhesive conidial surfaces. Expression of Mad1 in Saccharomyces allowed this yeast to adhere to insect cuticle. Expression of Mad2 caused yeast cells to adhere to a plant surface. Our study demonstrated that as well as allowing adhesion to insects, MAD1 at the surface of M. anisopliae conidia or blastospores is required to orientate the cytoskeleton and stimulate expression of genes involved in the cell cycle. Consequently, disruption of Mad1 in M. anisopliae delayed germination, suppressed blastospore formation and greatly reduced virulence to caterpillars. Disruption of Mad2 blocked adhesion of M. anisopliae to plant epidermis, but had no effects on fungal differentiation and entomopathogenicity. Thus regulation, localization and specificity control the functional distinction between Mad1 and Mad2, and enable M. anisopliae cells to adapt their adhesive properties to different habitats.


449. Expressing an insect-specific scorpion neurotoxin makes Metarhizium anisopliae hypervirulent to mosquitoes and caterpillars. Chengshu Wang and Raymond J. St. Leger. Department of Entomology, University of Maryland, College Park, MD 20742. stleger@umd.edu.


Insects cause $244 billion in damage to world agriculture every year and mosquito borne diseases are among the most devastating in modern times. Metarhizium anisopliae is being developed as a control agent against mosquitoes and other major pests, but its full potential has not been approached because of slow kill. Here we tested the ability of the promoter of the Metarhizium anisopliae hemolymph-induced collagen-like (Mcl1) gene to specifically target production of toxins into the hemocoel of infected insects. We expressed a codon-optimized synthetic gene encoding N- terminal secretary signals and an insect-specific neurotoxin (AaIT) from the scorpion Androctonus australis. The synthetic gene was introduced into a broad host range strain of M. anisopliae. Stable incorporation was confirmed by PCR and rapid hemolymph-specific expression confirmed by RT-PCR and assay of biologically active toxin. Improvements in pathogenicity were dramatic. Virulence was increased 22-fold against tobacco hornworm (Manduca sexta) caterpillars and nine-fold against adult yellow fever mosquitoes (Aedes aegypti), and was more rapidly effected. Furthermore, there were useful pre-lethal effects including reduced mobility and feeding as compared to hosts infected by the wild type. This work represents a significant step towards the development of hypervirulent insect pathogens for effective pest control.


450. Transposon tagging in Fusarium culmorum, a pathogen on cereals. M. Dufresne1, V. Balmas2, G. Ortu1, M.-J. Daboussi1 and Q. Migheli2. 1 Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France 2 Dipartimento di Protezione delle Piante, Università degli Studi di Sassari, Italy


Crown and foot rot of wheat is an important soilborne disease caused by several species of filamentous fungi. Fusarium culmorum (W.G. Smith) Sacc. is one of the most common incitants of this disease worldwide and is able to produce type B trichothecenes. Aiming at deciphering mechanisms involved in pathogenicity and mycotoxin production of F. culmorum, we have recently started two transposon-based mutagenesis to identify genes governing these characters without a priori knowledge on their function. We first used the impala element, which was already shown to transpose in a wide range of Ascomycete species. A collection of 300 revertant strains has been generated and tested for pathogenicity on wheat under glasshouse condition. Following two rounds of wheat assays, 7 strains, more or less altered in pathogenicity have been identified among 175 strains with a reinsertion event. To date, one strain has been characterised in detail. Loss of pathogenicity results from the insertion of impala in the close 5’ region of a gene encoding a putative HMG Co-A reductase, a function already described as involved in F. graminearum pathogenicity (Seong et al. 2006). In parallel, a systematic recovery of impala insertion sites has been started in order to determine its insertional preference. More recently, we introduced the double component mimp/impala to compare the tagging efficiency of both systems. Seong K et al.(2006) FGB 43:34-41.


451. Multiplication of the PEP-cluster-carrying conditionally dispensable chromosome by protoplast fusion in Nectria haematococca MPVI. Hamid Sadeghi Garmaroodi and Masatoki Taga. Department of Biology, Okayama University, Okayama 700-8530, Japan


Since the conditionally dispensable chromosomes (CDCs) in N. haematococca MPVI are known to be responsible for the higher fitness to the host plants of the strains harboring them, it is intriguing to examine the possibility of accumulation of CDC in the genome and its effect on the fitness of the fungus. In this study, we used protoplast fusion for producing strains with multiple copies of PDA1-CDC and analyzed the pathogenicity and homoserine (HS)- utilizing ability which are correlated with CDC. As the parents for electro- and PEG-mediated fusion, a lab strain bearing a single CDC marked with hygromycin resistance gene, Tr18.5 (Wassman and Van Etten, 1996), and a geneticin resistant transformant of the same strain were used. Using PCR, pulsed field gel electrophoresis and chromosome painting of fluorescence in situ hybridization, we detected at least two CDCs in the fusants. Cytological chromosome counting by fluorescence microscopy in parents and selected fusants showed that extra set(s) of A chromosomes in the fused protoplasts should have been deleted during the regeneration of the fused protoplasts to single-spored cultures. All fusants with multiple copies of CDCs in their genomes showed higher virulence to pea and ability to use HS in the media than the parents.


452. Protein arrays for transcription factors analysis in Magnaporthe grisea. Malali Gowda 1, Jixin Deng1, Yeon Yee Oh1, Hee-Sool Rho2, Cristiano Nunes1, Jin-Rong Xu3, Heng Zhu2, Thomas Mitchell1 and Ralph Dean1. 1Fungal Genomics Laboratory, North Carolina State University, Raleigh, NC 27606, USA. 2Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA. 3Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA.


Magnaporthe grisea is widely considered as a model fungus to study plant-fungal interactions. The signaling network in M. grisea is well established, which includes cAMP, MAP kinase and Ca/calmodulin pathways. However, understanding of downstream transcription factors that regulate the expression of these pathways is lacking. Through the whole genome annotation process, we have identified nearly 500 putative transcription factors in M. grisea, of which, 74 genes were found to have no intronic sequences in the coding regions. We further continued manual annotation and confirmed the expression of > 90% of these using expression data from EST, RL-SAGE and MPSS. We have cloned >80% of transcription factors from M. grisea into Gateway expression vectors, and are presently over expressing each in yeast to print Magnaporthe transcription factors array. These protein arrays will be used to assay the kinase specificity activity of putative transcription factors. Homologous gene-knockout and over-expression strategies will be deployed to map the transcription factor binding motifs and subsequently identify genes regulated by transcription factors using ChIP-chip hybridization and 454 sequencing approaches. We will present results from annotation and cloning efforts as well as preliminary phosphorylation assay.


453. The role of high affinity iron uptake systems in Ustilago maydis. Britta Winterberg1, Jan Schirawski1, Franziska Leßing1, Heiko Eichhorn1, Uwe Linne2 and Regine Kahmann1. 1 Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany 2 Philipps-University, Department of Biochemistry, D-35043 Marburg, Germany


The phytopathogenic basidiomycete Ustilago maydis induces tumors on maize. Within these tumors the diploid fungal spores develop. U. maydis has two high affinity iron uptake systems. The permease-based system is necessary for full virulence of U. maydis on its host plant maize. In addition, U. maydis is able assimilate iron via the siderophores ferrichrome and ferrichrome A. Using microarrays we identified genes that are regulated both by iron and the iron responsive transcription factor Urbs1. We investigated the putative function of these genes in siderophore biosynthesis. We constructed deletion mutants of sid2 and fer3, encoding two non-ribosomal peptide synthetases (NRPS), and fer4, fer5, and fer8, encoding a enoyl-CoA-isomerase, an acetylase and an oxidoreductase, respectively. HPLC analysis of culture supernatants revealed that the sid2 deletion mutant only produced ferrichrome A, while the other mutants solely synthesized ferrichrome. Based on predicted function of proteins involved in ferrichrome A synthesis we postulated a biosynthetic pathway that is currently under investigation. To analyze the role of siderophores during pathogenic development, plant infection assays with double deletion mutants of the NRPS Sid2 and Fer3 were performed. These mutants were as pathogenic as the wild type and produced viable spores. However, spore progeny were diploid indicating a meiosis defect. Additionally, double deletion mutants of sid2 and fer3 mutants are unable to grow on low-iron medium, suggesting that, in contrast to the permease-based system that is crucial for biotrophic development, the siderophore-based system is essential for germination and growth outside the plant.


454. A STE12-like transcription factor from the plant pathogen Colletotrichum lindemuthianum regulates adhesion and expression of a extracellular proteins. Bernard Dumas1, Joanne Wong Sak Hoi1, Nafees Bacha1, Claude Lafitte1, Gisèle Borderies2, Michel Rossignol2, Pierre Rougé1. 1UMR 5546 CNRS-Université Paul Sabatier Toulouse III, Castanet-Tolosan, France 2Plateforme protéomique de Toulouse, site IFR40, Castanet-Tolosan, France


In phytopathogenic fungi, STE12-like genes encode transcription factors essential for appressorium-mediated host penetration. However, their downstream targets are still largely unknown. We recently identified regulatory elements similar to those recognized by Ste12p in the promoter of a polygalacturonase gene in the bean pathogen Colletotrichum lindemuthianum, suggesting that STE12-like transcription factors could be involved in the regulation of genes coding cell wall degrading enzymes. This finding led us to isolate a STE12-like gene from C. lindemuthianum (CLSTE12) and to produce disruption mutants. Phenotypic analyses of the mutants revealed reduced pectinase activity and conidial adhesion to an artificial surface. Analysis of cell surface proteins allowed the identification of a major protein, Clsp1p (Colletrichum lindemuthianum Surface Protein 1), which was absent from the mutants. Accordingly, CLSP1 transcript accumulation was dramatically reduced in the clste12 mutants. Clsp1p belongs to a new family of fungal wall-associated proteins putatively involved in the interaction of fungal cells with abiotic or biotic surfaces.


455. Annotation of signaling pathways genes families in the ectomycorrhizal basidiomycete Laccaria bicolor reveals an expansion of a G-proteins encoding genes family. Sebastien Duplessis1, Laccaria genome consortium & DOE Joint Genome Institute2. 1UMR 1136 INRA/UHP Nancy 1 Interactions Arbres/Micro-organismes INRA Nancy, 54280 Champenoux France. 2DOE Joint Genome Institute Walnut Creek CA 94598 USA.


We have investigated the signaling pathways genes families in the genome sequence of the ectomycorrhizal basidiomycete L. bicolor. We focused on the known components of the G-protein mediated signaling pathways (i.e., G-proteins, GPCRs, PKAs, Adenylate cyclases) and MAP kinases that were previously described in other filamentous fungi. Our results show a large expansion of the gene family coding for heterotrimeric G-protein alpha subunits in L. bicolor with 39 putative members compared to other filamentous ascomycetes or basidiomycetes where only 3 to 4 copies had been described so far. Homologs of fungal G-alpha proteins known for their role in filamentous growth or mating were found in L. bicolor while no homolog of G-alpha related to virulence in phytopathogenic fungi was detected. In addition, two new classes of G-alpha were observed: one corresponding to shorter proteins and another one with similarities to G- alpha sequences described in Ustilago maydis (UmGPA4) and Aspergillus oryzae (AoGaoC). These new classes of G-alpha proteins supported by ESTs and expression profiling, may participate to fine-tune molecular crosstalk between the ectomycorrhizal fungus and tree roots during symbiosis development.


456. Genomic approaches to unravel Aphanomyces euteiches plant Interactions. Elodie Gaulin, Amin Madoui, Arnaud Bottin, Christophe Jacquet, Bernard Dumas. UMR CNRS-Université Paul Sabatier, Toulouse III, 24 Chemin de Borde-Rouge, 31326 Castanet-Tolosan, France, gaulin@scsv.ups-tlse.fr


Aphanomyces sp. ( Saprolegniaceae ) houses plant, animal and pathogens found in both terrestrial and aquatic habitats. Aphanomyces euteiches Drechs. causes seedling and root rot diseases on legumes and is the most pea (Pisum sativum ) serious disease in several countries. Because the strictly soil borne pathogen may survive for long periods in the soil (more than 10 years), the only existing control is to avoid cultivating peas in infested field for many years. While a huge genomic research effort were devoted to Oomycete such as Phytophthora, Aphanomyces received little attention and the mechanism by which it infects its hosts is largely unknown. A. euteiches is an interesting model to study plant-microbe interaction since it display differential specificity of interaction with the legume model Medicago truncatula . To gain a first insight into the transcriptome of A. euteiches a genomic approach have been undertaken. A total of >20000 EST were generated from 2 cDNA libraries. 7 000 consensus sequences were assembled and will be available through a public database. Annotation of ESTs revealed a number of genes that could function in virulence. Progress in this research will be presented.


457. MoRAC1 , a Magnaporthe oryzae Rho GTPase gene, is required for conidial morphogenesis and pathogenicity. Sung-Yong Yoo, Maruthachalam Karunakaran, and Yong-Hwan Lee. Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea.

