370. Optimizing gene silencing and overexpression in Phytophthora infestans using the PiBzp1 transcription factor, a gene required for appressorium formation and zoospore motility. Christina A. Bormann, Flavio A. Blanco, and Howard S. Judelson. Department of Plant Pathology, University of California, Riverside 92521 USA
Two priorities in studies of Phytophthora are identifying genes important in the disease cycles of these destructive pathogens, and improving methods for manipulating genes in transformants. For example, gene silencing or overexpression provides ways to characterize the role of genes in pathogenesis and validate their utility as targets for chemical inhibitors (i.e. fungicides). To help achieve these goals, we have been studying interactors of protein kinases induced during the spore cycle of P. infestans, the cause of late blight of potato and tomato. An interactor of a zoosporogenesis-induced kinase was found to encode a bZIP transcription factor, and experiments to misregulate the bZIP gene (PiBzp1) were initiated to test its role. Silenced transformants exhibited abnormal swimming behavior, failed to make appressoria, and were nonpathogenic. Using the bZIP gene as a model, optimal methods for both gene silencing and overexpression are now being identified. Constructs containing the bZIP coding region in sense or antisense directions between the ham34 promotor and terminator, or the entire native gene, are being evaluated. Moreover, four different transformation techniques are being tested, involving protoplasts, electroporation, particle bombardment, and Agrobacterium. Preliminary results show that protoplast transformation results in more efficient silencing than electroporation.
371. Feedback-regulation of the Neurospora transcription factor White Collar Complex by the circadian clock protein Frequency. Tobias Schafmeier, Krisztina Káldi, Andrea Haase, Johanna Scholz and Michael Brunner. Biochemistry Center, University of Heidelberg (BZH), Im Neuenheimer Feld 328 Heidelberg D-69120, Germany
Circadian clocks organize temporal expression of large numbers of genes in many organisms. They are self-sustained cellular oscillators dependent on interconnected transcriptional/translational feedback-loops of clock proteins. FREQUENCY (FRQ) is a central component of the circadian clock of Neurospora. frq RNA is rhythmically expressed and FRQ feedback-regulates its own expression by inhibiting its transcriptional activator, the White Collar Complex (WCC). Here we have investigated the mechanism of negative feedback of FRQ on expression of its own RNA. We show that WCC is concentrated in the nucleus while the localization of FRQ is predominantly cytosolic. Furthermore, WCC is expressed in excess over FRQ in the nucleus and the bulk of WCC does not interact with FRQ. This suggests that WCC is not directly inactivated by complex formation with FRQ. Rather, we show that FRQ controls the phosphorylation state of WCC.
372. Nitrogen metabolite and nitrate signalling. Mark X Caddick1, Igor Morozov1, Greg Fitzgibbon1, Ammar Razak1, Joseph Strauss2 and Meriel Jones1. 1 Biological Sciences, The University of Liverpool, UK. 2 Institut für Angewandte Genetik und Zellbiologie, BOKU-University, Vienna, Austria.
The GATA transcription factor AreA, which mediates nitrogen metabolite signalling in the filamentous fungus Aspergillus nidulans, is modulated by at least four distinct mechanisms. Our recent work has investigated two of these; the TOR kinase pathway and regulated transcript stability. In S. cerevisiae the TOR pathway acts via Ure2p to modulate the AreA orthologue Gln3p, and represents the predominant signalling mechanism. A. nidulans does not have a Ure2p orthologue but we have shown that the TOR pathway still contributes to nitrogen metabolite signalling through AreA. Previously we have shown that the areA transcript degrades rapidly in the presence of primary nitrogen sources (glutamine, ammonia) whilst remaining stable under conditions of nitrogen limitation, derepressing transcription of many genes involved in nitrogen metabolism. This 3' UTR-dependent degradation is preceded by rapid deadenylation. We have now demonstrated that nitrogen metabolites modulate the stability of further transcripts in these pathways, either accelerating or retarding decay. These include the niaD and niiA transcripts which are required for the reduction of nitrate to glutamine. Both transcripts degrade rapidly in the presence of glutamine and conversely, are stabilised by intracellular nitrate. Furthermore, when both glutamine and nitrate are present, these transcripts are stable, ensuring that nitrate and the toxic intermediate nitrite are always removed. Degradation of the niaD transcript is triggered by deadenylation, which is dependent on its 5' UTR. Nitrate inhibits poly(A) shortening even in the presence of glutamine. Therefore mRNA degradation, mediated through the poly(A) tail, is a fundamental part of the adaptive response to specific environmental signals.
373. Subcellular localization of velvet, a gene necessary for development and toxin production in Aspegillus nidulans. Suzanne M. Stinnett1, Eduardo Espeso2, and Ana Calvo1. 1Northern Illinois University, Biological Sciences, Dekalb, Illinois. 2Centro de Investigaciones Biológicas (C.S.I.C.), Microbiologia Molecular, Madrid, Spain
In Aspergillus nidulans, the velvet gene (veA) is required for morphological development and production of secondary metabolites. Previous studies have shown that deletion of veA results in a blockage in cleistothecia production. We have found that veA is also essential for the production of the mycotoxin sterigmatocystin and normal production of penicillin. Inspite of its importance, the veA mechanism of action remains unknown. In this study we investigated the subcellular localization of VeA to obtain clues about VeA molecular function. There is a conserved bipartite nuclear localization signal (NLS) motif present in the VeA N-terminal domain. With the goal of examining the functionality of this bipartite NLS, we generated A. nidulans veA fusion with the green fluorescent protein gene (gfp) and expressed them first in a yeast system. We observed that VeA was found in the nucleus and that such a transport is dependent on the alpha-importine protein SRP1. This interaction was also confirmed in a two-hybrid system assay. Currently we are examining the subcellular localization in A. nidulans and characterizing VeA NLS by expressing gfp fusions with the wild type veA allele and NLS veA mutant alleles.
374. Targeting stress-response genes for control of Aspergillus using antifungal natural compounds. Jong H. Kim1, Bruce C. Campbell1, Jiujiang Yu2, Deepak Bhatnagar2, and Thomas E. Cleveland2. 1Plant Mycotoxin Research Unit, Western Regional Research Center, USDA-ARS, 800 Buchanan St., Albany, California 94710 USA; 2 Food and Feed Safety Unit, Southern Regional Research Center, 1100 Robert E. Lee Blvd, New Orleans, Louisiana, 70124 USA
Signal transduction and stress-response genes of fungal pathogens play important roles in pathogenesis. Many genes in Saccharomyces cerevisiaeare interrelated to genes of many fungal pathogens. To discover stress-response genes critical for virulence, an in silico database of transduction/stress-response pathway genes of Aspergillus flavus was constructed based on orthologs of S. cerevisiae. Succesful functional complementation of an antioxidative stress gene from A. flavus, mitochondrial superoxide dismutase (sodA), in a sod2delta yeast mutant verified that S. cerevisiae could serve as a model system for functional genomics of A. flavus. Phenolics are released by plants during fungal infection and their detoxification is necessary for fungal pathogenesis. We developed a high throughput yeast bioassay to screen phenolics for potential antifungal or antiaflatoxigenic activities. Yeast genes affected by active compounds were identified, including a number of signal transduction and antioxidative stress response genes important to fungal tolerance. Then, compounds were used against A. flavus. A 100-fold synergism of strobilurin fungicides was achieved by targeting certain genes with phenolics.
375. Study of the bipartite promoter of the fet3-ftr1 loci of the basidiomycete Phanerochaete chrysosporium. Canessa, P., Larrondo, L., Agredo, M., Polanco, R. & Vicuña, R. Depto Genética Molecular y Microbiología, Fac Cs Biológicas, P Universidad Católica de Chile & Millennium Institute, Santiago, Chile.
Iron metabolism has been mainly studied in yeast. It has been shown that there are multiple systems of iron transport, which are regulated by the transcription factor Aft1. One of these is a bipartite iron transport system formed by a ferroxidase Fet3 and the permease Ftr1. Fet3 belongs to the family of multicopper oxidases, which includes laccases, ascorbate oxidases and ceruloplasmin. Ftr1 transports Fe (III) into the cell. To date, there are no studies describing an iron transport system involving Fet3 in basidiomycetes.
We have recently described MCO1 in P. chrysosporium, a multicopper oxidase that oxidases Fe (II) to Fe (III), as it has been described for Fet3. Interestingly, MCO1 presents similarities to the Fet3 protein. To verify if MCO1 corresponds to a Fet3, we explored the P. chrysosporium genome. We found a homologue to the yeast fet3 gene, just 0.8 kb away from its functional partner ftr1. We confirmed that fet3 is transcriptionally active and regulated by iron. They seem to harbor a shared promoter, containing two putative boxes that match the Aft1 binding site. Using EMSA and footprinting, a probe containing the two elements mentioned above is specifically recognized by at least one nuclear protein, been both elements protected. We founded that iron has an effect on the DNA-protein complex formation.
Financed by grant FONDECYT 1030495 & Millennium Institute (MIFAB).
376. The Glyoxylate Cycle in Penicillium marneffei. David Cánovas, Michael J. Hynes & Alex Andrianopoulos. Dept. of Genetics, University of Melbourne, Victoria 3010, Australia
The human oportunistic pathogen Penicillium marneffei is a thermally dimorphic fungus. The saprofitic form is filamentous in growth at 25 ºC, resembling other Penicillium species. The pathogenic form is an unicellular yeast at 37 ºC, which divides by fission. To cope with nutrient deprivation during the infection process, a number of fungi employ the glyoxylate bypass. Therefore, enzymes belonging to this pathway have been implicated in pathogenesis. The glyoxylate bypass is required to replanish TCA cycle intermediates during growth on gluconeogenic carbon sources. P. marneffei can utilise a variety of carbon sources, including acetate and fatty acids. To gain insight into the organism's ability to utilise these carbon sources during its pathogenic process, we studied one of the key enzymes and a regulator of the glyoxylate cycle. acuD (isocitrate lyase) is required for growth in both acetate and fatty acids. In yeasts and Aspergillus nidulans, induction of acuD is dependent on carbon source. Surprisingly, acuD is mainly regulated by temperature in P. marneffei, being strongly induced at 37 ºC (body temperature) even in the presence of glucose and weakly induced at 25 ºC only when acetate is present. A major Zn(II)2Cys6 regulator of isocitrate lyases in other fungi was also cloned and examined in P. marneffei.
