XXIV Fungal Genetics Conference at Asilomar

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 42^OC. The Δpmt2 mutant cannot send out germ tubes at 42^OC. The

Δpmt4 mutant has swollen hyphae and produces aberrant conidiophores at 42^OC. 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.

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

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 C^o), 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 Seidl^{1, 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 nimX^cdc2 to affect mitosis in Aspergillus nidulans. nimA affects the nuclear import of nimX^cdc2, while the snxA1 mutation is a cold-sensitive suppressor of the nimX2^cdc2 mutation. snxA1 leads to abnormal nuclear morphology at 17^oC. 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 17^oC 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 32^oC and upshifted to 44^oC for 3 hours, suggesting that the effects of snxA1 on nimA are not allele-specific. After 3 hours at 44^oC, 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

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 Schmitz^1,2. 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.

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.

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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 nimO^Dbf4. Steve James, James Barra, Megan Campbell, and Matthew Denholtz. Biology Department, Gettysburg College, Gettysburg, PA. sjames@gettysburg.edu

In Aspergillus nidulans, nimO^Dbf4 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 nimO^Dbf4 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?

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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.

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 Harispe^1,2,3, 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,