Rac proteins, highly conserved members of the Rho family GTPase, play important roles in control of polarized growth in many organisms but the specific function of these proteins in plant pathogenic fungi is yet to be explored. During large scale insertional mutagenesis through Agrobacterium tumefaciens-mediated transformation in the rice blast fungus Magnaporthe oryzae, we have identified a transformant where T-DNA was inserted in the gene, MoRAC1, encoding Rho type GTPase. Phenotype of the morac1 mutants exhibited pleiotropic effects on conidial development such as reduced conidiation, deformation in conidia and conidiophore development. The T-DNA insertion mutant phenotype was confirmed when creating null mutant for the MoRAC1 gene. morac1 mutants displayed defects in polarized growth and cell differentiation similar to T-DNA insertion mutant. Furthermore, morac1 mutant is also defective in appressorium formation and pathogenicity. Genetic complementation using the ORF of the MoRAC1 from fosmid clone fully recovered all defective phenotypes. Whole genome microarray analysis of morac1 suggests that nearly hundreds of the genes were regulated more than two folds up and down, strikingly some of the genes are highly associated with surface recognition, germtube and hyphal formation, conidiation, pathogenicity, and recycling of cell wall compounds. These results demonstrate that MoRAC1 is a crucial player for conidial morphogenesis, cell growth, and pathogenicity in M. oryzae.


458. Agrobacterium tumefaciens-mediated transformation (ATMT) in two Colletotrichum Species; C. falcatum and C. acutatum. Maruthachalam Karunakaran1, Hee-Sool Rho1, Soonok Kim1, N. Vijayan Nair2 and Yong-Hwan Lee1. 1Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. 2Biotechnology Laboratory, Sugarcane Breeding Institute, Coimbatore 641-007, India.


Agrobacterium tumefaciens-mediated transformation is becoming an effective system as an insertional mutagenesis tool in filamentous fungi. In this study, ATMT was developed and optimized for two Colletotrichum species; C. falcatum and C. acutatum, the causative agents of red rot of sugarcane and anthracnose of pepper respectively. A. tumefaciens strain pYL63e, carrying hygromycin phosphotransferase gene (hph) and green fluorescent protein gene, was used to transform the conidia of these two Colletotrichum species. The transformation efficiency was higher in C. falcatum than that of C. acutatum, and it was also correlated with the co-cultivation time and bacterial cell concentration. The presence of hph and GFP genes were verified by PCR and microscopic analysis. About 70% of the transformants had a single copy of the T-DNA insertion in both C. falcatum and C. acutatum. Southern hybridization indicated that T-DNA integrated randomly in both the fungal genome. The T-DNA flanking regions were identified in transformants through thermal asymmetrical interlaced PCR followed by sequencing. Our results suggest that ATMT can be used as a molecular tool to identify and characterize pathogenicity related genes of these two important Colletotrichum species.


459. Hexosaminidase and plant growth promotion by Trichoderma hamatum. Lauren S. Ryder and Christopher R. Thornton. School of Biosciences, University of Exeter, Washington Singer Labs., Perry Eoad, Exeter, UK, EX4 4QG


Trichoderma species are important agents in the biocontrol of root pathogens and are attractive alternatives to synthetic pesticides. In addition to biocontrol properties, certain strains display plant-growth-promotional activities. While the involvement of chitinases in the biocontrol activities of Trichoderma species has been studied in depth, little attention has been paid to the role of chitinases in the ‘nutritional chitinolysis’ of Trichoderma spp. A rhizosphere-competent strain of T. hamatum (strain GD12) has been identified that elicits a 5-fold increase in the root and photosynthetic biomass of A. thaliana and other plant species during growth in low nutrient status plant growing media such as peat. High levels of hexosaminidase activities occur during saprotrophic colonization of peat by GD12 suggesting that the fungus is mobilizing hexosamines present in the medium. Consequently, we set out to determine the role that hexosaminidases play in saprotrophism of the fungus and whether mobilization of hexosamines could account for the plant-growth-promotion witnessed during Trichoderma-plant interactions. A 1.9kb fragment of the extracellular enzyme hexosaminidase was identified using primers based on the T. harzianum exc2y sequence. Strains disrupted in hexosaminidase production were developed using an insertional mutagenesis strategy and preliminary phenotypic analysis shows that mutants display enhanced plant growth-promotion activities. The mechanism for this phenomenon is currently being determined.


460. Signaling for symbiosis and pathogenicity. Natalia Requena, Stephanie Heupel and Hannah Kuhn. Institute for Applied Microbiology: Plant-Fungal Interactions group, University of Karlsruhe, Hertzstrasse 16, 76187 Karlsruhe, Germany; natalia.requena@bio.uka.de


Plants have evolved to interact with different microorganisms and establish with them different types of associations ranging from parasitic to mutualistic. In doing so, the immune system of the plant has developed to recognize signal/pattern molecules from those microorganisms to discriminate between deleterious and beneficial partners. The establishment of the arbuscular mycorrhiza symbiosis belongs to the second group and plants forming this type of symbiosis, most vascular plants, recognize specifically their symbiotic partners modifying in consequence their genetic programs to accommodate them. The molecular bases of this recognition process are starting to be understood and point to common signaling pathways shared with other microbe-plant associations like the nitrogen fixation symbiosis or pathogenic interactions and to specific signaling pathways only but commonly elicited by all arbuscular mycorrhizal fungi. In searching for fungal proteins involved in this early recognition process we identified in the AM fungus Glomus intraradices a novel protein Gin1 with a two domain structure. We have recently identified that this protein is post- translationally modified by plant effectors to render an amino-terminus with a lipid moiety covalently bound. Interestingly, a putative orthologue of the amino-terminus exists in a few fungi including the plant pathogens Magnaporthe grisea and Giberella zeae. In order to identify whether this common protein plays a dual role in symbiotic and pathogenic interactions we have taken M. grisea as a model and studied the KO phenotype during plant infection and the effect of its replacement with the mycorrhizal fungal protein. (This project is financed by the DFG 1556/1-3; DFG 1556/4-1).


461. Differential gene expression between the biotrophic-like and saprotrophic mycelia of the Witches’ broom pathogen Moniliophthora perniciosa. Johana Rincones1, Leandra Scarpari1, Jorge Mondego1, Maricene Sabha1, Marcelo Carazzolle1, Joan Barau1, Lyndel Meinhardt2, and Gonçalo Pereira1. 1Lab. Genômica e Expressão, IB, UNICAMP, Campinas/SP, Brazil. 2 Sustainable Perennial Crops Laboratory, USDA–ARS, Beltsville/MD, USA. johana@lge.ibi.unicamp.br

In order to improve our understanding of the molecular mechanisms involved in the biotrophic development of M. perniciosa, we performed DNA microarrays and EST analyses of the biotrophic and saprotrophic stages of this fungus, grown in vitro. Reads from 3 cDNAs libraries (glucose- and cacao-induced saprotrophic mycelia and cacao-induced biotrophic mycelium) were clusterized into 1596 unigenes. DNA microarray analysis was performed for 2304 genomic DNA fragments, with probes prepared from mRNA of the biotrophic and saprotrophic mycelia. Our results suggest that both types of mycelia show different carbon metabolisms, with glycolysis and the glyoxalate shunt being repressed in the biotrophic phase. Moreover, the biotrophic stage showed induced transcripts indicative of amino acid starvation, as well as several characterized as pathogenicity factors, such as Glyoxal oxidase, ceratoplatanin, proteases and peroxidases. Differential expression of selected genes was confirmed by Real Time-PCR. This work is the first to analyze global gene expression between the infective and saprotrophic stages of a hemibiotrophic fungus. Support: FAPESP and CNPq.


462. Towards a transformation system for the arbuscular mycorrhizal model fungus Glomus intraradices. Nicole Helber and Natalia Requena, Plant-Fungal Interactions Group, Institute of applied Biosciences University of Karlsruhe, Germany


Arbuscular mycorrhiza (AM) is known as one of the evolutionary oldest plant symbiosis. However, the knowledge about the biology of AM fungi has remained relatively unstudied to date. This is due to their obligate symbiotic and biotrophic lifestyle. Furthermore, spores and hyphae are multinucleated which complicates their genetic analysis. To advance our understanding of symbiosis establishment and functioning we are trying to establish a transformation method for the model AM fungus Glomus intraradices. Therefore we are using Agrobacterium-mediated transformation and biolistic transformation. As target tissue we are using ungerminated or germinated spores. Initially we used GFP as marker to monitor transformants. However, because of the strong autofluorescence of the fungal tissue in the green channel we have constructed new vectors containing the dsRed gene fused to the NLS sequence of stuA from Aspergillus nidulans. The reporter gene is driven by the Glomus mosseae promoters GmPMA1 or Gmfox2. In control experiments we could show that Gmfox2 promoter is active in Aspergillus nidulans. First data using the biolistic approach inG. intraradices showed red fluorescence spots in some spores and hyphae. Although we have not proven yet that these red spots are located in the nuclei we regard this as a good starting-point for further investigations.


463. Silencing of Kex2 significantly diminishes the virulence of C. parasitica. Debora Jacob-Wilk1, Massimo Turina1,2, Pam Kazmierczak1 and Neal Van Alfen1. Department of Plant Pathology, University of California, Davis. 1 Department of Plant Pathology, University of California, Davis, CA. 95616, USA. 2 Current address: Istituto di Virologia Vegetale, CNR Torino, Italy.


Cryphonectria parasitica is the causal agent of chestnut blight, its infection with Cryphonectria hypovirus 1 (CHV1) results in hypovirulent strains of the fungus. The discovery that several of the CHV1 down regulated genes encoded for secreted proteins containing the Kex2 processing consensus in conjunction with the recent finding that CHV1 utilizes fungal vesicles for replication that co-localize with trans-Golgi network marker Kex2, led us to investigate the possible role of Kex2 in fungal pathogenicity. We report on the cloning and analysis of Cryphonectria parasitica Kex2 gene. C. parasitica Kex2 gene sequence analysis showed high homology and structural similarities to other kexin like proteins from different Ascomycetes. In order to assess cpKex2 possible role in fungal virulence, antibodies were raised and Kex2 silenced mutants were generated. Southern blot analyses of cpKex2 showed a single copy gene while Western blots showed Kex2 expression at every time point checked. Although growth rate was not considerably affected by silencing, silenced isolates had significantly reduced virulence and this reduced virulence was in correlation with Kex2 levels of enzymatic activity and relative mRNA transcript levels as measured by real time RT-PCR.


464. Penetration Resistance: The First Line of Defense Against Invasive Pathogens. Shauna Somerville, Monica Stein, Matt Humphry, Laurent Zimmerli, Melisa Lim. Carnegie Institute, Stanford, CA, 94305, U.S.A. SSomerville@stanford.edu


A majority of host plants are resistant to the majority of pathogens in their environment. The underlying mechanisms of this form of disease resistance are poorly studied. The Paul Schulze-Lefert lab (Cologne, Germany), Hans Thordal- Christensen (Copenhagen, Denmark) lab and our lab, working collaboratively, identified mutants of the model plant Arabidopsis thaliana that were compromised in their resistance to the powdery mildew fungus, Blumeria graminis f. sp. hordei, which is pathogenic only on barley. Several Arabidopsis genes, not previously implicated in plant resistance mechanisms, have been identified by this approach, including PEN1 (=SYP121) a syntaxin and functional homologue of the barley ROR2 gene; PEN2, a glycosyl hydrolase; and PEN3 (=PDR8), an ABC transporter. In addition, MLO2 was identified as the functional homologue of the barley MLO gene, which confers penetration resistance when inactivated. The plasma membrane localized MLO, PEN1 and PEN3 proteins accumulate locally at penetration sites in inoculated plant tissues. These observations suggest that plants mount effective, broad spectrum defenses designed to block pathogen invasion across the host cell wall and entry into cells. Over evolutionary time, virulent powdery mildew species, such as Golovinomyces cichoracearum, a pathogen of Arabidopsis, must presumably have acquired the ability to avoid these defenses by stealth or to be insensitive to them.


465. Gene expression analyses of Fusarium graminearum during infection of wheat using microarrays and reporter gene constructs. Donald M. Gardiner1, Amber E. Stephens1,2, Anca Rusu1, Jo K. Bursle1, Agnieszka Mudge1, Kemal Kazan1, Alan L. Munn2, John M. Manners1. 1Commonwealth Scientific and Industrial Research Organisation (CSIRO), Plant Industry, 306 Carmody Rd, St Lucia 4072, Queensland, Australia 2The Institute for Molecular Bioscience, The University of Queensland, St Lucia, 4072, Queensland, Australia Donald.Gardiner@csiro.au


Fusarium Head Blight is one of the most important diseases of wheat worldwide. In Australia, F. graminearum and the closely related fungus F. pseudograminearum, cause sporadic head blight epidemics and are also responsible for crown rot disease in wheat. These diseases can lead to substantial yield losses and accumulation of mycotoxins, particularly trichothecenes, which adversely affect human and animal health when consumed. We are using green fluorescent protein reporter constructs to investigate the temporal and spatial expression patterns of a number of candidate genes potentially involved in the infection process, particularly at the stem base. Candidates include the Tri5 trichothecene biosynthesis gene and a number of genes identified as being expressed in the early stages of crown rot disease using the F. graminearum microarrays and RT-qPCR.