377. The oxa1 Gene Controls Respiratory Complex Assembly and Lifespan in Podospora anserina through Interaction with the Essential rmp1 Gene. Carole H. SELLEM, Claire LEMAIRE and Annie SAINSARD-CHANET.
A causal link between deficiency of the cytochrome respiratory pathway and lifespan was previously shown in the filamentous fungus Podospora anserina. To gain more insight on the relationship between mitochondrial function and lifespan, we have constructed a strain carrying a thermosensitive mutation of the gene oxa1. OXA1 is a membrane protein conserved from bacteria to human. The mitochondrial OXA1 protein is involved in the assembly/insertion of several respiratory complexes. We show here that oxa1 is an essential gene in Podospora anserina. The oxa1ts mutant exhibits severe defects in the respiratory complexes I and IV correlated with an increased lifespan, a strong induction of the alternative oxidase and a reduction in ROS production. However, there is no causal link between alternative oxidase level and lifespan. We also show that in the oxa1ts mutant, the extent of the defects in complexes I and IV and the lifespan increase depends on the essential gene rmp1. The RMP1 protein, whose function is still unknown, can be localized in the mitochondria and/or the cytosolic compartment depending on the developmental stage. We propose that the RMP1 protein could be involved in the process of OXA1-dependent protein insertion.
378. Regulation of Neurospora Circadian Clock by an RNA Helicase Ping Cheng, Qun He, Qiyang He, Lixin Wang, Yi Liu. Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390 USA
The eukaryotic circadian oscillators consist of autoregulatory negative-feedback loops. FRQ, WC-1 and WC-2 are three known components of the negative feedback loop of the Neurospora circadian clock. FRQ represses its own transcription by interacting with the WC-1/WC-2 complex and inhibiting WC's role in transcriptional activation. Here we show that all FRQ associates with FRH, an essential DEAD box-containing RNA helicase in Neurospora. The budding yeast homolog of FRH, Dob1p/Mtr4p, is an essential cofactor of the exosome, an important regulator of RNA metabolism in eukaryotes. Down-regulation of FRH by inducibly expression of a hairpin RNA leads to low levels of FRQ but high levels of frq RNA and the abolishment of circadian rhythmicities. FRH is associated with the WC complex and this interaction is maintained in a frq null strain. Disruption of the FRQ-FRH complex by deleting a domain in FRQ eliminates the FRQ-WC interaction, suggesting that FRH mediates the interaction between FRQ and the WC complex. These data demonstrated that FRH is an essential component in the circaidan negative-feedback loop and reveal an unexpected role of an RNA helicase in regualting gene transcription.
379. A novel transcription factor GalA, homologous to the transcriptional activator of xylanolytic and cellulolytic systems XlnR, regulates D-galactose metabolism in Aspergillus nidulans. Ulla Christensen, Sara Hansen, Harm Mulder, Susan Madrid and Igor Nikolaev, Danisco Innovation, Langebrogade 1, P.O. Box 17, DK 1001, Copenhagen, Denmark
A BLAST search of the Aspergillus nidulans Database resulted, apart from a direct homologue, in two additional hits with high similarity to XlnR, a pathway-specific transcriptional activator of xylanolytic and cellulolytic system in Aspergilli. Here, we present data on one of them isolated from the contig 1.75, which is mapped on chromosome III. The coding sequence of this putative transcription factor is predicted as 2.4 kb long and is interrupted by one putative intron. Like XlnR, it contains a DNA binding domain represented by a Zn binuclear cluster found proximal to its N-terminus. Juxtaposing the physical and genetic maps of A. nidulans suggested that the predicted regulatory gene corresponds to the galA locus, which has been previously characterized to control expression of galactose kinase and galactose-1-phosphate uridyltransferase, the first two steps of D-galactose metabolism in fungi. A knockout of the aforementioned regulatory gene resulted in a lack of growth on D-galactose and galactitol indicating that the encoded transcription factor, called GalA, indeed, controls the expression of genes of both the Leloir and alternative pathways of D-galactose metabolism. GalA appeared to mediate transcription of, at least, the gal7 gene coding for galactose-1-phosphate uridyltransferase, as assessed by a Northern blot analysis. Other genes of the galactose utilization pathway were either constitutively expressed, like gal5 (encoding phosphoglucomutase) or gal10 (encoding UDP-galactose-4-epimerase), or only partially regulated, as ladA (encoding L-arabinitol-dehydrogenase required for galactitol conversion). Galactose transport apparently remained intact in the galA knockout, the result confirmed by a consumption rate of D-galactose similar to that of a wild type strain. A complete transcriptional profile of the genes related to D-galactose metabolism will be presented and a regulatory network will be discussed.
380. DNA Motifs in Promoters of Ivoa, Ivob and Other Conidiation Genes of Aspergillus nidulans. John Clutterbuck. Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, Scotland, U.K.
The ivoA and ivoB genes of A. nidulans are required for brownish pigmentation of the conidiophore. ivoA encodes an N-acetyl-6-hydroxytryptophan synthase containing AMP-binding, cytochrome P450 and two condensation domains as well as a phosphopantotheine attachment site. ivoB encodes a copper phenol oxidase.
Both are regulated by the brlA gene and possibly abaA. Their promoters include BRE and INR the corresponding recognition sites. In order to look for other possible motifs, these and the promoters of other conidiation genes (yA, wA, wetA, abaA, brlA, rodA and medA) have been analysed using a series of simple Perl scripts. It is evident that promoter DNAs are far from random in sequence and contain a many distinctive motifs in addition to those already known.
381. Neurospora crassa Mutants with Enhanced Blue Light-dependent Gene Transcription Laura Navarro-Sampedro and Luis M. Corrochano. Departamento de Genetica, Universidad de Sevilla, Spain
The gene con-10 of Neurospora is expressed during conidiation and after illumination of vegetative mycelia. Photoactivation of con-10 is transient and disappears after two hours of light. To isolate mutants with altered gene photoactivation we have used a strain with a fusion of the con-10 promoter to the gene conferring resistance to hygromycin. This strain is sensitive to the drug when the promoter is inactive, i.e. during vegetative growth either in the dark or under continuous light. We have isolated six mutants that grow in hygromycin under continuous light but not in the dark. The mutants showed an enhanced accumulation of the con-10/hygromycin mRNA after five hours of light compared to the parental strain. The mutations in two strains only affected the photoactivation of the con-10/hygromycin fusion. Four strains showed a high and sustained photoactivation of the endogenous con-10 and, in addition, con-6. This effect was specific since the photoactivation of genes al-2, vvd, and wc-1 was normal in the mutants. The threshold for con-10 and con-6 photoactivation was not altered suggesting that the phenotype is due to a higher transcriptional rate or mRNA stability after a light stimulus. Mutations in genes vvd, encoding a flavoprotein, rco-1, encoding a putative gene repressor, and rco-3, encoding a putative glucose sensor, also resulted in enhanced and sustained photoactivation of con-10 and con-6. Our results support a complex mechanism composed at least of VVD, RCO-1, and RCO-3 responsible for transient gene photoactivation.
382. The Gene for the Heat-shock Protein Hsp100 Is Induced by Blue Light and Heat-shock in Phycomyces blakesleeanus. Julio L. Rodríguez-Romero and Luis M. Corrochano, Departamento de Genética, Universidad de Sevilla, Spain
The Phycomyces hspA gene product is a 901 amino acid-protein member of the clpB/HSP100 family. HSP100 proteins are ATPases involved in the tolerance to high temperatures, proteolysis, and protein disaggregation. The hspA promoter contains three heat-shock elements that are presumably involved in the activation of hspA after a heat-shock. In addition, four sequences are present in the hspA promoter and in the promoter of the photoinducible genes carB and carRA, and are candidates for binding sites for light-regulated transcription factors. Blue light can activate transcription of the hspA gene by 10-fold with a threshold of 1 J/m2. The threshold for hspA photoactivation is 104 times higher than the thresholds for blue-light regulation of sporangiophore development and photocarotenogenesis, which suggests that there are differences in the photosensory systems for gene photoactivation and mycelial photoresponses. A heat-shock of 30 min at 34ºC or 42ºC activated the hspA gene by 160-fold. The differences in maximum hspA gene transcription by blue light and heat shock suggest the presence of different regulatory mechanisms.
Mutations in genes madA and madB impair all photoresponses, including the photoactivation of hspA but did not alter hspA activation by heat-shock. Mutations in car genes required for beta-carotene biosynthesis reduced or impaired hspA photoactivation. Our results suggest that mad and car gene products are required for hspA photoactivation in a complex photosensory system.
383. The Neurospora Circadian Clock is Composed of Multiple Oscillators. Renato Magalhaes de Paula, Zachary Lewis, Kyung Suk Seo, Andrew Greene and Deborah Bell-Pedersen*. Department of Biology, Texas A&M University, College Station, TX 77843
Circadian clocks coordinate daily changes in behavior, physiology, and gene expression in organisms. In Neurospora crassa, the clock regulates the production of conidia and the rhythmic expression of a number of genes and proteins. Using microarrays, we identified an evening-specific gene, ccg-16, that has rhythms in mRNA accumulation that persist in the absence of FRQ in constant darkness and in constant light. These data establish that ccg-16 is controlled by a novel oscillator that does not require FRQ. We call this oscillator the UFO for un-FRQ oscillator that regulates an evening-specific gene. The UFO appears to be coupled to the FRQ-based oscillator through the products of the wc-1 and wc-2 genes. Null mutations in either wc-1 or wc-2 abolish ccg-16 rhythms, and the phase of the ccg-16 rhythm is altered in a long-period frq7 mutant allele in cultures grown in constant darkness. Together, these data demonstrate that the circadian system of Neurospora involves multiple-coupled oscillators that contribute to the diversity and refinement of the output pathways.
384. Regulation of the pentose catabolic pathway genes of Aspergillus niger. M.J.L. de Groot1, L. Vandeputte-Rutten,2, C. van den Dool3, H.A.B. Wösten3, M. Levisson1, P.A. vanKuyk1, G.J.G. Ruijter1, and R.P. de Vries3. 1Fungal Genomics, Wageningen University, The Netherlands; 2Department of Crystal and Structural Chemistry, Utrecht University, The Netherlands; 3Microbiology, Utrecht University, The Netherlands.