466. A multi-locus sequence typing system for the chytrid, Batrachochytrium dendrobatidis. Russell Poulter, Margi Butler, Tim Goodwin. University of Otago, New Zealand. russell.poulter@stonebow.otago.ac.nz


Batrachochytrium dendrobatidis is a recently emerged pathogen of amphibia responsible for a world-wide pandemic, leading to numerous species extinctions. All isolates of B. dendrobatidis have very similar genomes and this restricts phylogenetic analyses and epidemiological studies. We will describe a multi-locus sequence typing system (MLST) based on the several inteins we have detected in the genome of this species. Intein encoding sequences are typically less constrained than most coding sequences and they are therefore well suited to analysing the phylogenies of homogeneous species. Inteins are proteins that are removed post-translationally from a host protein. This is an autocatalytic splicing process necessary for the maturation of the host protein. Inteins typically contain not only the necessary protein splicing domains but also a homing endonuclease (HEG). The HEG enables the intein encoding sequence to spread through the gene pool of a species and potentially to also undergo horizontal transmission to other species. The HEG activity is therefore seldom used and the HEG could be completely inactive without adversely affecting the protein splicing function. This is the explanation of the relatively unconstrained sequence of inteins.


467. Characterization of Yeasts Associated with Green Tea Leaves. Pham Tran To Nhu, Do Quyen, Ngo Duc Duy, Hoang Quoc Khanh. Institute of Tropical Biology, Viet Nam Academy of Science and Technology 1 Mac Dinh Chi St., Dist. 1, Ho Chi Minh City, VIETNAM


This paper is described yeast strains isolated from tea leaves collected in Bao Loc and Gia Lai provinces, Viet Nam. Five yeast strains of BL1, BL2, BL3, BL4 and GL were identified by using conventional (morphology, physiology, biochemistry) and molecular phylogenetic studies. The vegetative reproduction occurs in these strains by budding. Lipase and acid production from glucose were negative. Fermentation of sugar such as glucose, galactose, and maltose were positive. All strains revealed the capacity of gelatin liquefaction, except BL2. These strains assimilated carbon compounds glucose, galactose, sucrose, L-sorbose, glycerol, sorbitolâ€| and nitrogen compounds (NH4)2SO4, ethylamine, L-lysine. Based on the sequence analysis of the 26S-rDNA D1/D2 domains and internal transcribed spacer (ITS) regions, then compared with the databases of the Genbank NCBI, the results show that BL1 and GL belonged to the genus Candida, near Candida tropicalis and Candida fermentati respectively. Two strains of BL2 and BL4 were known as Rhodotorula mucilaginosa and Rhodotorula sp. CBS 8885. Finally, BL3 was the nearest Rhodosporidium fluviable. These strains (BL2, BL3 and BL4) were further investigated the production of carotenoid.


468. Signaling in Botrytis cinerea – a focus on small GTPases and early steps of infection. Leonie B. Kokkelink and Paul Tudzynski. Westfaelische Wilhelms University, Institute of Botany, Schlossgarten 3, D-48149 Muenster, Germany


The necrotrophic ascomycete B. cinerea causes gray mold diseases on various crop plants. Up to the present, several components of signaling pathways were identified that are involved in pathogenicity. So far there is little knowledge about the impact of small GTPases belonging to the Ras superfamily on pathogenicity in B. cinerea and about connections to other signaling pathways. We are currently investigating several small GTPase encoding genes from B. cinerea: two genes encoding Rho type GTPases, bccdc42 and bcrac, and two Ras homologues, bcras1 and bcras2. By different approaches like pathogenicity assays, epidermis penetration experiments, germination assays and microscopy we determine the role of the small GTPase deletion strains in pathogenicity and early infecton stages. bcras1 and bcrac deletion mutants are viable, but show a severe phenotype characterized by an undirected hyphal growth with abnormal branching pattern, a lack of sporulation and impaired infection ability. In contrast, the Δbccdc42 mutants grow properly on rich media and are able to form conidia. This strain is able to infect plants, though the infection progress is delayed. Furthermore, we are interested in the interconnections and hierarchies to other major signaling pathways (e. g. cAMP signaling / MAP kinases). For instance, phosphorylation experiments are carried out to elucidate the connection between Ras proteins and MAPK.


469. A secondary metabolite is involved in recognition of the blast fungus Magnaporthe grisea by resistant rice cultivars. Jérôme Collemare1, Sabrina Bouras1, Heidi U. Böhnert1, Isabelle Fudal1, Zhongshu Song2, Russell J. Cox2, Didier Tharreau3 and Marc-Henri Lebrun1. 1UMR2847 CNRS-Bayer Cropscience, Physiologie des plantes et des champignons lors de l’infection, Bayer Cropscience, 14-20 rue P Baizet, 69263 Lyon Cedex 09, France. 2School of Chemistry, University of Bristol, Cantock’s close, Clifton, Bristol BS8 1TS, UK. 3UMR BGPI, CIRAD-INRA-AGROM, TA41/K, 34398 Montpellier Cedex 05, France


Recognition of the fungal plant pathogen Magnaporthe grisea by resistant rice cultivars is controlled by interactions between fungal avirulence genes (AVR) and their corresponding plant resistance genes (R). Most fungal AVR genes encode small peptides secreted into host tissues during infection. ACE1 from M. grisea differs from other AVR genes as it encodes a polyketide synthase fused to a non-ribosomal peptide synthetase, an enzyme involved in the biosynthesis of a secondary metabolite. This AVR gene controls the production of a signal recognized by rice cultivars carrying Pi33 resistance gene. ACE1 is specifically expressed in mature appressoria during penetration of the fungus into rice leaves. The protein Ace1 is only detected in the cytoplasm of appressoria and not in infectious hyphae differentiated inside infected epidermal cells. Ace1-ks0, a non-functional ACE1 allele obtained by site-directed mutagenesis of an amino acid from polyketide synthase KS domain essential for its enzymatic activity, is unable to confer avirulence. This result suggests that the avirulence signal recognized by Pi33 is not the Ace1 protein, but the secondary metabolite synthesized by Ace1. In order to characterize this metabolite, ACE1 was expressed in M. grisea under the control of a constitutive promoter. ACE1 was also expressed under the control of an inducible promoter in Aspergillus oryzae and in Fusarium venenatum. Secondary metabolites produced by these transgenic strains are currently analyzed by LC-MS-MS (coll. Certon, Metcalf and Drivon, Bayer CropScience, France). At the ACE1 locus, we identified 14 genes predicted to encode enzymes involved in secondary metabolism, including two enoyl-reductases and a binuclear zinc-finger transcription factor. These genes have the same expression pattern as ACE1 defining a cluster of co-expressed genes, suggesting that they are involved in the same biosynthetic pathway. The inactivation of these genes in an avirulent isolate is underway to assess their role in the biosynthesis of the metabolite recognized by Pi33 resistant rice cultivars.


470. Functional analysis of Botrytis cinerea NEP-like proteins. Yaite Cuesta Arenas, Alexander Schouten, Peter Vredenbregt, Mirjam Dieho , Beatrice Uwumukiza & Jan van Kan. Wageningen University, Laboratory of Phytopathology, Wageningen, The Netherlands

Necrosis and ethylene inducing proteins (NEP) have been described in bacteria, oomycetes and fungi and have been proposed to act as phytotoxin in dicotyledons but not in monocotyledons. However, the mechanism of action is not known. Botrytis cinerea contains two NEP-like proteins, BcNEP1 and BcNEP2, which have been produced in Pichia pastoris.Infiltration of purified proteins into Nicotiana benthamiana leads to induction of ethylene in a dose-dependent manner. BcNEP2 is less active in inducing ethylene than BcNEP1.BcNEP1 and BcNEP2 contain a number of cysteines that may form disulfide bridges, as well as several potential posttranslational modification motifs. In order to study which amino acid residues are important for phytotoxic activity, site directed mutagenesis was performed and mutant proteins were expressed transiently in N. benthamiana through Agrobacterium tumefaciens (ATTA). Also through this approach, GFP was coupled to the C-terminus of BcNEP1 protein. The fusion protein retained phytotoxic activity and will be used for localization studies.


471. Defense of Aspergillus fumigatus against reactive oxigen species mediated by Afyap1. Franziska Lessing, Olaf Kniemeyer and Axel A. Brakhage. Leibniz Institute for Natural Products and Infection Biology – Hans-Knoell-Institute, Friedrich Schiller University, Jena, Germany Contact: franziska.lessing@hki-jena.de


With the increasing number of immunocompromised individuals Aspergillus fumigatus has become one of the most important opportunistic fungal pathogens. During infection A. fumigatus is confronted with a number of defence mechanisms in the host, particulary neutrophiles and macrophages, which kill conidia by producing reactive oxygen species. We identified a homologe of the AP1 like transcription factor Yap1 from yeast in A. fumigatus which we designated Afyap1. In yeast, Yap1p was found to be a global regulator for oxidative stress response and required for the protection of the cell against H2O2 and other reactive oxygen species. Yap1 is transported in and out the nucleus under nonstressed conditions. The nuclear export is inhibited by oxidative stress and Yap1 induces the transcription of target genes. Nuclear loaclisation of an Afyap1-eGFP fusion in A. fumigatus was dependent on the presence of H2O2 and diamide. To identify new targets of Afyap1, we compared the proteome pattern from H2O2 induced and uninduced wild-type mycelia and a Afyap1 deletion strain by 2D-gel analysis.


472. Investigating the roles of reactive oxygen species during Mycosphaerella graminicola infection of wheat. Siân Deller, John Keon, John Antoniw, Kim Hammond-Kosack, and Jason Rudd.. Wheat Pathogenesis Programme, Plant- Pathogen Interactions Division, Rothamsted Research, Harpenden, Herts., AL5 2JQ, UK


Mycosphaerella graminicola (anamorph Septoria tritici) is a hemibiotrophic ascomycete pathogen of wheat leaves. Infection reduces crop yield via the appearance of chlorotic / necrotic lesions, which reduce the photosynthetically active leaf area. In the early stages of infection, following penetration through stomata, the fungus grows in the leaf intercellular space without producing any specialised feeding structures or causing visible disease symptoms. In the later infection stages, fungal biomass increases, hyphal nutrition becomes necrotrophic and localised host programmed cell death occurs (1). Septoria leaf blotch disease of wheat is currently regarded as the most economically damaging pathogen of wheat in the UK and Western Europe. Microscopy with reactive oxygen species (ROS)-specific stains has shown that hydrogen peroxide and superoxide are present in infected leaves, and that intensity of ROS stains increases with disease progression. In particular ROS are present on and within the asexual fruiting bodies, pycnidia. The involvement of ROS in the development of fungal sexual fruiting bodies has been reported (2, 3). Expression profiling of M. graminicola during infection of susceptible wheat genotypes has shown a cluster of genes, many ROS-associated, that have greatly increased expression as disease progresses and symptoms become visible. This project aims to better understand the molecular basis of the involvement of ROS in the disease-causing ability of M. graminicola on wheat. EST microarray datasets have been produced which have compared fungal gene expression under different growth conditions (4, 5). The microarray has also been employed to investigate to what extent application of exogenous ROS alone can induce gene expression patterns similar to those seen at the later stages of plant infection. Access to the sequenced genome allows targeted Agrobacterium-mediated gene deletion to be used for the investigation of fungal genes involved in the production of ROS or the oxidative stress response. Deletion analysis will allow comparison between transformant strains and wild-type IPO323 via in vitro and in planta characterisation and use of the EST microarray. 1.Keon, J., Antoniw, J., Carzaniga, R., Deller, S., Hammond-Kosack, K., & Rudd, J. (2007) MPMI (In press). 2.Malagnac, F., Lalucque, H., Lepere, G., & Silar, P. (2004) Fungal Genetics and Biology 41, 982-997. 3.Lara-Ortiz, T., Riveros-Rosas, H., & Aguirre, J. (2003) Molecular Microbiology 50, 1241-1255. 4.Keon, J., Antoniw, J., Rudd, J., Skinner, W., Hargreaves, J., & Hammond-Kosack, K. (2005) Fungal Genetics and Biology 42, 376-389. 5.Keon, J., Rudd, J. J., Antoniw, J., Skinner, W., Hargreaves, J., & Hammond-Kosack, K. (2005) Molecular Plant Pathology 6, 527 - 540. This project receives financial support from Syngenta and the Biotechnology and Biosciences Research Council (BBSRC) of the UK. Rothamsted Research receives grant aided support from the BBSRC.