The aim of this study was to obtain a better understanding of the pentose catabolism in Aspergillus niger and the regulatory systems that affect it. To this end, we have cloned and characterised the genes encoding the A. niger L-arabitol dehydrogenase (ladA) and xylitol dehydrogenase (xdhA), and produced the enzymes in Escherichia coli to determine the substrate specificity.
Expression analysis of the pentose catabolic pathway genes resulted in a model in which an L-arabinose specific regulator activates the expression of all genes required for the conversion of L-arabinose to D-xylulose-5-phosphate. In addition, XlnR regulates the first step and, to a lesser extent, the other steps of the conversion of D-xylose into D-xylulose-5-phosphate. This model explains all reported data for the expression of pentose catabolic genes, arabinolytic genes and xylanolytic genes. It also takes into account the overlapping substrate specificities of the pentose catabolic enzymes that effect the accumulation of L-arabitol, the inducer of the L-arabinose catabolic pathway genes and the arabinolytic genes.
385. Hog Map Kinase Controls Stress Response in T. harzianum. Delgado-Jarana, J.1, Sousa, S.1, Monte, E., Rey, M.2, Llobell, A1. 1Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de Sevilla. Av. Americo Vespucio s/n. 41092 Sevilla. Spain. 2Newbiotechnic, S.A. Parque Industrial de Bollullos A-49 (PIBO). 41110 Bollullos de la Mitación. Sevilla. Spain.
Trichoderma harzianum is a soil-borne fungus, able to colonize the rhizosphere displaying antagonistic activities against phytopathogenic fungi and developing mycorrhiza-like interactions with plant roots. In the soil, T. harzianum is exposed to a highly variable environment with respect to the availability of nutrients, temperature, pH, oxygen and water. All these changes have to be sensed by the fungal cell to adapt its metabolism and to develop the appropriate response.
We have characterized the T. harzianum hog1 gene, which encodes a MAPK involved in stress response. Hog1 is phosphorylated under oxidative stress, hyperosmotic shock, presence of heavy metals and other conditions. We have analysed hyperactive mutants (carrying F314S mutation) and a silenced strain (using a hairpin construct) with respect to stress resistance and cross protection, showing the key role of Hog1 in stress response. hog1 was able to complement hog1delta mutation in yeast, but not hog1delta pbs2delta which may be related to different sequence features and/or functional divergent role of Hog1 protein in both systems. On the other hand, we have carried out arrays hybridization using to study hyperosmotic shock transcription profile. From these data, a clear view of the genes induced under stress, and the potential co-regulation in plant-fungus association can be established.
386. Mutations in the gene encoding the mitochondrial DNA polymerase affect differentially the Podospora anserina lifespan in different genetic contexts. Michelle Dequard-Chablat, Veronique Contamine, Marguerite Picard, and Robert Debuchy. Institut de Genetique et Microbiologie, UMR8621, Orsay, France.
Podospora anserina has a limited lifespan, which is measured as the length of mycelium from ascospore germination. In our laboratory culture conditions, a wild-type strain has a lifespan of 10 cm and displays alterations of the mitochondrial genome at the time of death. A mutation in the AS1 gene, encoding a cytosolic ribosomal protein, reduces the lifespan to 1.4 cm and leads to the accumulation of a specific deletion of the mitochondrial genome at the time of death. A screen for suppressors increasing the lifespan of the AS1 mutant strain led to the isolation of 4 different mutations in the gene encoding the mitochondrial DNA polymerase (polG). All these mutations map in the C-ter part of the polG, an evolutionnary non-conserved region of unknown function. In an AS1 mutant strain, these suppressors increase the lifespan to a value higher than the mean longevity of a wild-type strain, while in an AS1+ strain, the polG suppressors decrease the lifespan to a value below the lifespan of a wild-type strain. We are currently performing a physiological and genetic analysis of these mutants and of other site-directed mutations in evolutionnary conserved positions of the polG. The genetic interactions of these mutations with previously obtained suppressor mutations mapping outside the polG gene open the possibility to identify the polG partners.
387. Gene Regulation During Nitrogen Stress and Invasive Plant Growth in the Rice Blast Pathosystem. Nicole Donofrio and Ralph A Dean. NCSU, 851 Main Campus Drive, Raleigh, NC
Previous research on Magnaporthe grisea, causal agent of the rice blast disease, suggests that during certain stages of its invasive growth cycle, the pathogen is in a nitrogen-limited environment. To gain insight into M. grisea physiology during nutrient stress and invasive growth, we examined global gene expression changes under nitrogen starvation conditions in culture. Using the whole genome, oligo microarrays were designed in collaboration with Agilent Technologies, and contain 13,666 M. grisea genes. Approximately nineteen hundred genes showed significantly increased expression during nitrogen starvation. Among these, we found an increase in expression level of nitrogen scavenging genes, such as the global nitrogen regulator in M. grisea, NUT1. Furthermore, we found several genes with known roles in pathogenicity, including PTH11 and MPG1, to be highly expressed during nitrogen starvation after both 12 and 48 hours. We confirmed the expression patterns of 20 up-regulated genes using reverse transcription PCR, providing a confirmation of our microarray results. To determine whether nitrogen regulatory genes were also expressed during infection, we used RT-PCR to examine expression patterns of several genes during invasive growth of M. grisea in rice. These data will be presented, along with preliminary data from targeted deletion lines of several genes differentially regulated during nitrogen stress.
388. Nitrogen metabolite represion of arginine catabolism genes in Aspergillus nidulans is mediated by negatively acting factor AREB. Agnieszka Dzikowska1 , Piotr Weglenski1 and Claudio Scazzocchio2 . 1Department of Genetics Warsaw University, ul. Pawinskiego 5a 02-106 Warsaw, Poland; 2Institut de Génétique et Microbiologie, Université Paris-Sud, UMR 8621, Bat. 409, 91405 Orsay CEDEX, France
The arginine catabolism genes agaA and otaA , coding for arginase and ornithine transaminase (OTAse) respectively, are specifically induced by arginine and repressed by ammonia. In A. nidulans nitrogen metabolite repression is mediated by transcriptional activator AREA from GATA family. areA 600 loss of function mutant does not grow on arginine as a nitrogen source but we have shown that agaA and otaA expression does not depend on AREA. Arginase and OTAse enzymatic activities are fully inducible in areA600 mutant. The same was shown for mRNAs. It is possible that an arginine permease gene(s) can be a target for AREA activator. Preliminary data suggest that there are two arginine transport systems and only one depends on AREA activator. We have also shown that another negatively acting factor from GATA family (AREB) participates in the ammonia repression of agaA and otaA . In areB loss of function mutant the ammonia repression of both genes is changed at the level of enzyme activity and at the level of mRNA.
389. Characterization of a sugar sensing pathway in Neurospora crassa. Xin Xie, Heather H. Wilkinson, and Daniel J. Ebbole. Program for the Biology of Filamentous Fungi, Dept. of Plant Pathology and Microbiology Texas A&M University, College Station, TX
Approximately 19% of 1385 Neurospora crassa genes represented on a cDNA microarray are regulated following a shift from medium containing glucose to carbon starvation medium. Glucose transport activity and expression of a high-affinity glucose transporter gene is induced in response to this shift. We previously hypothesized that one mechanism for sensing glucose involves the use of glucose transporter homologs as glucose receptors. The rco-3 gene is a regulator of sugar transport and conidiation in N. crassa and we isolated suppressors that remedy the sorbose resistance phenotype of the rco-3 mutant. A previously characterized mutant defective in the dgr-1 gene phenotypically resembles rco-3. To test whether dgr-1 is involved in the rco-3 signaling pathway, epistasis relationships among rco-3, dgr-1 and the suppressors were examined. To further investigate the relationship between rco-3 and dgr-1 and to help assess the extent to which they may constitute a common regulatory cascade, the transcriptional response to glucose status in rco-3 and dgr-1 mutants was examined via microarrays. rco-3 and dgr-1 are both required for appropriate regulation of genes involved in the TCA cycle, the glyoxylate cycle and gluconeogenesis. We conclude that rco-3 and dgr-1 form part of a common pathway to regulate the cellular response to carbon source.
390. Cyclic-AMP dependent kinases facilitate inactivation of nitrogen regulation by newly formed Ure2p amyloid in S. cerevisiae. Herman K. Edskes, Benedetta M. Naglieri, Reed B. Wickner. Laboratory of Biochemistry and Genetics, NIDDK, NIH
The Saccharomyces cerervisiae Ure2 protein regulates the transcription factor Gln3p that targets genes needed for the uptake and utilization of poor nitrogen sources. Thus in the presence of a good nitrogen source like ammonium the Dal5 permease, capable of taking up the poor nitrogen source allantoate, is not expressed. However, in approximately one in a million cells the nitrogen regulation system (NCR) is inactivated due to Ure2p aggregating as infectious (prion) amyloid filaments. Overexpression of Ure2p greatly enhances initiation of amyloid formation. Deletion of MKS1, a negative regulator of the Rtg1p and Rtg3p transcription factors that direct transcription of glyoxylate and tricarboxylic acid cycle genes, prevents activation Dal5p by newly seeded Ure2p amyloid but not by established Ure2p filaments. Deletion of RTG3 is epistatic to deletion of MKS1. However, glutamate, a potent repressor of RTG1/3 directed transcription does not affect Dal5p activity in a MKS1 deletion strain. Deletion of both TPK1 and TPK3, two cyclic-AMP dependent kinases, also prevents activation of Dal5p by newly seeded Ure2p amyloid. Again this effect is overcome when RTG3 is deleted.
391. VVD's role in entrainment of the Neurospora crassa circadian clock. Mark Elvin & Christian Heintzen. Faculty of Life Sciences, The Michael Smith Building, The University of Manchester, Manchester, M13 9PT, UK
To provide an accurate depiction of external time, Neurospora's circadian clock is synchronized to the rhythmic environment via a process called entrainment. Light and temperature are important entrainment cues. We previously identified VVD as a small PAS/LOV protein that is clock-controlled, rapidly light induced and, although not essential for circadian rhythmicity, influences light sensitivity and the phase of the circadian clock. Our recent work has focused on elucidating VVD's significance for the circadian clockwork by investigating the molecular and phenotypic differences between vvd knockout and wild-type strains in various light-dark entrainment conditions. Data will be presented that illuminate the role of VVD in entrainment of the Neurospora circadian clock to light-dark cycles and we propose a molecular mechanism by which VVD facilitates light-dark entrainment.
392. Inactivation of the histidine-kinase Bcos1 of Botrytis cinerea has pleiotropic effects on fungal growth, development, and virulence. Sabine Fillinger1, Weiwei Liu1, Laurent Legendre2, Pierre Leroux1, Jai Santosh Polepalli2, and Muriel Viaud3. 1UPMC, INRA Versailles, Route de Saint-Cyr, 78026 Versailles, France. 2Centre for Horticulture and Plant Sciences, University of Western Sydney, Locked bag 1797, Penrith South DC, NSW 1797, Australia, 3 PMDV, INRA Versailles, Route de Saint-Cyr, 78026 Versailles, France
Filamentous ascomycetes contain large numbers of histidine-kinases (HK) most of which are highly conserved
among species (Catlett et al, 2003; Eukaryot. Cell 2, 1151)). We have inactivated the HK of group III, Bcos-1, in the
economically important plant pathogen Botrytis cinerea. The Bcos-1 gene was previously involved in fungicide
resistance (Cui et al., 2002; FGB 36, 187). A detailed phenotypic analysis shows that Bcos-1 deletion has pleiotropic
effects on the fungus. Besides the expected fungicide resistance and osmosensitivity also the asexual development is
altered (reduced growth, no conidiation). However, the mutants do not present higher sensitivities to other stress-conditions, but are more resistant to menadione (vitamine K3) provoking oxidative stress. Osmosensitivity and
reduced protoplast-formation moreover suggest alterations in cell-wall composition. Pathogenicity tests show that
Bcos-1 mutants are severely impaired for plant infection. The BcOs1 histidine kinase therefore constitutes a major
pathogenicity factor of B. cinerea.
Presumably it regulates a signal-transduction cascade involved in osmosensing comparable to the HOG (high-osmolarity glycerol) pathway of Saccharomyces cerevisiae. In contrast to the yeast Sln1 sensor histidine kinase, BcOs1 does not have a detectable transmembrane domain. Using a BcOs1-GFP fusion we can show the fluorescence localised in the cytoplasm. The cytoplasmic localisation of BcOS1 raises the question if the histidine-kinase is the sensor protein of external osmotic pressure or if this function is taken over by an additional sensor protein. Transcriptome analyses on 3032 unigenes are being carried out under various conditions between the deletion mutants and the parental strain in order to combine gene-expression data and phenotypes.
393. Regulation of the biosynthesis of the toxin, sirodesmin, in the ascomycete Leptosphaeria maculans. Ellen M. Fox, Donald M. Gardiner and Barbara J. Howlett. University of Melbourne, Melbourne, VIC, Australia
Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). A cluster of 18 genes with predicted roles in the biosynthesis of sirodesmin PL has been cloned. Attempts are being made to determine the roles of individual genes in the cluster via RNA interference, as well as by targeted mutagenesis. The knock down of sirZ, a putative transcription regulator of the cluster, resulted in a dramatic reduction in sirodesmin production. Additionally genes elsewhere in the genome that regulate sirodesmin biosynthesis are being sought. An assay, which exploits the antibacterial properties of sirodesmin, has been developed to screen for sirodesmin deficient mutants. This screen has already yielded five mutants, which are currently being analysed.
394. In vitro sporulation of the obligate plant pathogen Ustilago maydis. Maria D. Garcia-Pedrajas and Scott E. Gold. Department of Plant Pathology, University of Georgia, Athens, GA
The repression of a subset of genes normally expressed in budding cells, upon switch to filamentous growth may be critical for programming morphogenesis and virulence in the corn pathogen Ustilago maydis. To identify these filament downregulated genes and to better understand the role of wild-type budding cells in the life and disease cycle of U. maydis in nature, we used suppression subtractive hybridization (SSH). We screened 5,500 cDNA clones by an iterative sequencing and hybridization process. Greater than 88% of the differentially expressed clones detected corresponded to 48 different genes. Differential expression (*2 fold) was confirmed for 37 of these genes by northern blot analysis. We have produced deletion mutants in several of the highly differentially expressed genes. The effect of these mutations on morphology and/or pathogenicity will be discussed. Additionally, analysis of the putative promoter region of these filament downregulated genes identified a potential regulatory sequence, which led us to the isolation of a new regulator (ust1) of morphogenetic processes in U. maydis. Deletion of ust1in wild-type budding cells and in solopathogenic haploid and diploid backgrounds resulted in filamentous growth, with areas devoid of cytoplasm. Additionally, highly melanized thick-walled structures, closely resembling teliospores produced in plant tumors, were produced in vitro in these mutants. We are currently in the process of fully characterizing this newly identified transcription factor and its role as a repressor of sporulation.
395. Expression of Fost20, a PAK Kinase Gene from the Phytopathogenic Fungus Fusarium oxysporum. M. A. García-Sánchez, B. Ramos, N. Martín-Rodrígues, A. P. Eslava and J. M. Díaz-Mínguez. Area de Genética, Centro Hispano-Luso de Investigaciones Agrarias (CIALE) Universidad de Salamanca 37007, Salamanca, Spain
Fusarium oxysporum is a soil-borne plant pathogen that causes wilt disease in a variety of crops. The fungus enters the plant through the roots and to spread throughout entire vascular system, causing characteristic wilt symptoms. To perform these processes the fungus must perceive chemical and physical signals from the host plant and respond with the appropriate metabolic and morphological changes required for disease development. Many of these responses have been shown to be dependent on conserved signal transduction pathways.
In eukariotic cells, a family of serine/threonine protein kinases (PAK kinases) are involved in various cellular signaling and developmental processes. To understand the role of PAK kinases in activating the MAP kinase pathway, we isolated and characterized the Ste20 homologous in F.oxysporum, named fost20. The fost20 mutants were reduced in conidiation but had no obvious defect in pathogenicity and/or virulence. Although it is not clear how nutritional conditions affect these genes, one possibility is that the MAP kinase pathway involved in nutrient or pH sensing may be affected in fost20 mutants. Thus, we have analysed whether there is any differential expression of these genes under nutrient starvation conditions, and pH stress using Real-Time PCR and Norhern analysis.
396. Culture-dependent alternative transcription initiation of the enolase-encoding gene (enoA) in Aspergillus oryzae. Katsuya Gomi1, Yuuichi Yamaki1, Takeshi Akao2, Tomomi Toda3, Masayuki Machida3. 1Graduate School of Agricultural Science, Tohoku University, Sendai, 2National Research Institute of Brewing, Higashi-Hiroshima, 3National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Enolase generally plays a pivotal role in the glycolysis and is therefore one of the most highly expressed proteins in most organisms. We have cloned an enolase-encoding gene (enoA) of Aspergillus oryzae and identified a cis-element of 15-bp located between -195 and -181 relative to the translation start codon responsible for the transcriptional regulation of the enoA by promoter deletion analysis and gel mobility shift assay1). Surprisingly, recent comparative analysis using ESTs and whole genomic sequence data of A. oryzae suggested the existence of alternative transcription initiation sites in the enoA dependent on culture conditions. In the absence of carbon source or in carbon-limited cultures, transcription of the enoA is initiated mainly at -530 upstream of the start codon and results in the formation of transcript with an intron of approximately 440-bp in the 5f-untranslated region (5fUTR) removed. In contrast, the enoA is transcribed from -50 relative to the start codon, which is located within the intronic sequence in the 5fUTR, in the presence of carbon sources such as glucose. To our knowledge, this is the first finding that alternative transcription initiation sites are utilized depending on culture conditions in the genes involved in the primary metabolic pathway in filamentous fungi, except for the developmental regulatory gene, brlA, of A. nidulans2).
1) T. Toda et al., Curr. Genet., 40, 260-267 (2001). 2)R. Prade and W. Timberlake, EMBO J. , 12, 2439-2347 (1993).
397. Both carbon and nitrogen regulate expression of genes involved in nitrogen translocation between partners in the arbuscular mycorrhizal symbiosis. Manjula Govindarajulu1, Philip E. Pfeffer2, Yair Shachar-Hill3, Peter J. Lammers1. 1Dept. Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA. 2USDA-ARS Eastern Regional Research Ctr., 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA. 3Plant Biology Dept., Michigan State University, East Lansing, MI 48824-1312, USA.
Arbuscular mycorrhizal fungi (AMF) supply nutrients including nitrogen to host plants in return for carbon derived from photosynthesis. Analysis of N and C flux using stable isotopes supports a model whereby AMF shuttle nitrogen to their host plants via assimilation of inorganic N into arginine and translocation of this amino acid to fungal tissue within the host plant where the catabolic arm of the urea cycle releases N as ammonia to the host plant with no loss of fungal C. We have measured fungal mRNA levels for key N enzymes and transporters in both extraradical (ERM) and intraradical mycelium (IRM) tissues using quantitative real-time PCR. Glutamine synthase (GS) was shown to be expressed in both ERM and IRM tissues isolated from the carrot hairy root co-culture system. GS transcripts were significantly higher when NH4+ was substituted for NO3- in the ERM compartment of an in vitro split plate culture system. Consistent with predictions from the model, the same conditions lead to significantly higher mRNA levels in the IRM tissues for two urea cycle enzymes [urease accessory protein (UAP), ornithine aminotransferase (OAT)] and an ammonium transporter (AMT). Conversely, expression of the fungal AMT, OAT and UAP genes was down-regulated in IRM tissues as glucose levels in the medium dropped. This was reversed by supplemental glucose demonstrating that regulation of key fungal N genes is dependent on carbon availability. The same pattern of down regulation of fungal AMT, OAT and AMT was observed when pot-grown Medicago sativa plants colonized by Glomus intraradices were placed in the dark to limit carbon availability. These results show that nutrient exchange in the symbiosis is tightly controlled and suggests a mechanism for regulation of coupled nutrient exchanges between the symbionts.