473. Expression of GFP in the obligate biotrophic oomycete Plasmopara halstedii using electroporation and a newly developed mechanical method (Löchern). Timo Hammer, Otmar Spring, Marco Thines. University of Hohenheim, Institute of Botany 210, 70593 Stuttgart, Germany, t-hammer@uni-hohenheim.de


Transformation of several oomycete species has been achieved during the past few years. Among these are plant pathogens of the genera Pythium and Phytophthora, which can be cultivated on media. In case of the obligate biotrophic pathogens belonging to the downy mildews (Peronosporaceae), no transgenic expression of any gene has been reported. In the model system Plasmopara halstedii and Helianthus annuus we have therefore tried several methods to obtain Pl. halstedii strains expressing GFP as a first starting point for further investigations into plant-pathogen interactions. Using electroporation and Löchern, a novel technique for transforming obligate biotrophic organisms we could achieve GFP expression in sporangia and sporangiophores of Pl. halstedii. Löchern involves injuring pathogen-infected leaves with a thin needle and at the same time applying a solution of the transformation vector on the leaf surface. Because of the easy handling and the possibility to circumvent the need for regeneration on media, Löchern could become an important technique in transforming obligate biotrophic pathogens.


474. Identification of virulence genes in Fusarium oxysporum by large-scale transposon tagging. M. Sánchez López-Berges1, M. Dufresne2, C. Hera1, H. abd el Wahab2, M.I.G. Roncero1, M.J. Daboussi2, A. Di Pietro1. 1Departamento de Genética, Universidad de Córdoba, Campus Rabanales Ed. C5, 14071 Córdoba, Spain. E- mail: ge2snlpm@uco.es 2Institut de Génétique et Microbiologie, Université Paris-Sud, 91405 Orsay Cedex, France.

Forward genetic screens are efficient tools to dissect complex biological processes such as fungal pathogenicity. A transposon tagging system was developed in the vascular wilt fungus Fusarium oxysporum by inserting a novel modified version of the impala element upstream of the Aspergillus nidulans niaD gene, thus allowing easy monitoring of transposition events and isolation of revertants showing wild type growth on nitrate. In a pilot study, 1500 Nit+ revertants were screened for altered virulence using a rapid infection assay on apple slices. Seven mutants exhibiting reduced virulence were identified and their phenotype was confirmed by infection assays on tomato plants. In 5 of them, the genomic region flanking the insertion site was characterized and the transposon insertion confirmed by Southern blot analysis. In 4 mutants, the insertion was found upstream of a putative ORF, whereas in the remaining mutant it was located within an ORF. Targeted knockout of the tagged genes is currently in progress to confirm their role in virulence.


475. Detoxyfication of progesterone in fungi Fusarium oxysporum and Ustilago maydis by progesterone-induced enzymes. Anna Poli, Helena Lenasi. University of Ljubljana, Faculty of Medicine, Institute of Biochemistry Ljubljana, Slovenia


Progesterone was previously shown to induce in filamentous fungi Rhizopus nigricans (phylum Zygomycota, class Zygomycetes, order Mucorales) and Cochliobolus lunatus (phylum Ascomycota, class Acsomycetes, order Plesporales) a multienzyme steroid hydroxylating system. It was also observed that progesterone signalling was mediated via receptors located in the plasma membrane and inside mycelia. It is generally believed that the induced enzyme system is involved in the detoxyfication process. In the present study we tried to answer the question whether this progesterone action is general in fungi. For our investigation we chose plant pathogens from phyla Ascomycota and Basidiomycota: Fusarium oxysporum (class Acsomycetes, order Hypocreales), Ustilago maydis (class Ustilaginomycetes, order Ustilaginales). In liquid growth medium progesterone was applied. Steroids were extracted from mycelia and from the medium in different time intervals. The content of progesterone and progesterone derivatives were analysed either by TLC or HPLC and identified by gas chromatography/mass spetrometry. Fusarium oxysporum transformed progesterone mainly into 11- hydroxyprogesterone which appeared mostly in the medium, whereas Ustilago maydis transformed progesterone into a reduced product of A-steroid ring in addition to some hydroxylated products. The biological role of progesterone-induced enzymes is most likely transformation of toxic progesterone into less toxic products.


476. The Phytophthora sojae avirulence gene Avr1a, encoding an RxLR effector protein, displays copy number polymorphism. Dinah Qutob 1, Brett M. Tyler 2,and Mark Gijzen 1. 1 Agriculture and Agri-Food Canada, London,ON, Canada. 2 Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA.


We have completed the map-based cloning and identification of the Phytophthora sojae avirulence gene, Avr1a. Mapping of Avr1a required more than 1500 F2 individuals from two separate crosses, illustrating the relatively low recombination frequency of the region. Problems in physical mapping occured even after completion of the P. sojae genome sequence, because the Avr1a gene does not occur on a large assembly contig. Physical mapping was aided by comparison to a syntenic region in Phytophthora ramorum. Eventually, we identified an open reading frame within the Avr1a delimited region in P. sojae that encodes a secreted RxLR-dEER protein of 121 amino acids. Transcriptional analysis demonstrated that the gene is expressed during infection, and that differences in the expression level could be correlated to Avr1a phenotype. Transient expression of this gene in soybean triggered an Rps1a -specific cell death response, confirming that it corresponds to Avr1a. Preliminary evidence suggests that multiple copies of Avr1a occur as a repetitive unit in certain P. sojae strains, including the isolate that was used for whole genome sequencing (P6497), thus accounting for the poor assembly of this region. Overall, our results can be integrated into a model whereby Avr1a copy number polymorphisms lead to transcriptional and translational differences that either activate or evade effector-triggered immunity on Rps1a soybean plants.


477. Identification of Phytophthora sojae Avr3a by Expression Profiling. Jennifer Tedman-Jones, Jonathan Eckert, and Mark Gijzen. Agriculture and Agri-food Canada, Southern Crop Protection and Food Research Centre, London, Ontario, Canada


Past studies have shown that some Phytophthora sojae isolates have lost expression of a particular avirulence (Avr) gene to evade recognition by soybean cultivars carrying the corresponding resistance (R) gene. To identify genes whose expression is associated with a particular avirulence trait, two F2 bulks were generated from an existing P. sojae population (Race2: Avr3a/Avr5/Avr3c crossed with Race7: avr3a/avr5/avr3c) based on the virulence phenotype of individual progeny on cultivars carrying Rps3a, Rps5 or Rps3c. The expression profiles of 15,800 transcripts were measured from total RNA, from infected soybean seedlings, for the two bulks and the two parents, using Affymetrix gene chips. The microarray data was mined to identify avirulence-associated transcripts. Transcripts that showed an association were further analyzed by RT- PCR of total RNA from individual F2 progeny. RT-PCR analysis revealed one transcript that showed 100% association to avirulence, conditioned by Avr3a and Avr5, among the F2 progeny and parents. This transcript encodes a small secreted peptide containing RXLR-dEER motif typical of oomycete avirulence genes. The corresponding gene is located in a region of the genome with a high density of genes encoding secreted peptides with the RXLR-dEER motif. Co-bombardment of the avirulence gene candidate with the promoter:reporter gene construct, 35S:beta-glucuronidase (GUS), results in a marked reduction in GUS activity in soybean plants carrying Rps3a, but not those carrying Rps5. This data shows the protein product of this candidate is specifically recognized by RPS3a in the soybean host, and therefore most likely encodes the P. sojae avirulence gene Avr3a.


478. Identification of secreted proteins from appressoria of Colletotrichum higginsianum by analysis of expressed sequence tags. Jochen Kleemann, Hiroyuki Takahara, Kurt Stueber, and Richard O'Connell. Max Planck Institute for Plant Breeding Research, Koeln, Germany. oconnel@mpiz-koeln.mpg.de

The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease on brassica crops and the model plant Arabidopsis. Melanized appressoria initially pierce the host cuticle and cell wall to form specialised biotrophic hyphae that develop inside living epidermal cells. The aim of this study was to identify proteins secreted by appressoria, which may function to overcome host defense responses and reprogram host cells for biotrophy. A random- primed cDNA library was prepared from mature appressoria formed in vitro and expressed sequence tags (ESTs) derived from sequencing 980 clones were assembled into 520 unique sequences. These were then screened for putative open reading frames containing signal peptides using the SignalP prediction program. Although based on a relatively small dataset, including some N-terminally incomplete sequences, this analysis identified 61 proteins with predicted signal peptides that are likely to be cotranslationally imported into the endoplasmic reticulum. Sequence similarity searches with BLASTP revealed that 37 of these had significant homology to proteins of known function, including some soluble secreted proteins and integral membrane proteins that have been implicated in fungal pathogenicity. A further 24 putative secreted proteins were either homologous to fungal hypothetical proteins or lacked any database matches and are candidates for being novel effectors. The temporal expression of selected candidates during fungal development in vitro and in planta was examined using RT-PCR.


479. Translating and Coping with Stress: eIF2α kinases and the Cross-Pathway Control System of Aspergillus fumigatus. Christoph Sasse1, Elaine M. Bignell2, Stanley Kim3, Gerhard H. Braus1, Sven Krappmann1. 1Georg-August University Goettingen, Grisebachstr. 8, D-37077 Goettingen, Germany; 2Imperial College London, UK; 3 The Institute for Genomic Research, USA


Aspergilli represent unique pathogens with Aspergillus fumigatus being the predominant perpetrator. We are interested in nutritional requirements sustaining propagation and supporting virulence. Fungal amino acid (aa) biosynthesis is regulated on various levels; besides pathway-specific systems, one global regulatory network has evolved that acts on aa metabolism as a whole. In its very components, this Cross-Pathway Control is made up by an eIF2 kinase sensing aa deprivation and translating it into increased levels of the transcriptional activator protein CpcA, which in turn elevates transcription for the majority of amino acid biosynthetic genes. As deduced from profiling studies, the scope of the CpcA-directed transcriptome exceeds amino acid biosynthesis. To elucidate the role of the CPC signal transduction cascade, the gene of the sensor kinase CpcC was cloned. In contrast to cpcAdelta mutants, strains deleted for cpcC are not impaired in virulence, indicating that the basal expression level of CpcA is necessary and sufficient to support pathogenesis. Western blot analyses indicate that the cpcC-encoded kinase is not required exclusively to phosphorylate the eIF2 subunit. Upon inspection of the A. fumigatus genome, the presence of a related gene (ifkB, for initiation factor kinase) could be revealed. The ifkB gene was deleted in and corresponding mutant strains are currently evaluated.


480. Gene expression of dermatophyte Trichophyton rubrum during assimilation of keratin in mimetic infection. Maranhão, FCA, Paião, FG, Martinez-Rossi, NM. Departamento de Genética, Faculdade de Medicina de Ribeirão Preto-USP Av Bandeirantes 3900, 14049-900 Ribeirão Preto-SP, Brazil E-mail: fcam@usp.br


T. rubrum is a cosmopolitan and antropophilic fungus, the most prevalent in superficial mycoses, growing preferentially in keratin substrates as skin and nail. This work evaluated the pH changes during T. rubrum growth in keratin (after 6, 12, 24, 48, 72h and 7 days) at initial pH 5.0. T. rubrum promoted a medium pH increase reaching 8.3 in keratin, after 72h. However, this effect was not observed when the fungus was cultivated in glucose as carbon source, where the pH was maintained in approximately 5.0. Also, we identified 576 T. rubrum transcripts differentially expressed by Subtractive Suppression Hybridization using mycelia cultivated in keratin (72h) as tester and in glucose as driver. Clusters were recovered with CAP3 sequence assembly program, yielding 40 contigs and 215 singlets. The ESTs sequences were analyzed against the protein databases in GenBank by BLASTx search. Putative proteins encoded by genes obtained under keratin condition showed similarity to several fungi proteins involved in basic metabolism, growth and virulence, i.e., transporters ABC-MDR, MFS and ATPase of copper, NIMA interactive protein, pol protein, virulence factors serine-protease subtilisin and metalloprotease, cytochrome p450, GlcN-6-phosphate deaminase and HSP30. The induction expression of 214 genes was confirmed by macro array dot blot, and the upregulation of T. rubrum genes of subtilisin, metalloprotease and pol protein were also validated by northern blot. Our results show the SSH efficacy to reveal genes upregulated in keratin and suggest a relationship between these proteins and T. rubrum pathogenicity. Financial Support: FAPESP, CAPES, CNPq, FAEPA


481. Genomic discovery and evolutionary patterns of the effector proteins in Phytophthora. Rays H.Y. Jiang1,2,3, Francine Govers1, Michael C. Zody2, Chad Nusbaum2, Sucheta Tripathy3, and Brett M. Tyler3. 1Laboratory of Phytopathology, PSG, Wageningen University, Wageningen, The Netherlands 2Broad Institute of MIT and Harvard, Cambridge MA, USA 3Virginia Bioinformatics Institute, Virginia Polytechnic and State University, Blacksburg VA, USA


Biotropic oomycetes and fungi secrete a variety of molecules, many of which presumably promote infection. Molecules with a role in virulence or pathogenicity are termed effectors. In Phytophthora, a genus that belongs to the oomycetes and comprises many notorious plant pathogens, several effectors have been identified that all possess a RXLR- dEER domain. Bioinformatic mining of the genome sequences of Phytophthora sojae and Phytophthora ramorum revealed around 350 RXLR-dEER effector-like genes in the each of the two species. These genes belong to the most rapidly evolving genes in the genome. They show a high level of sequence divergence between species as well as extensive lineage specific expansion. Moreover, the genomic regions where RXLR-dEER genes are located show significant enrichment of retrotransposons, and nearly always show features associated with genome rearrangements. Despite of their high sequence divergence, all the proteins in the large RXLR-dEER super-family seem to be related. A single ancestral molecule has most likely evolved into this super-family by rapid duplication and divergence. The rapid pace of evolution of the RXLR-dEER proteins underlines the potential role of these proteins in the interaction with host plants, whereas the conserved RXLR-dEER domain points to a functional feature shared by the members of this super- family.