398. Nonsense-mediated mRNA decay in yeast regulates global gene expression through a network of direct and indirect targets. Qiaoning Guan and Michael R. Culbertson. Laboratories of Genetics and Molecular Biology, University of Wisconsin, Madison, WI 53706
Nonsense-mediated decay (NMD) is a eukaryotic mechanism that rapidly degrades messenger RNAs containing premature termination codons (PTC). Recently, several genome-wide expression profiles in yeast and mammals indicate that NMD may serve a post-transcriptional regulatory role in controlling the steady-state levels of several hundred wild-type mRNAs. Similar physiological categories of the natural substrates of NMD have been identified in the yeast Saccharomyces cerevisiae and humans. Although several molecular mechanisms have been proposed for how NMD targets specific mRNAs for altered decay, many of the mRNAs whose abundance is influenced by NMD do not show altered decay rates. We are investigating a model of direct and indirect targeting by the NMD pathway that includes transcription factors and the genes they regulate, resulting in a complex network of changes in mRNA abundance. mRNAs coding for transcriptional activators and repressors are candidates for direct NMD targets, although some of these can be indirect targets if they function in a regulatory network of multiple transcription factors. The direct target mRNAs exhibit a change in half-life when NMD is inactivated. Other genes whose expression is regulated by a targeted transcription factor become substrates of NMD as an indirect effect. Indirect targets exhibit a change in mRNA abundance due to the change in rate of transcription. We have determined the decay kinetics of a list of mRNAs coding for transcription factors in isogenic NMD+ and nmd- strains, and have identified both direct and indirect targets. The transcription factor FZF1 mRNA contains an upstream open reading frame (uORF) which overlaps with its protein-coding region. Eliminating the uORF by mutating its start codon from AUG to AGG results in diminished sensitivity of the FZF1 mRNA to NMD, suggesting that the uORF plays a role in but is not completely responsible for the targeting of the FZF1 mRNA. To fully understand how the mRNA abundance including FZF1 and other transcription factors is directly affected by NMD, we are using visual analysis of meiotic tetrads expressing GFP fusions to delineate the destabilization elements of these mRNAs. Further discrimination between direct and indirect targets of NMD coupled with an analysis of transcriptional regulatory circuits will allow us to generate a blue print of the global gene expression regulation in the subset of yeast genes whose expression is influenced by the NMD pathway.
399. Differential gene expression during perithecial development in Gibberella zeae (anamorph Fusarium graminearum). Heather E Hallen, Weihong Qi, Frances Trail. Michigan State University, Department of Plant Biology, East Lansing, MI 48824
Gibberella zeae (anamorph Fusarium graminearum) is the causal agent of Fusarium head blight, a disease of wheat, corn and other crop plants causing an estimated three billion dollar loss in the US alone during the 1990's. The fungus overwinters on crop debris and ascospores are discharged in the spring to infect the new crop. As sexual development and spore discharge are thus crucial features of the infection cycle, we are investigating the genes involved in different stages of sexual development. We designed and used a cDNA microarray representing 2067 genes to investigate differential expression. Currently, we are conducting genome-wide studies using Affymetrix gene chips representing all predicted genes, ORFs and ESTs of Fusarium graminearum. Knockouts on genes likely to be involved in sexual development and ascospore discharge are underway.
400. Investigating abnormal phenotypes associated with RNA silencing enzymes and RNA-dependent RNA polymerases in Aspergillus nidulans. T. M. Hammond1, Y. Reyes Domínguez2, L. R. Milde1, J.W. Bok1, C. Scazzocchio2 and N. P. Keller1. 1Plant Pathology, University of Wisconsin, Madison, WI. 2Institut de Génétique et Microbiologie, Université Paris-Sud, France.
Of the three Aspergilli with fully sequenced and publicly available genomes, A. fumigatus, A. nidulans, and A. oryzae, A. nidulans encodes the fewest enzymes commonly attributed to RNA silencing related processes. For example, A. nidulans is predicted to have only one ‘Dicer', one Paz and Piwi domain (PPD) protein and two RNA dependent RNA polymerases (RDRPs), while the other two sequenced Aspergilli, and most other studied filamentous ascomycetes, are predicted to have at least two Dicers, two PPD proteins, and three RDRPs. The reduced number of these types of genes in A. nidulans suggests that it is an ideal model organism to study the biological function of these genes in filamentous fungi. In previous studies we have shown that the predicted A. nidulans PPD protein (RsdA) is required for RNA silencing during the vegetative cycle but that its two RDRPs (RrpA and RrpB) are not required for the same process. Interestingly, the simple experiments required to determine that the one predicted Dicer (DcrA) is required for RNA silencing in A. nidulans have not yet been completed because of an abnormal level of difficulty encountered during our attempts to obtain a dcrA deletion strain. In addition to our continual pursuit of a dcrA deletion strain, we are currently investigating two peculiar phenotypes associated with RNA silencing related genes but not thought to be directly related to RNA silencing during the vegetative cycle. These include a ‘slow-growth' phenotype associated with dcrA over-expression and a ‘selfing proficient' but ‘crossing deficient' phenotype associated with rrpB deletion.
402. Galactokinase activity is essential for high cellulase transcript levels during growth on lactose in Hypocrea jecorina. L. Hartl, B. Seiboth, C.P. Kubicek. Institute of Chemical Engineering, TU Vienna
H. jecorina (Trichoderma reesei) is a producer of important extracellular enzymes, e.g. cellulases and hemicellulases. The H. jecorina galactokinase encoded by gal1 catalyses the first step of the D-galactose degrading Leloir pathway. Strains deleted in gal1 show reduced growth on D-galactose and reduced cellulase transcript levels while growing on lactose.
In Kluyveromyces lactis galactokinase is bifunctional: it catalyses D-galactose phosphorylation, and also signals D-galactose-induction to the GAL genes via interaction with Gal80p.
To define the role of the H. jecorina galactokinase in cellulase induction the following strains were constructed by transforming a gal1 deleted strain: Strains expressing (i) an enzymatically inactive galactokinase by deletion of two essential amino acids (Gal1-SA), (ii) a solely enzymatically active galactokinase by using the galK from E. coli and (iii) the gal1 gene under the pki1 promoter. Only strains expressing galK or the H. jecorina galactokinase under the pki1 promotor showed increased growth rates on D-galactose indicating that the Gal1-SA had lost its galactokinase activity. Cellulase induction by lactose was dependent on galactokinase activity since induction was found in strains expressing the E. coli galactokinase or the H. jecorina galactokinase under the pki1 promotor but not in the Gal1-SA expressing mutant.
403. Molecular cloning and characterization of two hydrophobins and a Cerato ulmin-like protein from the entomopathogenic fungus Beauveria bassiana. Diane Holder, Eun-Min Cho, and Nemat O. Keyhani. University of Florida, Microbiology and Cell Science, Bldg 981, Museum Rd. Gainesville, FL 32611
Hydrophobins are small predominantly hydrophobic, amphipathic proteins that function in a broad range of growth and developmental processes in fungi. They are involved in the formation of aerial structures, the attachment of fungal cells to surfaces, and act in signaling in response to surface cues and pathogenesis. Beauveria bassiana is an important entomopathgenic fungus used as an arthropod biocontrol agent. To examine the feasibility of using phage display technology to clone cDNAs encoding hydrophobins, we performed biopanning experiments using a variety of affinity resins, including N,N'-diacetylchitobiose, fucose, lactose, maltose, and melibiose-coupled agarose beads. After 5 rounds of iterative biopanning, cDNAs corresponding to three B. bassiana hydrophobins were isolated, one of which displayed high homology to the toxin, Cerato-ulmin. Suppressive subtractive hybridization (SSH), used to isolate transcripts induced during fungal growth on chitin, also resulted in the isolation of one of the B. bassiana hydrophobins. These results reveal the differential regulation of the isolated hydrophobins and indicate that phage display may represent a novel approach to cDNA cloning of hydrophobins.
This work was support it part by NSF grant # EF-0412137 (NOK).
404. Antisense frequency (qrf) RNA and its role in the circadian clock of Neurospora crassa. Suzanne Hunt, Zoulikha Mohammed, Matthew Mayho, Marion Hogg & Susan Crosthwaite. Faculty of Life Sciences, The Michael Smith Building, The University of Manchester, Manchester, M13 9PT, UK
Rhythmic levels of frequency (frq) messenger RNA are essential for maintaining circadian rhythmicity in the filamentous fungus Neurospora crassa. Apart from mRNA transcripts encoding the FRQ protein, antisense frq (qrf) transcripts are also transcribed and these have been shown to affect the response of the clock to light (Kramer et al., 2003 Nature 21:948-952). Altering the expression of qrf does not affect the clocks ability to entrain to temperature cycles. However, preliminary data indicate that when temperature pulses are administered at different times of the circadian day the response of qrf-defective strains differs from the wild type. frq and qrf transcripts are cis-encoded and complementary over large regions. Therefore, we investigated whether or not their levels might be regulated via the formation of double-stranded RNA and present data on the circadian properties of quelling-defective mutants.
405. Identification of a Natural Antisense Transcript of aflR, the Transcriptional Regulator in the Aflatoxin Biosynthesis Pathway in Aspergillus flavus. Carrie Jacobus1 , Gary Payne 2 , and Niki Robertson 1,3. 1Department of Genetics. 2Department of Plant Pathology. 3Department of Botany. NCSU Raleigh, North Carolina.
Natural antisense transcripts (NATs) are involved in gene expression in several eukaryotic systems. Cases of transcriptional interference, RNA masking, and double-stranded RNA dependent mechanisms have all been shown to involve NATs. Recently, antisense transcripts have been identified in the filamentous fungus Neurospora crassa that are complementary to frq, a key component of the circadian clock. An antisense transcript was identified in a cDNA library of another filamentous fungus, Aspergillus flavus. This species among others in the genus Aspergillus produces aflatoxin, the most potent naturally occurring carcinogen. The antisense transcript identified in the cDNA library corresponds to aflR, which is the transcriptional regulator of aflatoxin biosynthesis. This NAT, aflRas, overlaps with the 5' end of aflR and extends into its promoter. RT-qPCR analysis was used to compare aflR and aflRas levels with respect to aflatoxin production at different time points. In addition, an over-expression construct utilizing the alcA promoter to drive expression of alfRas was transformed into A. flavus. These over-expression transformants will provide insight as to the function of aflRas. (Presented at poster number 538)
406. Characterization of 14-3-3 homolog in the rice blast fungus, Magnaporthe grisea. Jun Seop Jeong and Ralph A. Dean. North Carolina State University, CIFR(Center For Integrated Fungal Research).
14-3-3 protein is a small acidic protein that is found universally in eukaryotes. In contrast to animals and plants, only one or two homologs are usually found in fungi. Despite the extensive researches in yeast species, little is known in regard to its role in filamentous fungi. The protein is implicated in signal transduction, cell cycle, nutrient utilization, vesicular trafficking, growth, and development. However, due to the considerable sequence divergence and functional overlap of the gene family, the precise role 14-3-3 proteins play in cellular processes remains obscure. In the rice blast fungus, one 14-3-3 homolog is annotated (MgFTT1) in the current Magnaporthe grisea genome database (http://www.broad.mit.edu/ annotation/fungi/magnaporthe/). Further analysis revealed the presence of, at least, one more homolog in the genome. To investigate the function of MgFTT1, gene knockout mutants were created. The phenotypic consequences of targeted deletion will be presented.
407. Global patterns of gene regulation associated with the development of ectomycorrhiza between birch and Paxillus involutus. Tomas Johansson, Antoine Le Quéré, Derek Wright, and Anders Tunlid. Microbial Ecology, Lund University, Sweden
The formation of ectomycorrhizal (ECM) root tissue is characterized by distinct developmental stages including pre-infection/adhesion, mantle and Hartig net formation. The global pattern of gene expression along the development of an ECM association between birch (Betula pendula) and Paxillus involutus was analysed using cDNA microarrays. In comparison to non-symbiotic tissue, 251 fungal genes (of 1,075 in total) and 138 plant genes (of 1,074 in total) were differentially regulated during the ECM tissue development and clusters of coregulated plant or fungal were identified. For example, during mantle and Hartig net development, an upregulation of plant genes known to be involved in plant defense responses elicited during fungal pathogen challenge occurred. Notably, this response was significantly reduced in fully developed ECM tissue. Among fungal genes several homologues encoding proteins involved in the respiratory chain were upregulated during mantle and Hartig net formation. However, the relative expression level of these genes decreased during later stages of ECM tissue development. Within fully developed ECM tissue, there was significant upregulation of fungal genes related to protein synthesis and components of the cytoskeleton assembly machinery. This study highlights the complex molecular interactions which occur between symbionts during the development of ECM associations.
408. Analysis of Two-component Histidine Kinases, and Several Downstream Effectors in the Filamentous Fungus, Neurospora crassa. Carol A. Jones, Suzanne Phillips, and Katherine A. Borkovich Department of Plant Pathology, University of California, Riverside, CA, USA 92521
Two-component systems consist of proteins that contain a histidine kinase and/or a response regulator domain. Histidine kinases (HKs) signal via a phosphorelay. In complex two-component signaling, the HK is of the hybrid type, meaning that the HK and the RR domains are within the same protein. The HK is autophosphorylated in response to an environmental signal; this phosphate is then transferred to the response regulator (RR) domain, which then gets transferred to a histidine phosphotransferase (HPT), and then onto a second RR. In various organisms the HK pathway helps regulate environmental responses to light, nutrient and oxygen levels, osmolarity, and other factors. In eukaryotes, HKs are involved in the activation of mitogen-activated protein kinase (MAPK) cascades and/or transcriptional regulation.
The completion of Neurospora crassa genome has revealed eleven putative HKs, one HPT, and two RR genes. The number of HKs found in Neurospora is considerably larger than that of the sequenced yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. I am in the process of producing Neurospora mutants that lack these eleven putative hybrid HKs using targeted gene replacement. Upon constructing these mutants I will perform cellular and developmental phenotypic analysis of the mutants to determine the functions of these genes. Additionally, multiple-gene knockout strains will be produced of phylogenetically-related genes in cases where no phenotype is observed. I have produced four homokaryotic knockouts (without a robust phenotype), and am in the process of generating double mutants of the two HKs which have the highest relatedness in their sensor domains.
The phenotypes of HK mutants can be compared to the HPT, and the RR mutants. Analysis of one of the RR, rrg-1, has begun. One or more of the HKs are likely to regulate RRG-1. Analysis of the HKs and RRs will help provide insight into two-component signal transduction pathways and may contribute to the development of new antifungal agents for mammalian and plant pathogens.
409. One of the two Dicer-like proteins in the filamentous fungi Magnaporthe oryzae genome is responsible for hairpin RNA-triggered RNA silencing and related siRNA accumulation. Naoki Kadotani, Hitoshi Nakayashiki, Yukio Tosa, Shigeyuki Mayama. Laboratory of Plant Pathology, Kobe University, Kobe, Japan.
Dicer is an RNase III-like enzyme playing a key role in the RNA silencing pathway. Genome sequencing projects have demonstrated that eukaryotic genomes vary in the numbers of Dicer-like (DCL) proteins from one (human) to four (Arabidopsis). Two DCL genes, Magnaporthe Dicer-like (MDL)-1 and -2, have been identified in the genome of the filamentous fungus Magnaporthe oryzae . Here we show that the knockout of MDL-2 drastically impaired gene silencing of enhanced green fluorescence protein (eGFP), by hairpin RNA, and reduced related siRNA accumulation to non-detectable levels. In contrast, mutating the other DCL, MDL-1 exhibited a gene silencing frequency similar to wild-type and accumulated siRNA normally. The silencing-deficient phenotype and loss of siRNA accumulation in the mdl-2 mutant was restored by genetic complementation with the wild-type MDL-2 allele. These results indicate that only MDL-2 is responsible for siRNA production and no functional redundancy exists between MDL-1 and MDL-2 in the RNA silencing pathway in M. oryzae. Our findings contrast a recent report in the filamentous fungus Neurospora crassa where two DCL proteins are redundantly involved in the RNA silencing pathway but are similar to the results obtained in a more distantly related organism Drosophila melanogaster where an individual DCL protein has a distinct role in the siRNA/miRNA pathways.
410. In vitro analysis on the assembly mechanisms of the Aspergillus CCAAT-box binding factor. Masashi Kato and Tetsuo Kobayashi. Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
The CCAAT-box is one of the most common cis-elements present in the promoter regions in eukaryotes. It has been shown to be important in the high-level expression of many genes in Aspergillus species. The factor which binds to the CCAAT-box, so-called the Hap complex, belongs to the NF-Y family and consists of three subunits, HapB, HapC and HapE. Previously we have shown that the number of HapE is strictly dependent on the number of HapE, suggesting the number of HapC could adjust that of HapE by forming stable heterodimers prior to assembly of the Hap complex1). In this study, we performed a reconstitution study with the recombinant subunits and 35S-labeled in vitro translated subunits. Significant amounts of the translated HapE were recovered in the insoluble fraction while the other two subunits were in the soluble fraction. However, HapE was recovered in the soluble fraction when the recombinant HapC was added in the translation reaction. These results suggest that the HapC subunit plays a chaperon-like role specific to the HapE subunit. Furthermore, pull-down assays of the labeled subunits with recombinant GST or MBP fusion subunits were also carried out to investigate the mode of subunit assembly of the Aspergillus Hap complex.
1) M. Kato et al. FEBS Lett. 512, 227-229 (2002)
411. Specific distribution in basidiomycete Lentinula edodes hymenophore of the transcripts of various L. edodes genes. S. Katsukawa1, S. Kaneko2, Y. Tanaka1, Y. Sakuragi1, T. Yamazaki1, Y. Miyazaki3 and K. Shishido1. 1)Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan; 2)Mitsubishi-Kagaku Institute of Life Sciences, Tokyo, Japan; 3)Department of Applied Microbiology, Forestry and Forest Product Research Institute, Tsukuba, Japan.
By using in situ RNA-RNA hybridization, we analyzed the distribution in Lentinula edodes hymenophore of the transcripts of several L. edodes genes, obtaining the following results. (1) Large amounts of the transcripts of ribonucleotide reductase small subunit gene (rnr2) and UMP-CMP kinase gene (uck1) are present in both hymenium and outer region of trama.The hymenium is the part for production of basidiospores and the outer region of trama is the region branching out into subhymenium (on the top of which hymenium is formed). (2) The ras transcript is present mostly in outer region of trama, while the transcript of trimeric G-protein alpha-subunit gene (ga1) is mostly in hymenium. The results suggest that rnr2 and uck1 genes play a role mainly in the nucleotide biosynthesis essential for production of basidiospores and for divergence of trama cells into subhymenium cells. The ras and ga1 play a role in divergence of mycelial cells and in spore-production respectively. Analysis of the transcripts of RecQ-type DNA helicase gene (recQ) and other developmental regulated genes is in progress.
412. Loss-of-function mutations in the Aspergillus nidulans xprG/phoG gene are associated with the loss of extracellular protease activity and a repressible acid phosphatase. Margaret E. Katz, Karen-Ann Gray, Stella M.H. Bernardo and Brian F. Cheetham. Molecular and Cellular Biology, University of New England, Armidale, NSW AUSTRALIA,
Two types of mutations in the A. nidulans xprG gene have been identified. The xprG1 mutation is semi-dominant and associated with increased extracellular protease activity, decreased ability to utilise some nitrogen sources and dark conidia. Strains carrying the recessive xprG2 mutation are protease-deficient and have pale conidia. The xprG gene is tightly linked to sarB, a gene involved in nitrogen metabolism. A clone containing the phoG gene was isolated by complementation of the xprG2 mutation. To confirm that phoG and xprG are allelic, a phoG- knockout was generated and proved to have a phenotype indistinguishable from the xprG2 mutant. Sequence analysis showed that xprG1 is a missense mutation while xprG2 is a frameshift mutation in the 9th codon of the gene. The xprG/phoG gene did not complement the sarB7 mutant and no mutations were found in the xprG coding region of a sarB7 mutant.
It has been reported that the phoG gene encodes or regulates nonrepressible acid phosphatase activity in A. nidulans and mutations in a N. crassa homologue, vib-1, show reduced nonrepressible acid phosphatase activity. In contrast, we have shown that loss-of-function mutations in the xprG/phoG gene are accompanied by the loss of a phosphate-repressible acid phosphatase.