482. The Galpha-subunit BCG1 – An activator of Ca2+/calcineurin-dependent signaling in Botrytis cinerea. Julia Schumacher 1, Muriel Viaud2, and Bettina Tudzynski1. 1Institut für Botanik, Westf. Wilhelms-Universität Münster, Schlossgarten 3, D-48149 Münster, Germany 2Unité de Phytopathologie et Méthodologies de la Détection, INRA, Route de Saint-Cyr, F-78086 Versailles, France

The alpha-subunit BCG1 of a heterotrimeric G-protein plays an important role during the infection of host plants by the gray mold fungus Botrytis cinerea: BCG1 triggers the transition from primary infection to secondary invasive growth, is essential for the production of the phytotoxic secondary metabolite botrydial and for the secretion of proteases. Based on the fact that the expression of several BCG1 target genes is also dependent on the Ca2+/calmodulin-dependent calcineurin-phosphatase, we suggest that BCG1 is an upstream regulator of calcineurin, in particular in regulation of secondary metabolism. In order to identify the transcription factor downstream of BCG1 and calcineurin, we cloned the B. cinerea homologue of CRZ1 (Calcineurin-Responsive Zinc finger), the mediator of calcineurin function in yeast. The analysis of gene expression in the corresponding deletion mutants confirmed our suggestion that this transcription factor acts downstream of calcineurin. Furthermore, we propose a model in which the interconnection between BCG1 and calcineurin is mediated by BcPLC1, a phosphatidylinositol-specific phospholipase C. Mutants with educed transcript levels of bcplc1 showed the same alteration in gene expression as bcg1- and bccrz-mutants.


483. The cAMP-signaling cascade is required for full pathogenicity in Botrytis cinerea. Julia Schumacher, Christina Huesmann, and Bettina Tudzynski. Institut für Botanik, Westf. Wilhelms-Universität Münster, Schlossgarten 3, D-48149 Münster, Germany


It was shown that the highly conserved cAMP signaling pathway plays a crucial role during pathogenesis in the plant infecting fungus Botrytis cinerea. The upstream components of this cascade were analysed in detail: There are three different alpha-subunits of heterotrimeric G-proteins and two of them, BCG1 and BCG3, are activators of the adenylate cyclase BAC, the producer of cAMP. The characterization of the corresponding deletion mutants showed that this pathway regulates morphogenesis (sugar concentration-dependent colony morphology), germination of conidiospores (reduced sugar dependent germination) and virulence on bean plants (retarded infection process). To study the cAMP-pathway in more detail, we focused on the cAMP-dependent protein kinase (PKA), the main effector of the adenylate cyclase and cAMP. In the B. cinerea genome, two catalytic subunit encoding genes (pka1 and pka2) and one gene encoding the regulatory subunit (pkaR) were identified. While the pka1- mutants show a general growth defect and reduced virulence similar to bac-mutants, pka2-mutants have no obvious phenotype. In contrast, mutants with a “dominant-active” PKA, resulting from the knock out of the regulatory subunit, revealed a slow growth rate and repressed sporulation. Interestingly, some features of the bac-mutant differ from those of the pka1-mutant suggesting crosstalks to other signaling pathways.


484. Comparisons of the morphology and pathogenicity of Fusarium graminearum strains PH1 and Gz3639. S. Bec1, D. Van Sanford2, L. Vaillancourt1. 1Department of Plant Pathology, 2 Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY


Fusarium graminearum is a major causal agent of Fusarium Head Blight (FHB) of small grains. The genome sequences of two F. graminearum strains isolated from wheat heads, PH1 (NRRL 31084) and Gz3639, have been assembled and released. In spite of the high level of overall genome sequence homology, the two strains differ significantly in colony morphology, growth rate, production of asexual spores, fertility, and mycotoxin profiles. Apart from deoxynivalenol (DON), which is produced by both strains, PH1 also produces 3-ADON while Gz3639 belongs to the 15-ADON chemotype. We used the point inoculation method of infecting wheat heads at anthesis to compare pathogenicity and aggressiveness of these two strains at different inoculum concentrations on three wheat varieties. The wheat varieties differ in the degree and type of resistance they express against FHB. Both of the Fusarium graminearum strains produced similar disease symptoms at high (5 x106 and 5 x 105 spore/ml) inoculum concentrations. However, significant differences in disease severity and the rate of symptom development became evident at lower (5 x103 spore/ml) spore concentrations, with PH1 being more aggressive than Gz3639. It has recently been reported that 3-ADON chemotype strains (like PH1) are more aggressive on wheat. Our goal is to analyze the progeny of a cross between PH1 and Gz3639 in order to test the hypothesis that the higher level of aggressiveness displayed by PH1 is due to production of 3-ADON.


485. Discovering differentially regulated genes in the lichen symbiosis. Suzanne Joneson1, and Francois Lutzoni1. 1Duke University, Biology Department, Box 90338, Durham NC 27708, USA.


Lichens are the symbiotic association of fungi (mycobionts) with green algae and/or cyanobacteria (photobionts). Although one fifth of all known fungi form obligatory lichens with photobionts, we know nothing of the genetic or molecular mechanisms underlying this life-style. Here we present the first investigations into differentially expressed genes in early lichen development including pre-contact, and initial contact of the symbionts. Using the fungus Cladonia grayi and the green alga Asterochloris sp., we have sequenced over 2000 fungal and algal clones upregulated in response to resynthesized symbiosis. We used BLAST and FASTA algorithmic searches of databases containing nucleotides, proteins, and conserved protein domains to characterize our sequences. The data obtained provides insights into similarities between C. grayi’s response to it’s photosynthetic host, and that of phytopathogenic fungi to their plant hosts. This dataset represents the first global survey of fungal and algal gene sequences involved in lichen symbiosis, and a summary of these genes and their putative functions will be presented. The results of this study will allow us to identify candidate genes of early lichen development for future research.


486. Different secreted lipases are virulence factors of F. graminearum. Nguyen Nam L., M. Fehrmann, S. Salomon, W. Schäfer. University of Hamburg, Biocenter Klein Flottbek, Ohnhorststr. 18, Hamburg, Germany

Fusarium graminearum (teleomorph Gibberella zeae) is the major cause of Fusarium head blight on wheat, barley and other small grain cereals and of ear rot on maize. The outcome of infection varies between total destruction of the host and minor damage. Virulence of F. graminearum is the consequence of a collection of additively acting virulence factors. Only the trichothecene mycotoxins were known to be secreted virulence factors. We identified recently a secreted fungal lipase as a new virulence factor (Voigt et al. 2005), in addition to the already known secreted trichothecenes. Knowledge about the role of all virulence factors in promoting virulence and their distribution in different isolates will contribute to the understanding of the cereal – Fusarium interaction and offer new approaches to resistance breeding. We identified 17 putatively secreted lipase genes in the genome of F. graminearum. We cloned and characterized 5 of these genes. Three of them act as virulence factors, whereas a forth secreted lipase is not involved in fungal virulence. A fifth lipase seems to uphold vital functions in the fungal cell, as we were unable to disrupt the gene. We show the infection behaviour of the lipase-mutants on wheat and a transcriptional analysis of the lipase genes in culture and during wheat head infection. Voigt, C.A., Schäfer, W., and Salomon, S.: A secreted lipase of F. graminearum is a novel virulence factor during infection of cereals. Plant J. 42, 364–375 (2005).


487. Distinct amino acids of Phytophthora infestans AVR3a condition activation of R3a hypersensitivity and suppression of cell death. Jorunn I. B. Bos, Angela Chaparro-Garcia, Lina M. Quesada, Sophien Kamoun. Dept. of Plant Pathology, Ohio State University-OARDC, Wooster, OH, USA


The AVR3a effector of Phytophthora infestans is a member of the RXLR family of cytoplasmic effectors that exhibits dual effector functions. AVR3a induces hypersensitivity mediated by the resistance protein R3a and suppresses cell death induced by P. infestans INF1 elicitin. Two polymorphic forms of AVR3a that differ in two amino acids in the mature region occur in P. infestans. While AVR3aKI can mediate both R3a activation and cell death suppression, no effector function has been identified for AVR3aEM. To gain insights in the molecular basis of AVR3a activities, we performed structure-function analyses of both forms of AVR3a. We used near saturation high-throughput random mutagenesis screens based on functional expression in Nicotiana benthamiana to identify Avr3a mutants with altered activation of R3a hypersensitivity. Out of more than 6500 AVR3aEM mutants tested, we identified about 150 gain-of-function mutants that induced R3a hypersensitivity. About 10 residues, mostly charged amino acids, were frequently substituted in these mutants. These AVR3aEM gain of function mutants did not always gained the ability to suppress cell death suggesting that distinct residues condition R3a hypersensitivity and cell death suppression. A similar loss of function screen of 4500 AVR3aKI mutants resulted in smaller number of mutants with altered activity. These results point to a model that involves the interaction of AVR3a with a host protein and is not consistent with the recognition of AVR3a through an enzymatic activity.


488. Roles of MFS and ABC transporters in cercosporin resistance and biosynthesis in the fungus Cercospora nicotianae. Alongkorn Amnuaykanjanasin and Margaret E. Daub. Department of Plant Biology, North Carolina State University, Raleigh, NC, U.S.A.


Cercosporin, a photoactivated toxin produced by Cercospora spp., is highly toxic to a wide range of organisms due to its production of reactive oxygen species. The producing fungus has self-resistance to its own toxin. However, a C. nicotianae mutant disrupted in the transcriptional regulator gene CRG1 is sensitive to cercosporin and also produces approximately a half of the toxin produced by wild type (WT). This mutant (crg1) also showed increased green fluorescence in the mycelium, indicative of accumulation of the endogenous, reduced form of cercosporin. We hypothesized that efflux pump transporters may not be functioning properly in crg1. A subtractive library was obtained of genes differentially expressed between crg1 and WT. From this library we focused on genes encoding major facilitator superfamily (MFS) and ATP-binding cassette (ABC) transporters to determine their possible roles in self-resistance and production of cercosporin. We cloned and sequenced two MFS transporter genes (CnCFP and MFS2), the former having homology to cercosporin facilitator protein (CFP) reported to be the cercosporin efflux pump in C. kikuchii. Overexpression of these two MFS genes in the crg1 mutant did not increase either cercosporin resistance or production. We are currently disrupting three ABC transporter genes (ABC1, ABC2, and ABC3) in the WT. We also determined that the crg1 mutant produces cercosporin in complete medium (CM) liquid culture, but none in potato dextrose broth (PDB). Quantitative RT-PCR data indicated that whereas all of the transporter genes studied were down-regulated in crg1 grown in PDB, the three ABC transporters were up-regulated in CM.


489. Elicitation of systemic induced resistance by Trichoderma virens. Charles Kenerley, Dept. Plant Pathology & Microbiology, Texas A&M University, College Station, Texas


We have isolated a small hydrophobin-like protein (SM1) and an 18 amino acid peptaibol (TVB group) from Trichoderma virens and have shown that they are elicitors of systemic disease resistance in several plants. The native, purified SM1 elicits plant defense responses and systemic resistance against a foliar pathogen, Colletotrichum. The protective activity of SM1 is associated with the accumulation of reactive oxygen species and phenolic compounds, and increased levels of transcription of the defense genes regulated by salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET), as well as genes involved in the biosynthesis of sesquiterpenoid phytoalexins. Similar as in dicots, colonization of maize roots by T. virens induces systemic resistance (ISR-like) in leaves challenged with C. graminicola. This protection is associated with induction of JA and green leaf volatiles biosynthetic genes. We have demonstrated that SM1 is required for activation of ISR in maize as plants grown with sm1 deletion strains exhibit the same levels of systemic protection as plants without T. virens. Conversely, plants grown with sm1 over-expressing strains displayed an increased protection compared to the wild type strain. The application of two synthetic peptaibol isoforms to cucumber seedlings induce systemic protection against the bacterial pathogen Pseudomonas syringae pv. lachrymans, stimulate production of antimicrobial compounds in cotyledons, and upregulate the expression of defense-related genes. Additionally, cucumber plants co-cultivated with fungal strains disrupted in tex1 show reduced ability to produce phenolic compounds with inhibitory activity towards the bacterial pathogen as compared to those grown with the WT.