The xprF and hxkC genes encode atypical hexokinases involved in the regulation of extracellular proteases. The xprG2 mutation suppresses loss-of-function mutations in the xprF and hxkC genes. XprF, HxkC and XprG contain putative nuclear localisation sequences and gfp-tagged XprF has been shown to be located predominantly in the nucleus. The yeast two-hybrid system is being used to test the hypothesis that the xprF and xprG genes encode interacting proteins.
413. Functional Analysis of Aspergillus nidulans alpha-COP. Eun-Joung Song, Ki-Hyun Kim and Hee-Moon Park. Chungnam National University, Korea
Our previous results showed that a mutation in alpha-COP, component of COPI vesicle, is responsible for the temperature dependent osmo-sensitive phenotype of Aspergillus nidulans and Saccharomyces serevisiae, which is mainly due to the defect in cell wall biogenesis. In S. cerevisiae the interaction between alpha-COP and epsilon-COP is required for the functional COPI vesicle and thus for normal cell wall biogenesis. We cloned a gene for alpha-COP/SodVICp interactor, epsilon-COP, in A. nidulans using Yeast Two-Hybrid Screening. Interacting domain analyses between two proteins revealed that each C-terminal domain of alpha-COP and epsilon -COP plays a crucial role in their interactions. Especially, 283-288 region of epsilon -COP was essential. It was also confirmed that the N-terminal WD40 domain of alpha-COP plays as cis-acting element in interaction with epsilon -COP mediated by the C-terminal domain of alpha-COP as the previous results of S. cerevisiae. We also tested whether the temperature dependant osmo-sensitive phenotype of alpha-COP mutant can be rescued by the epsilon-COP overexpression which is known to stabilize alpha-COP mutant.
414. Inducer-dependent nuclear localization of a Zn(II)2Cys6 transcriptional activator, AmyR. Tetsuo Kobayashi and Masashi Kato. Department of Biological Mechanisms and Functions, Nagoya University, Nagoya, Japan
AmyR is a Zn(II)2Cys6 transcriptional activator that regulates expression of the amylolytic genes in Aspergillus species. Subcellular localization studies of GFP fused AmyR revealed that the fusion protein preferentially localized to the nucleus in response to isomaltose, the physiological inducer of the amylolytic genes. The C-terminal domains of AmyR, designated as MH3 (residues 419-496) and MH4 (residues 516-542), were essential for sensing the inducing stimulus and regulating the subcellular localization, while the nuclear localization signals were identified within the N-terminal Zn(II)2Cys6 DNA binding motif. The MH2 domain (residues 234-375) located in the middle of AmyR was required for transcriptional activation of the target genes. Cytoplasmic localization of AmyR under non-inducing conditions does not seem to be caused by masking of the AmyR nuclear localization signal (NLS), since addition of an unrelated NLS to the NLS-mutated AmyR derivative retained isomaltose-dependent nuclear localization.
415. Post-translational regulation of AreA, the global transcriptional activator of nitrogen metabolism in Aspergillus nidulans. Koon Ho Wong, Richard B. Todd, Meryl A. Davis and Michael J. Hynes. Department of Genetics, University of Melbourne, Australia.
In Aspergillus nidulans, the global nitrogen regulatory gene areA, encoding a positively acting GATA transcription factor AreA, is required for activating genes involved in nitrogen metabolism. When a good nitrogen source like ammonium or glutamine is available, areA-dependent genes are expressed at a low level, while the expression of these genes is up regulated when only a poor nitrogen source, like alanine, is present (nitrogen limiting condition). This differential expression is controlled by areA transcript stability and the interaction of AreA with a negative regulator NmrA. A further increase in gene expression, which is not mediated by these two regulatory mechanisms, is observed under nitrogen starvation and correlates with AreA accumulation in the nucleus. Nuclear accumulated AreA is rapidly exported upon addition of a nitrogen source via the CrmA exportin. We therefore sought to investigate the importance of post-translational modifications on nuclear localization and function of AreA.
We have shown that AreA is multiply phosphorylated and its phosphorylation status differs under nitrogen sufficient, limiting or starvation conditions. A number of conserved potential phosphorylation sites on AreA have been mutated and assessed for their roles in AreA function. In addition, AreA contains a highly conserved small ubiquitin-like modifier (SUMO) modification site adjacent to a putative CrmA exportin binding motif. We have deleted the gene encoding the SUMO peptide and mutated the putative sumoylation site on AreA to address the involvement of sumoylation in the regulation of AreA.
416. Contribution of ammonia to the expression of pelB and PL secretion during pathogenicity of C. gloeosporioides. H.Kramer-Haimovich1, E.Servi1, Y.Okon2, T. Katan1, J. Rollins3 and D. Prusky1. 1Department of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, 2Department of Plant Pathology and Microbiology, Hebrew University, Faculty of Agriculture, Rehovot 76100, Israel, 3 Department of Plant Pathology, University of Florida, Gainesville, Florida 32611, USA.
Tissue alkalinization resulting from the accumulation of ammonia predisposes avocado fruit to attack by C. gloeosporioides by enhancing transcript expression of pelB and the secretion of pectate lyase a virulence factor expressed during fungal colonization. However recently obtained data suggests that nitrogen also affects pelB expression. Natural pH environment occurring in the fruit triggers the initiation of ammonia secretion, its accumulation at the infection court and the increase of the host pH during the necrotrophic attack. Ammonia secreted induced PL secretion by the pathogen. C. gloeosporioides nit- mutants lacking the capability to secret ammonia, could not secret PL at inducing pH conditions and showed reduced pathogenicity. However, infiltration of avocado fruits with ammonia enhanced C. gloeosporioides attack. Present results suggest that the natural pH changes occurring in the host tissue during ripening, may induce the secretion of ammonia by the pathogen, and the ammonification and alkalinization of tissue both together contributes to the expression of pelB, secretion of PL and decay development.
417. Analysis of differential gene expression across regions of the Aspergillus oryzae mycelium. Kumiko Masai1, Jun-ichi Maruyama1, Harushi Nakajima2, Kazutoshi Sakamoto3, Osamu Akita3, Katsuhiko Kitamoto1. 1Department of Biotechnology, University of Tokyo, Tokyo, Japan. 2Department of Agricultural Chemistry, Meiji University, Kawasaki, Japan. 3National Research Institute of Brewing, Higashi-Hiroshima, Japan.
Regions of differentiation in filamentous fungi mycelia can be observed during growth on solid substrates. This suggests that each of these regions may have different functions during the life cycle of the fungus and accordingly, their characteristics being controlled by gene(s) specifically expressed in each region. Here, we propose a mycelial-scale gene expression analysis approach to determine the gene(s) responsible for this phenomenon in the industrially important filamentous fungus Aspergillus oryzae. Upon observation of a mycelium of A. oryzae, three distinct regions were detected. They were designated as tip, white and basal, based on their visual characteristics. Tip cells growing at the edge of the colony comprised the tip region, whereas aerial hyphae and developed conidiophores were observed in the white and basal regions, respectively. Differential expression of genes in the three regions was detected by RT-PCR in the preliminary analysis. Microarray analysis using the A. oryzae cDNA chip was performed to identify the genes that are specifically expressed in each region. Genes involved in the translation process were abundant in the tip region, whereas, genes encoding transporters were expressed at high levels in the basal region. This result supports the assumption that the genes expressed in each region reflect the characteristics observed in each region. Thus, this study has laid the basic foundation in the investigation of what makes each region unique. Further characterization of each region and functional analysis of selected genes will be presented.
418. The MpkB MAP kinase plays a central role in signaling for the sexual development of Aspergillus nidulans. Sei-Jin Lee§1, Ji-Young Kang, Mira Jin, Dong-Min Han2, Keon-Sang Chae and Kwang–Yeop Jahng*1. Division of Biological Sciences, Institute for Basic Sciences, Institute for Molecular Biology and Genetic Engineering, 1Korea Basic Science Institute Jeonju Center, Chonbuk National University, Chonju 561-756, Republic of Korea, 2Division of Life Sciences, Wonkwang University, Iksan 570-749, Republic of Korea
Two genes encoding a MAP kinase homologue, designated as mpkB and mpkC, were isolated from Aspergillus nidulans by PCR with degenerated primers. The amino acid sequence of MpkB showed 92% - 93% identity to the MAP kinases of diverse phytopathogenic fungi that are involved in differentiation and pathogenicity. The amino acid sequence of MpkC showed 77% - 85% identity to the stress activated protein kinases (SAPKs) of filamentous fungi that play a role in responding to environmental stresses. Deletion of the mpkB gene caused failure in forming the sexual organ cleistothecium in any condition that could induce sexual development, suggesting that MpkB may be essential for transmitting the signals for sexual development. In addition, deletion mutant of mpkB showed a slower hyphal growth and an aberrant conidiophore morphology. Deletion and over-expression mutants of mpkC showed no detectable phenotypes under any conditions provided by various external stresses. Although the mpkB and mpkC genes were constitutively transcribed through entire life span, transcripts of both genes showed varieties in size and abundance according to the developmental stages. The mpkB transcript was hardly detected in the deletion mutant of veA in contrast to highly accumulated in the over-expression mutant of veA. Deletion of mpkB caused a drastic reduction of medA and steA transcript accumulation during sexual reproduction as well as the stuA transcript during both asexual and sexual developments. Taken together these results, we propose that the MpkB MAP kinase has a central role in diverse signaling pathways that mediate sexual development in A. nidulans.