490. RNA Silencing Functions as an Antiviral Defense Mechanism in the Chestnut Blight Fungus Cryphonectria parasitica. Xuemin Zhang, Gert Segers, Qihong Sun and Donald Nuss*. Center for Biosystems Research University of Maryland Biotechnology Institute Shady Grove Campus 9600 Gudelsky Dr. Rockville, MD 20850 *E-mail: nuss@umbi.umd.edu

RNA silencing pathways are conserved in animals, plants and fungi. Although RNA silencing has been shown to play a crucial role as an antiviral defense mechanism in plants and to influence virus replication in animal cells, the interaction between mycoviruses and RNA silencing in fungi is not well understood. We recently reported that the p29 protein encoded by the virulence-attenuating hypovirus CHV1-EP713 can suppress RNA silencing in the natural host, the chestnut blight fungus Cryphonectria parasitica. (Segers et al., 2006, Eukaryotic Cell, 5:896-904). We now report the cloning and characterization of hypovirus-derived small RNAs (vsRNAs) from CHV1-EP713 infected C. parasitica. The vsRNAs were distributed along the viral genome and were derived from both the positive and negative viral RNA strands with a ratio of 3:2. These results suggest that hypovirus vsRNAs are derived from both structured regions of the positive strand and from viral double-stranded RNA, which contrasts with reports that vsRNAs are derived primarily from the positive strand RNA of several plant viruses. We also report the cloning and disruption of an Argonaut and two Dicer genes from C. parasitica. While the disruption mutants did not exhibit any obvious phenotypic changes relative to the wild-type strain, CHV1-EP713 infection resulted a dramatic debilitation of the DCL-2 disruption mutant. The identification of hypovirus derived vsRNAs and demonstration of enhanced virus-mediated symptom expression upon disruption of the C. parasitica RNA silencing pathway provides the first experimental evidence that RNA silencing serves as an antiviral defense mechanism in fungi.


491. Effect of ToxA treatment on wheat leaf gene expression. Iovanna Pandelova1, Todd Mockler 1, Lynda Ciuffetti1. 1Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR


The fungal wheat pathogen Pyrenophora tritici-repentis (PTR) produces several proteinaceous host-selective toxins (HSTs). The HSTs of P. tritici-repentis represent compounds that evoke a susceptibility response in the host in a single-gene dependent manner. Understanding the nature of events triggered by HSTs in the host will provide valuable insight into the nature of host disease susceptibility. ToxA is one of the proteinaceous HSTs produced by PTR that has been well characterized. ToxA treatment of a sensitive cultivar produces necrotic symptoms similar to those produced by the pathogen. Previous studies in our laboratory suggest that ToxA is imported by an endocytotic process and then localized to chloroplasts. In vitro studies suggest that ToxA interacts with the chloroplast protein ToxABPI thought to play a role in thylakoid formation or regulation of photosystem II. The downstream events of this interaction and other processes involved in the susceptible response in the host are likely to require differential gene expression. In the present study, we provide microarray data analysis for genes expressed in ToxA- and mock-treated leaves of a ToxA-sensitive cultivar over time. We used Wheat Genome Arrays from Affymetrix. Normalization and filtering of the data was done with Bioconductor software packages and relational database queries. Differentially expressed genes were identified between each treatment at each time point with three different statistical methods.


492. Internalization of Ptr ToxA is facilitated by the RGD-containing, solvent exposed loop. Viola Manning1, Sara Hamilton1, P. Andrew Karplus2, and Lynda Ciuffetti1. 1Department of Botany and Plant Pathology, 2Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR


Pyrenophora tritici-repentis produces host-selective toxins (HSTs) that induce the symptoms of tan spot of wheat. Expression and secretion of Ptr ToxA (ToxA), the first proteinaceous HST to be isolated and characterized, results in necrotic lesions on ToxA-sensitive leaves. Necrosis also occurs when ToxA-sensitive leaves are treated with purified toxin. Internalization of ToxA into sensitive, but not insensitive mesophyll cells suggests that internalization of toxin is required for activity. The cloning of the ToxA gene and its subsequent expression in E. coli results in an active HST and provides a method to investigate the structure/function relationship of ToxA. Crystal structure analysis indicates that an RGD-containing loop in ToxA is available for protein-protein interaction and heterologously expressed proteins with mutations in this loop are less active. We sought to determine if the reduced activity of these mutant proteins was due to decreased internalization. By monitoring toxin internalization with a protease protection assay we were able to determine that the decrease in toxic activity was due to a decrease in internalization and the amount of internalized protein correlates with the intensity of symptoms induced.


493. Medical Mycology in the graduate and undergraduate curriculum. Thomas Volk University of Wisconsin-La Crosse, Biology


Although fungi are increasing in importance as more patients become immunocompromised and acquire fungal infections, Medical Mycology is not a common course in undergraduate or graduate programs, with fewer than 40 full-fledged Medical Mycology courses currently taught in North America. However, Medical Mycology can be an interesting and popular course if taught and “marketed” to students in certain ways. This presentation is the story of a successful Medical Mycology course at the University of Wisconsin-La Crosse (about 9000 students) where enrollment one semester peaked at 38 students. I started with an enrollment of 13 students in Spring 1997; there have been about 260 students in the past 11 years. Through word of mouth and “advertising,” enrollments have steadily increased, especially among pre-med students, when they realize they will receive very little training in medical mycology in medical school. My course is taught mostly from the fungus’ point of view, although we do discuss the symptoms and cures for each disease, as well as mycetismus and mycotoxicosis. The laboratory is the crux of the course; each student makes a permanent reference slide collection of approximately 45 species of pathogenic and contaminant fungi, mostly deuteromycetes, using PVLG mounts of slide cultures or tape mounts. Students learn the methodology to identify just about any unknown deuteromycete, a very important and sought-after job skill. We have a poster session where each student presents a poster on a self-chosen topic, similar to my fungal web pages at TomVolkFungi.net . Most importantly, it’s a fun course to teach, and students enjoy it a lot.


494. Fusarium verticillioides GAS1, a gene encoding glycolipid anchored surface protein, is involved in growth and conidiation. Uma Shankar Sagaram, Brian D. Shaw, and Won-Bo Shim. Department of Plant Pathology & Microbiology, Texas A&M University, College Station, TX77843-2132, USA

Fusarium verticillioides is an important pathogen of maize that causes ear rot and produces mycotoxins fumonisins. To date, our knowledge about the pathogenicity and the regulation of fumonisin biosynthesis in F. verticillioides is limited. Here, we present the molecular characterization of GAS1, which encodes a putative 540-amino acid protein. Gas1 protein belongs to a glycolipid anchored surface protein (GASP) family. F. verticillioides GAS1 was identified as an expressed sequence tag (EST) up-regulated in the acidic culture condition. GAS1 was shown to be involved in fungal cell wall biogenesis and virulence in F. oxysporum and A. fumigatus. GAS1 deletion mutants (gas1-h and gas1-g) grew restricted but fluffier when compared to the wild type on solid media. However, no defect with regard to mycelial mass production and filamentous growth was observed when the strains were grown in liquid media. The deletion strains produced significantly fewer conidia than its wild type progenitor. Microscopic studies as visualized by concanavalin-A staining indicated that the deletion of GAS1 altered the cell wall carbohydrate composition/deposition process. Interestingly, the deletion of GAS1 did not affect pathogenicity of F. verticillioides when tested on maize seedlings and stalks. Complementation of gas1-h with GAS1 restored growth, conidiation and cell wall abnormality phenotypes. Our results suggest that GAS1 is associated with growth, development and conidiation in F. verticillioides but not pathogenicity. The role of GAS1 in fumonisin biosynthesis will also be discussed the presentation.


495. Loss of pathogenicity converts a pathogenic Fusarium oxysporum isolate to a potential biocontrol agent in Arabidopsis thaliana. Hye-Seon Kim1, Kirk Czymmek2 and Seogchan Kang1. 1Department of Plant Pathology, The Pennsylvania State University, University Park, PA 16802,USA. 2Department of Biological Sciences, University of Delaware, Newark, DE 19716,USA.


The soil-borne fungal pathogen Fusarium oxysporum can cause vascular wilt and root rot diseases in many crop species. To facilitate the investigation of the molecular and cellular mechanisms underpinning its pathogenesis and host responses, a new model system was developed using Arabidopsis thaliana as a host. In vivo observations of root colonization were facilitated by transformation of F. oxysporum for constitutive cytoplasmic expression of fluorescent protein reporters. In combination with these reporters, we investigated the role of the cAMP-dependent protein kinase A (CPKA) in pathogenicity. Targeted knock-out mutagenesis of CPKA caused the loss of pathogenicity in Arabidopsis and the failure to colonize the vascular system and also led to reduced vegetative growth and conidiation in culture. It was observed that the coinoculation of the wild-type strain with a cpka mutant significantly reduced the disease severity and the colonization of the vascular system by the wild-type strain, indicating that the cpka mutant protects Arabidopsis plants from infection. Molecular cytological tools and gene expression analyses are currently being used to study the role of CPKA during root pathogenesis, how the loss of this gene function protects plants from a virulent strain, and whether the loss of function of other pathogencity genes convert pathogenic isolates to potential biocontrol agents.


496. Habitat-defining genes and synteny of conditionally dispensable (CD) chromosomes in Nectria haematococca. Marianela Rodriguez-Carres, Dai Tsuchiya, Jose Luis Goicoechea, Rod Wing, and Hans D. VanEtten. Department of Plant Science, University of Arizona, Tucson, AZ 85721.


Many studies of extra chromosomes in plant and animals have described them as detrimental or parasitic, and containing non-coding or non-expressed DNA. However, in fungi many of these chromosomes are required for pathogenicity but not for axenic growth, and as such they have been called conditionally dispensable (CD). The wide range of habitat of the plant pathogenic fungus Nectria haematococca is partially attributed to the presence of CD chromosomes. One of these CD chromosomes, the 1.6Mb PDA1- CD chromosome, contains a cluster of genes for pea pathogenicity and rhizosphere colonization. In the current study a physical map of the PDA1-CD chromosome was constructed in order to evaluate synteny between this CD chromosome and another CD chromosome, the MAK1-CD. The MAK1-CD chromosome has a similar size to the PDA1-CD chromosome, and contains a gene (MAK1) for pathogenicity on chickpea. Results show that about 463kb (>89% sequence identity) are shared by both CD chromosomes but not found anywhere else in the genome. In addition, each chromosome has ~1 Mb of unique DNA that is unshared. Anchoring of the PDA1-CD map to the genome of N. haematococca allowed the identification of additional putative habitat-defining genes present on both CD chromosomes. Southern hybridizations of pulsed-field gels and sequence analyses of the syntenic regions reveals features reminiscent of recombination/translocation events, and a mosaic composition of the genes in these CD chromosomes. Taken together, these results indicate that: CD chromosomes are a reservoir of unique DNA, advocates for a common ancestor, and suggests possible mechanisms that lead to the formation of CD chromosomes.


497. Comparative analysis of the MAT locus to understand speciation and sexual cycle transitions: Cryptococcus neoformans vs. Filobasidiella depauperata. Marianela Rodriguez-Carres, Keisha Findley and Joseph Heitman. Duke University Medical Center, Department of Molecular Genetics and Microbiology, Durham, NC 27707.


Cryptococcus neoformans and Cryptococcus gattii are dimorphic basidiomycetes that cause respiratory and neurological disease in animals and humans, following spore inhalation from the environment. Sexual reproduction in these fungi is controlled by a bipolar system in which a single mating type (MAT) locus specifies compatibility. The MAT loci in C. neoformans and C. gattii, which are thought to have evolved from a common ancestral locus, share several features reminiscent of sex chromosomes in multicellular eukaryotes. They are unusually large (>100 kb), do not recombine during meiosis, and contain many genes clustered together, most of which function during mating whereas others may not. The alpha mating type (MAT alpha) is overwhelmingly prevalent, both in nature and clinical isolates, and has been associated with increased virulence in certain genetic backgrounds. The abundance of MAT alpha isolates could be partially attributed to their ability to undergo monokaryotic fruiting which is a variant of the sexual cycle that involves same-sex mating and results in the production of basidiospores. Consequently it has been proposed that the evolution of the MAT locus could be closely linked to the pathogenic potential of C. neoformans and C. gattii. To gain insight into the evolution of the MAT locus and monokaryotic fruiting, we are cloning the MAT locus from the closest known relative Filobasidiella depauperata. This fungus is homothallic, exclusively filamentous and found in associations with insects. Preliminary results suggest that several genes linked to the MAT locus in Cryptococcus are unlinked in F. depauperata. This is intriguing, as it has been proposed that the ancestral MAT locus of C. neoformans might have evolved through a chromosomal translocation that clustered all of the mating genes in one region. Furthermore, morphological characterization of F. depauperata shows that clamp connections are absent, and monokaryotic basidiospores and hyphae are present, features that resemble monokaryotic fruiting of C. neoformans.


498. Recovery and functional analysis of a putative pathogenicity gene in the Dutch Elm Disease fungus Ophiostoma novo- ulmi. Karine Plourde, Louis Bernier. Laval University, Québec, Canada.

The ascomycete fungus Ophiostoma novo-ulmi is the main causal agent of Dutch Elm Disease. We used insertional mutagenesis to tag genes that may contribute to parasitic fitness in this fungus. We recovered several transformants with a significantly reduced pathogenicity phenotype compared to the aggressive wild-type reference strain H327. Analysis of a genomic library for one of these mutants allowed us to identify 4 genes next to the mutation site, including a locus with high homology with the Aspergillus fumigatus ubiquitin-protein ligase E3. To evaluate if the mutation modified the expression of these genes during infection of the host plant by O. novo-ulmi, the mutant and its wild-type progenitor were inoculated to elm saplings under controlled conditions. We used quantitative RT-PCR to estimate fungal biomass and gene expression in elm leaves collected at 7, 14, 21 and 42 days after inoculation. This allowed us to verify if differences in the expression level between strains were associated with differences in their ability to colonize elms. In addition, wood xylem samples collected 42 days after inoculation were used for histopathological analyses. Preliminary observations showed a stronger defense response (suberisation, phenol production) in plants inoculated with the wild-type strain than with the mutant. Overall, our results suggest that activity of the ubiquitin-protein ligase E3 locus, or one of its immediate neighbors, is important for the virulence of O. novo-ulmi towards elms.


499. Functional Analysis of MPEX7, the putative peroxisome-targeting signal type 2 (PTS2) receptor gene of Magnaporthe oryzae. Jaeduk Goh, Hyojeong Kim and Yong-Hwan Lee. Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.


Peroxisomes are single-membrane-bound organelles that function in multiple metabolic pathways and, as recently demonstrated, influence appressorium-mediated penetration of pathogenic fungi to host plants. A putative peroxisomal targeting signal type-2 (PTS2) receptor gene, MPEX7, of the rice blast fungus Magnaporthe oryzae was identified through our Agrobacterium tumefaciens -mediated transformation project. mpex7 mutants generated by a T-DNA insertion or a targeted gene replacement showed significant reductions in agar-invasive hyphal growth, conidiophore differentiation and conidiation, while displaying normal rates in conidial germination and hyphal extension. They formed normal-appearing appressoria, however, the appressoria of the mpex7 mutants failed to cause disease in plant cells, due to inability to develop penetration hyphae. mpex7 mutants caused lesions only if inoculated through wound sites. Moreover, the appressoria proved to be defective in turgor pressure generation and delayed in lipid degradation during appressorial maturation. Thus, our results demonstrate that MPEX7 is required for the formation of functional appressorium and pathogenicity of M. oryzae.


500. The effectors Avr2 and EPIC2B secreted by two unrelated pathogens target the tomato defense protease Rcr3. Jing Song, Joe Win, Miaoying Tian, Hsin-Yen Liu and Sophien Kamoun Department of Plant Pathology, The Ohio State University-OARDC, Wooster, OH 44691, USA


Current models of plant-pathogen interactions stipulate that pathogens secrete effector proteins that disable plant defense components known as virulence targets. Occasionally, the perturbations caused by these effectors trigger innate immunity via plant disease resistance proteins as described by the “guard model”. This model is nicely illustrated by the interaction between the fungus Cladosporium fulvumand tomato. C. fulvum secretes a protease inhibitor Avr2 that targets the tomato cysteine protease Rcr3. In plants that carry the resistance protein Cf2, Rcr3 is required for resistance to C. fulvum strains expressing Avr2, thus fulfilling one of the predictions of the guard model. The model has two other predictions that have not been tested. First, Rcr3 could be disabled by different pathogens. Second, Rcr3 may directly contribute to basal defense. In this study we tested these two predictions using a different pathogen of tomato, the oomycete Phytophthora infestans. This pathogen secretes an array of protease inhibitors, one of which, EPIC2B, was shown to inhibit tomato cysteine proteases. Here, we showed that, similar to Avr2, EPIC2B binds and inhibits Rcr3 using co-immunoprecipitation and activity profiling assays. We also found that the rcr3-3 mutant of tomato that carries a premature stop codon in Rcr3 exhibits enhanced susceptibility to P. infestans suggesting a role for Rcr3 in basal defense. In conclusion, our findings fulfill the predictions of the guard model and suggest that the effectors Avr2 and EPIC2B secreted by two unrelated pathogens of tomato target the same defense protease Rcr3.


501. The initiation of map-based cloning of an avirulence gene from Pyrenophora teres. Zhaohui Liu1, Justin D. Faris 2, Michael C. Edwards2, Timothy L. Friesen2. 1Department of Plant Pathology, North Dakota State University, Fargo ND, 58105 2USDA-ARS Cereal Crops Research Unit, Northern Crop Science Laboratory, 1307 18th Street North, Fargo ND, 58105.


The fungus Pyrenophora teres, causal agent of barley net blotch, is an economically important pathogen in most barley-growing areas. In a previous study, we identified and mapped a fungal gene associated with avirulence on Tifang barley that was already designated AvrHar. This study used a P. teres mapping population consisting of parental isolates 15A (avirulent on Tifang) and 0-1 (virulent on Tifang). In the present study, an attempt is being made to clone AvrHar using a map-based approach. Initially, the fungal population used in the mapping of AvrHar was enlarged from 78 progeny to a total of 278 progeny to create a map with increased resolution. A single AFLP marker closely linked to the avirulence gene was identified and TAIL-PCR was used to extend the fragment from 250 bp to 1.7kb for use as a probe. A 20X BAC library was constructed from parental isolate 0-1 and a 3X fosmid library was constructed from parental isolate 15A. The AFLP probe allowed the identification of 14 positive clones from the 0-1 BAC library. A single BAC clone was sequenced and shown to contain a large portion of Gypsy-like LTR- retrotransposon repeats at both ends. A 60kb region in the middle of the BAC contained several predicted genes. A PCR marker based on one of the genes located approximately 50 kb from the original AFLP was found to co-segregate with the AFLP marker, indicating that AvrHar has not been reached. An additional four BAC clones have been identified using the new marker. Twelve fosmid clones spanning approximately 90 kb outside the BAC clone sequence have been characterized. Recombination in this region is lower than expected and several walking steps may be necessary. Additional steps are being taken to span AvrHar.


502. Putative G protein-coupled receptors GPRC and GPRD are involved in development and morphogenesis in Aspergillus fumigatus. Alexander Gehrke1, Thorsten Heinekamp1 and Axel A. Brakhage1,2. 1Leibniz Institute for Natural Product Research and Infection Biology - Department of Molecular and Applied Microbiology – Hans-Knoell-Institute, 2Friedrich-Schiller-University, Jena; eMail:alexander.gehrke@hki-jena.de

The opportunistic human-pathogen Aspergillus fumigatus was subject to recent studies on cAMP signal transduction with regard to morphogenesis and virulence. To date, one of the most important questions is still unanswered: what are the external signals and the corresponding proteins sensing those ligands or stimuli which enable the fungus to grow in a wide variety of different ecological niches? In a first approach, two genes encoding putative G protein-coupled receptors gprC and gprD, designated as carbon source-sensing receptors, were deleted in A. fumigatus. The physiological characterisation of the mutants revealed altered growth on solid media. However, various growth conditions, which included the use of different carbon- and nitrogen-sources, did not restore the defect of the mutants. Virulence of the mutant strains, as tested in a low-dose murine infection model, was attenuated. The function of the putative GPCRs was further investigated by analysing fluorescent protein-fusions in vivo by confocal microscopy.


503. A+T-rich isochores as niches for avirulence and pathogenicity genes in the genome of Leptosphaeria maculans. I. Fudal1, B. Profotova1, S. Ross1, L. Gout1, F. Blaise1, M.L. Kuhn1, M. Eckert1, L. Cattolico2, S. Bernard-Samain2, M.-H. Balesdent1, T. Rouxel1. 1INRA-PMDV, Route de St Cyr, 78016 Versailles, France. 2Genoscope, 2 rue Crémieux, 91057 Evry, France.


Leptosphaeria maculans develops very specific “gene-for-gene” interactions with its host plant, Brassica napus, where fungal avirulence (AvrLm) genes are the counterpart of plant resistance (Rlm) genes. A 1.1 Mb region encompassing avirulence genes AvrLm1 and AvrLm6 was sequenced and shown to display a particular organization, with five 20–70kb G+C-equilibrated, ORF-rich isochores intermingled with 3 large (170-450 kb) A+T-rich isochores mainly composed of degenerated and truncated retrotransposons. These A+T-rich regions contain only seven isolated genes (one every 90 kb on average), of which four are predicted to encode small-secreted proteins. These include AvrLm1 (205 aa, one cysteine), AvrLm6 (144 aa, 6 cysteines), and two single-copy genes encoding for two cysteine-rich proteins termed LmCys1 (233 aa, 8 cysteines) and LmCys2 (247 aa, 8 cysteines). Except for LmCys1, all these genes have a low G+C content, similar to that of their genomic environment. Contrasting with genes occurring in GC-equilibrated regions of the genome, AvrLm1, AvrLm6, LmCys1 and LmCys2 are specific of L. maculans ‘brassicae’, and absent from other species or sub-species of the L. maculans-L. biglobosa species complex, or from the closely related species, Stagonospora nodorum. These four genes are up-regulated upon plant infection, with AvrLm1, AvrLm6 and LmCys2 being expressed at a low level, but over-expressed up to 800-x in the first stages of plant infection. Functional analyses, including RNAi silencing or complementation, suggested a role for LmCys1 and LmCys2 in pathogenicity of L. maculans and confirmed that A+T-rich isochores are specific niches for genes involved in pathogenicity or avirulence. B. Profotova and M. Eckert were funded by Marie-Curie Fellowship HPMT-CT-2001-00395 ‘FUNGENE’ I. Fudal and S. Ross were funded by EU contract QLK5-CT-2002-01813 ‘SECURE’


504. The Phytophthora infestans avirulence gene PiAvr4 encodes an RxLR-DEER effector protein. Pieter M.J.A. van Poppel1, Jun Guo1,2, Peter J.I. van de Vondervoort1, and Francine Govers1. 1 Laboratory of Phytopathology, Wageningen University, The Netherlands 2 IVF-CAAS Beijing and Northwest A & F University, Yangling Shaanxi, China


The oomycete pathogen Phytophthora infestans causes late blight, an important disease in potato world wide. P. infestans secretes numerous effector molecules, some of which are recognized by host plants carrying resistance (R) genes. These effectors then act as avirulence (Avr) factors and elicit a hypersensitive response (HR) that arrests growth of the pathogen. Through a combined approach of genetic mapping and transcriptional profiling (cDNA-AFLP) we isolated an Avr gene that shows a gene-for-gene interaction with R4. This gene, named PiAvr4, is highly expressed in germinated cysts and encodes a 287 amino acid protein with a putative signal peptide and an RxLR-DEER motif. The Avr4 protein belongs to a large family of P. infestans effector proteins that are highly divergent but share the RxLR-DEER motif. This motif is thought to play a role in delivery of effectors into the host cell. Transformation of PiAvr4 into P. infestans isolates virulent on R4 plants, resulted in complementation, i.e., the transformants elicited HR and thus became avirulent on plants carrying R4. In planta expression of PiAvr4 using agroinfection-based Potato Virus X (PVX) expression vectors caused an HR specifically on R4 plants but not on r0 plants. However, the HR was only observed when a signal peptide sequence was included in the construct and not when the mature protein was produced. Apparently, ER modification and/or secretion of Avr4 are required for recognition of Avr4 as avirulence factor. Direct virus inoculation of plants with PVX expressing PiAvr4 with and without signal peptide sequence gave the same results as the agroinfection inoculation.


505. The plasma membrane H+-ATPase LmPMA1 is involved in pathogenicity of Leptosphaeria maculans on oilseed rape. E. Remy, M. Meyer, M.H. Balesdent, F. Blaise, M. Chabirand, J.P. Narcy, J. Roux, T. Rouxel INRA, Unité PMDV, Route de Saint Cyr, F-78026 Versailles cedex, France


A collection of 5000 Agrobacterium-mediated random insertion transformants of the stem canker fungus, Leptosphaeria maculans, has been generated. One of these, m210 is severely affected in pathogenicity whereas it is morphologically similar to the WT isolate in vitro and shows no growth or sporulation defect. It induces a typical hypersensibility reaction on oilseed rape leaves and cannot colonize the stem. Electron microscopy experiments further suggested that m210 is depleted in its germination ability on oilseed rape leaf surface. The T-DNA was inserted 274 bp upstream the start codon of one ORF homologous to the highly conserved fungal gene PMA1, which encodes the predominant and essential plasma membrane H+-ATPase. Quantitative RT-PCR showed that LmPMA1 is expressed constitutively at a high level both in vitro (germinating conidia, mycelia) and in planta, whereas the T-DNA insertion induced a 50% reduction of expression of LmPMA1 in all tested conditions. Complementation experiments further confirmed that this gene is responsible for the m210 phenotype. The basic function of plasma membrane H+-ATPase is to create an electrochemical proton gradient for (i) uptake of nutrients and (ii) regulation of the intracellular pH. Its expression is under the control of environmental pH and morphogenic development. However, no modification of LmPMA1 expression levels were observed when investigating these two factors in L. maculans, although m210 was affected in its tolerance to various environmental pH. We suggest that the separation of transcriptional regulation boxes from the gene start by the T-DNA insertion led to a deregulation of the expression in m210.


506. Monitoring the induction of trichothecene mycotoxins of Fusarium graminearum by using GFP during wheat head infection and in culture. Maier, F.J., S. Jaeger, M. Boenisch, and W. Schaefer, University of Hamburg, Biocenter Klein Flottbek, Ohnhorststr. 18, Hamburg, Germany

The fungal pathogen F. graminearum is the most common causal agent of Fusarium head blight (FHB) of small grain cereals and of cob rot of maize. The threat posed by this fungus is due to yield decreases and mycotoxin contamination. It has been shown that trichothecenes influence virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific (Maier et al. 2006). To evaluate the induction of the trichothecenes, the gfp gene was fused to the Tri5 promoter and integrated into the genome by homologous recombination. The resulting mutants exhibit a gfp gene driven by the endogenous Tri5 promoter and a fully functional Tri5 gene. Now we are able to monitor the induction of the trichothecene pathway under real time conditions with a cellular resolution. Furthermore, the method needs no time consuming and expensive toxin measurement. By monitoring gfp expression, we evalute the performance of the trichothecene pathway: - during infection of wheat - in culture with plant derived substances, fungicides and miscellaneous chemicals in 96 well plates The results of the current research will be presented. Maier et al. 2006. Involvement of trichothecenes in fusarioses of wheat, barley, and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and aggressiveness. Mol. Plant Pathol. 7:449-461.


507. Constitutive expression of the secreted lipase Fgl1 partly restores pathogenicity in apathogenic Fusarium graminearum Gpmk1 MAP kinase disruption mutants. Salomon, S., C.A. Voigt, A. Gácser, N.J. Jenczmionka, C. Kröger, S. Frerichmann, and W., Schäfer. University of Hamburg, Biocenter Klein Flottbek, Ohnhorststr. 18, Hamburg, Germany


Mitogen activated protein (MAP) kinases regulate virulence in several pathogenic fungi e.g. Claviceps purpurea, Cochliobolus heterostrophus, Magnaporthe grisea, and Fusarium graminearum. Up to now it is unclear which virulence factors are regulated via this signal transduction pathway. We reported that Gpmk1 MAP kinase disruption mutants of F. graminearum are apathogenic and cannot infect wheat spikes (Jenczmionka et al., 2003, Curr. Genet.). Additionally, we showed that Gpmk1 MAP kinase disruption mutants exhibit an altered induction of several hydrolytic enzymes (Jenczmionka and Schäfer (2005, Curr. Genet.), among them the secreted lipase FGL1. This lipase is a major virulence factor of F. graminearum (Voigt et al., 2005, Plant J.). Here, we show the regulation of the FGL1 gene in dependence on the Gpmk1 MAP kinase in culture and during plant infections. We constructed Gpmk1 MAP kinase deficient F. graminearum strains that express the FGL1 gene additionally under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter to examine the influence of expression control of the virulence gene FGL1 by Gpmk1 during wheat infections. These strains are not longer apathogenic on wheat, but in contrast to the wild type strain, they cannot spread out through the entire spike.


508. Improving Maize and Sorghum breeding to turcicum leaf blight in Uganda by fungal population genetics and plant genomics. Tom Martin, Christina Dixelius. Department of Plant Biology and Forest Genetics, Box 7080, Swedish University of Agricultural Sciences, Uppsala, Sweden.


Turcicum leaf blight (TLB) caused by the fungal pathogen Exserohilum turcicum is a significant factor contributing to major maize (Zea mays) and sorghum (Sorghum bicolor) yield losses in sub-Saharan Africa. Population studies can be a powerful tool in order to further understand the variation of the pathogen. E. turcicum isolates derived from sorghum and maize hosts will be assessed using AFLP in order to determine population structure and variation in Uganda. Plant molecular defense responses to E. turcicum will be analysed. mRNA extractions from interacting fungal and plant cells using laser microdissection will be used in microarrays containing ESTs from maize and sorghum. This will identify up and down regulation of gene expression during pathogen attack thus indicating defense response. Candidate genes will be investigated in order to identify novel resistance genes. Molecular characterization of resistance and polygenic resistance to TLB will lead to the development of markers to support marker assisted breeding programs in East Africa.


509. HOG-like MAP kinase of Fusarium graminearum regulates osmotic stress and virulence. Cornelia M. Gregel, Nicole J. Jenczmionka , Wilhelm Schäfer, and Siegfried Salomon. University of Hamburg, Biocenter Klein Flottbek, Ohnhorststr. 18, Hamburg, Germany.


In the course of evolution eukaryotic cells developed a complex network of signal transduction mechanisms to respond to environmental changes. Mitogen-activated protein (MAP) kinases are frequently integrated in these pathways to induce the phosphorylation of many proteins with regulatory functions resulting in a plethora of cellular consequences as proliferation, differentiation, cell cycle arrest, and apoptosis. MAP kinases are ubiquitous enzymes, e.g. in the yeast Saccharomyces cerevisiae five MAP kinases are identified that modulate cell growth, differentiation, and stress response. Analysis of the sequenced Fusarium genome revealed, like in Magnaporthe grisea, the existence of only three conserved MAP kinase genes. Two previously identified enzymes (MGV1 (Hou et al., 2002, Mol Plant Microbe Interact.) and Gpmk1 (Jenczmionka et al., 2003, Curr. Genet.)) are involved in the regulation of mating, hyphal growth, pathogenicity, conidation, and nutrient sensing. We report on the characterization of the third MAP kinase of F. graminearum, GOV1, that shows a high homology to the well-characterized yeast protein Hog1, which is implicated in osmotic stress signalling and osmoadaptation. To analyze the function of this Osmo-MAP kinase we generated disruption mutants. The involvement of this MAP kinase in the response to osmolarity stress, viability, conidiation, and pathogenicity will be presented and discussed.


510. Exploring potato microRNA and siRNA in order to mediate resistance to Phytophthora infestans : novel approaches to the late blight disease control in potato. Ramesh Raju Vetukuri1, Christina Dixelius1, Eugene Savenkov1. 1Department of Plant Biology and Forest Genetics, Box 7080, Swedish University of Agricultural Sciences, Uppsala, Sweden.


The devastating potato late blight is caused by the pathogen Phytophthora infestans. As a first step to develop disease resistance, we are in the process of picking out the most suitable P. infestans genes as targets for a miRNA- inactivation approach. Functional analysis of virulence in P. infestans revealed several candidate genes. Recently, it has been shown that a G-protein alpha-subunit (encoded by the Pigpa1 gene) plays diverse roles in pathogenesis-related development of P. infestans (Latijnhouwers et al., 2004). To obtain resistance to P. infestans, we will design miRNA and siRNA constructs to specifically target P. infestans candidate virulence genes, namely conservative regions of the Pigpa1 gene and the RxLR motif. On the other hand, several miRNA and siRNA constructs will be designed using a potato miRNA precursor as a backbone. These constructs will target several genes in multi-gene families of the pathogen.


511. The YSST screen identifies secreted proteins of the corn pathogen Colletotrichum graminicola. Jorrit-Jan Krijger, Ralf Horbach, Holger B. Deising and Stefan Wirsel; Martin-Luther-Universität, Institut für Agrar- und Ernährungswissenschaften, Halle, Germany;

stefan.wirsel@landw.uni-halle.de


The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on its host Zea mays. Following germination and penetration of epidermal cells, the fungus enters a short biotrophic phase. Thereafter it switches to the necrotrophic phase which is characterized by hyphal proliferation and killing of the host tissue. Macroscopically this gives rise to leaf anthracnose in which acervuli containing conidia develop. During both phases, secreted proteins may play important roles to establish the interaction. Our interest is focused on proteins that are involved in suppression of host defense and in reprogramming of host metabolism. To identify genes encoding such proteins, we employ the Yeast Secretion Signal Trap (YSST) in Saccharomyces cerevisiae that takes advantage of a 5´-truncated invertase gene on a vector to identify cDNAs carrying a start codon that is followed by a signal sequence. A YSST-cDNA-library was constructed from RNA extracted from in vitro-grown mycelium that was induced with corn leaf-extract. To date, we identified 28 unigenes, about one-third of which encode enzymes, mostly hydrolases, and two-thirds encode non-enzymatic proteins. Several of the latter show homology to proteins of unknown function from other phytopathogenic Ascomycota that were annotated in genome projects. RT-PCR experiments indicate changes in expression of some of these genes during progression through the growth phases in planta.


512. New insight into antifungal response pathways from a Candida albicans protein kinase mutant set. Jill R. Blankenship, Saranna Fanning, Jessica J. Hamaker, and Aaron P. Mitchell. Department of Microbiology, Columbia University Medical Center, New York, NY 10032


Environmental response pathways of pathogens have proven time and again to be critical for infection; the ability to survive macrophage assault or persist during drug therapy requires adaptation to diverse environmental changes. Protein kinases (PKs) play critical roles in environmental response pathways in all organisms. This rationale prompted us to focus on PK genes of the fungal pathogen C. albicans as an entry point into dissection of key environmental response pathways. Of 115 predicted PK genes in the genome, we have made insertion mutants for 68. Another 34 may be essential, although our “UAU1” mutagenesis methodology limits the strength of this conclusion. We have focused on the response of C. albicans to the cell wall inhibitor caspofungin, a clinically successful antifungal that inhibits glucan synthase activity. Our analysis has led to two unexpected conclusions. First, the highly conserved protein kinase Ire1 is required for the response to caspofungin. Ire1 activates the unfolded protein response to counteract stress in the endoplasmic reticulum, and we propose that caspofungin induces the relevant stress through induction of cell wall protein synthesis. Second, our indication thus far is that Ire1 may be essential for C. albicans viability: an insertion near the 3’ end of the ORF permits viability, but insertions elsewhere cannot be constructed. Independent verification of this conclusion will suggest that the secretory system is particularly taxed in this organism even under normal laboratory growth conditions.


513. Antifungal Targeting of Signaling in Dermatophytes (Trichophyton rubrum). Nalu TA Peres1, Henrique CS Silveira1, Nilce M Martinez-Rossi1 and Rolf A Prade2, 1Department of Genetics, Ribeirão Preto School of Medicine, University of São Paulo, and 2Department of Microbiology and Molecular Genetics, Oklahoma State University E-mail: prade@okstate.edu


Dermatophytes such as Trichophyton rubrum select proteins as the preferred carbon and nitrogen source and polar growth is established at the expense of a delicate osmotic balance. During vegetative growth hyphal-tips are swollen and branching is delayed, which is easily recuperated by adding as little as 25 mM of sodium chloride. Antifungal drugs that interfere with osmotic signaling, e.g., ambruticin, enhance hyphal-tip swelling, resulting in rupture of cell walls and membranes with leakage of cell contents leading to cell death. Here we describe a series of ambruticin resistant mutants with a pleiotropic phenotype such as enhanced conidiation, altered pigmentation and modified vegetative growth rates. We also characterize the molecular structure of antifungal targets and its relation to pathogenicity. The antifungal ambruticin interferes with osmotic signaling, turning a non-essential target into a lethal activity.


514. Ecological niche modeling of Coccidioides sp. in Western North American deserts. R. C. Baptista-Rosas1, A. Hinojosa2 and M. Riquelme3. 1Arid Land Ecosystem Management, School of Sciences, Autonomous University of Baja California (UABC); 2Geographic Information Systems and Remote Perception Laboratory and 3 Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Mexico.


Coccidioidomycosis is an endemic infectious disease in Weste