419. A MAP Kinase Pathway Essential for Mating and Contributing to Vegetative Growth in Neurospora crassa. Dan Li, Piotr Bobrowicz, Heather H. Wilkinson and Daniel J. Ebbole. Department of Plant Pathology and Microbiology, Texas A & M University, College Station, Texas 77843, United States
MAP kinases homologous to Saccharomyces cerevisiae Fus3p/Kss1p have been identified in several plant pathogenic fungi and found to be required for pathogenicity and sexual reproduction. To better understand the role of MAP kinase signaling in development in Neurospora crassa, and to identify downstream target genes of the pathway, we isolated, cloned, and disrupted the FUS3 homolog mak-2 and the STE12 homolog, pp-1. The mak-2 and pp-1 deletion mutants have reduced growth rate, produce short aerial hyphae, and fail to develop protoperithecia. In addition, ascospores carrying null mutations of either gene are inviable. Subtractive cloning was used to isolate several genes that display reduced expression in the mak-2 mutant. Expression of some of these genes is protoperithecia-specific and three of them are part of a gene cluster potentially involved in the production of a polyketide secondary metabolite. The ORF encoding a putative polyketide synthase in this gene cluster was partially deleted and phenotype of the mutant was carefully studied. Microarray analysis was used to extend the analysis of gene expression in mak-2 and pp-1 mutants. The role of the MAP kinase pathway in both sexual and asexual development as well as secondary metabolism is consistent with the dual regulation of the mating process and pathogencity/invasive growth observed in many fungal pathogens.
420. Cold induced gene expression in Botrytis cinerea. Amnon Lichter, Ayelet Ezra, H-W. Zhou, Orit Dvir and Anna Danshin. ARO, The Volcani Center, Israel
Cold storage is the major postharvest tool to prevent fungal decay. As evident from its potential to develop on grapes during extreme cold storage and cause gray mold, Botrytis cinerea is well adjusted to low temperature. This condition may require optimization of cellular processes as well specialized functions which are redundant at high temperature. The growth kinetics of Botrytis cinerea were determined at a temperature of zero compared to 20C, in different media and with and without conidial germination at zero. We have approached the mechanism of cold fitness by molecular cDNA subtraction following exposure of germinating spores to low temperature. A subset of clones obtained by this procedure were sequenced and screened for differential expression at low or high temperature using cDNA probes. A subset of those clones were verified to be differentially expressed by northern analysis. The limited number of clones with known sequence homology permit a first glance into molecular mechanisms that makes this fungus a successful postharvest pathogen.
421. Identification of genes differentially expressed in response to acidic induction in Trichophyton rubrum. Henrique C.S. Silveira, Mônica S. Ferreira-Nozawa, Antonio Rossi and Nilce M. Martinez-Rossi. Universidade de São Paulo, FMRP, Ribeirão Preto, Brazil.
Trichophyton rubrum, a filamentous fungus that causes infections in human skin and nails, is recognized as cosmopolitan and is one of the most frequently encountered dermatophytes. Although several factors contribute to the pathogenicity of dermatophytes, the successful initiation of infection depends on the capacity of the infecting fungus to sense and overcome the acidic pH of the skin. Thus, it is important to understand the metabolic responses that govern homeostatic and extracellular pH sensing in dermatophytes. Here, we describe genes differentially expressed in response to acidic pH by employing the suppression subtractive hybridization (SSH) approach. The tester and driver cDNAs were obtained from mycelium respectively derepressed at pH 5.0 and at pH 8.0. Out of a total of 300 cDNA clones analyzed by dot-blot macro-arrays, we confirmed 96 clones differentially expressed at pH 5.0, which represented 21 different genes. Based on BLAST homology, the clones selected from these libraries are largely of fungal origin. The hypothetical proteins represent 57% of the total, and the putative proteins identified are involved in metabolism, cell differentiation, cell cycle, and resistance mechanisms. These studies should provide data that will be useful for identifying the metabolic machinery essential for initiating infection by T. rubrum.
Financial support: FAPESP, CNPq, FAEPA and CAPES.
422. Identification of potential regulatory RNAs. Tami McDonald, Zhihong Zhang, and Fred Dietrich. University Program in Genetics, Duke University, Durham, North Carolina USA.
Gene regulation is accomplished in fungi by several mechanisms. One intriguing class of gene regulators is comprised of regulatory RNAs. There are three main mechanisms of regulation by regulatory RNAs: Promoter competition, in which two nearby or overlapping promoters compete for the polymerase or transcription factors; Transcription interference, in which transcription of an upstream RNA facilitates read-through across the promoter of the downstream gene; and RNA-mediated control in which RNA transcripts bind to regions to prevent transcription. We are investigating the use of 5' SAGE to identify additional small RNA species in fungi. This method allows high throughput identification of transcription start sites. While this method is used primarily for identification of the 5' transcription start sites of protein coding genes, we have also observed start sites corresponding to transcription of what are possibly small, previously un-identified RNA molecules. We are investigating several of these potential regulatory molecules, including one in the upstream region of the mitochondrial transporter ODC2, suggesting an upstream microORF, and several tags clustering on the strand of DNA complementary to ASE1, a cell-cycle regulated microtubule-associated protein (MAP) found at spindle fibers, suggesting a reverse microORF. Preliminary northern data suggests that a RNA product is formed by microORF upstream of ODC2.
423. Expression and characterization of Trichoderma reesei beta-xylosidase in Aspergillus oryzae. Sandra T. Merino, Hanshu Ding, and Joel R. Cherry, Novozymes Biotech, Davis, CA.
L-Arabinose residues, in furanose form, are widely distributed in plant tissue heteropolysaccharides, such as arabinans, arabinogalactans and arabinoxylans. The presence of arabinose as side groups can restrict enzymatic hydrolysis of hemicellulose. The T. reesei beta-xylosidase has been shown to have alpha-L-arabinosidase activities against small synthetic model substrates (Margolles-Clark et al., 1996, Applied and Environmental Microbiology, Vol. 62, No. 10, p. 3840-3846). We describe the cloning and expression of the T. reesei beta-xylosidase gene in the host, Aspergillus oryzae. Broth samples were analyzed for activity and the results show activity on the substrate xylan and even higher activity on p-nitrophenyl-xylose when compared with a positive control.
424. The Aspergillus niger Unfolded Protein Response Element. Harm Mulder, Igor Nikolaev and Susan Madrid. Danisco Innovation, Copenhagen Denmark.
Eukaryotic cells respond to the accumulation of unfolded proteins in the ER by activating a pathway known as the unfolded protein response (UPR), that culminates in the induction of a set of genes with functions affecting nearly every stage of the secretory pathway. The promoters of UPR target genes contain an unfolded protein response element (UPRE), which confers the stress inducibility to the gene, via an interaction with the transcription activator HACA. In the promoters of the ER-stress responsive genes bipA, cypB, pdiA, prpA, tigA and hacA a consensus sequence was identified, which was located close to the transcription start site of the gene ( in vitro selection procedure, an optimal binding site for HACA was isolated. This sequence, ACACGTGTCCT, resembles the UPRE but lacks the spacer nucleotide, and in vivo it behaves as a more potent cis-acting element. Two UPREs were identified in the upstream region of the hacA gene itself, an interesting finding given the fact that ER stress is associated with the appearance of shorter hacA transcript. The mechanism behind this truncation of the hacA mRNA upon ER stress is still unknown. However, the presence of those two UPREs could indicate a possible role for HACA itself in its transcriptional regulation under ER stress, a hypothesis we are currently investigating.
425. RNA silencing as a tool for exploring gene function in ascomycete fungi. Hitoshi Nakayashiki, Shugo Hanada, Nguyen Bao Quoc, Naoki Kadotani, Yukio Tosa, Shigeyuki Mayama. Laboratory of Plant Pathology, Kobe University, Kobe, 657-8501 Japan.
RNA silencing provides potentially versatile reverse genetic tools for exploring gene function in the post-genomics era. We have developed a pHANNIBAL-like silencing vector, pSilent-1, for ascomycete fungi, which carries a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression with a spacer of a cutinase gene intron from the rice blast fungus Magnaporthe oryzae. Application of pSilent-1 to two M. oryzae endogenous genes, MPG1 and polyketide synthase-like gene resulted in efficient silencing of the genes. RNA silencing was also induced by a pSilent-1-based vector in Colletotrichum lagenarium at a slightly lower efficiency than in M. oryzae, indicating that this silencing vector should provide a useful reverse genetic tool in ascomycete fungi.
426. Redundant and distinct roles of two PKA catalytic subunits in Aspergillus nidulans. Min Ni, Sara Rierson, Jeong-Ah Seo and Jae-Hyuk Yu. University of Wisconsin, Madison WI 53706 USA
Previously, PkaA, a cAMP-dependent protein kinase (PKA) catalytic subunit, was shown to play an important role in transducing a heterotrimeric G protein mediated vegetative growth signaling in Aspergillus nidulans. We have identified and characterized the pkaB gene encoding the second PKA catalytic subunit. Although the absence of pkaB alone did not cause clear phenotypic alterations, deletion of both pkaA and pkaB is found to be lethal, indicating that PkaB and PkaA constitute the sole PKA catalytic subunits. Overexpression of pkaB increased hyphal proliferation in wild type, and partially restored colony growth in pkaA deletion mutant, suggesting that PkaB also functions in vegetative growth signaling as a secondary PKA subunit. While an additional copy of pkaB rescued spore germination defects resulted from deletion of pkaA on glucose medium, it blocked spore germination in the medium without carbon source, indicating that PkaB may negatively regulate spore germination via sensing carbon sources. Furthermore, deletion of pkaA or up-regulation of pkaB resulted in the reduced tolerance of vegetative hyphae against oxidative stress, suggesting that PkaB and PkaA play opposite roles in responding to certain stresses. Taken together, we propose that PkaB is the secondary PKA catalytic subunit that plays a redundant role in vegetative growth signaling, but a distinct role in regulating germination, development, and stress response in A. nidulans.
427. Gene expression upregulated by a conidiogenesis patterning regulator Acr1 in Magnaporthe grisea. Marie Nishimura. National Institute of Agrobiological Sciences, Tsukuba, 305-8602, JAPAN.
Rice blast disease, caused by the filamentous fungus Magnaporthe grisea, is one of the most devastating diseases to global rice production. To understand the regulation mechanism of conidiation in M. grisea, a morphological mutant, acr1, has been studied. Acr1 is a functional homolog of MedA, a conidiophore-developmental regulator in Aspergillus nidulans. The acr1 mutants produce chains of elongated conidia in a head-to-tail array, while the wild type conidia are produced in a sympodial array. In acr1, appressoria production is greatly reduced and the appressoria are defective in plant penetration. The reductions in the appressorium formation and penetration rates in the mutant are not complemented by the addition of exogenous cAMP. To investigate the genes upregulated by Acr1, an oligo DNA microarray, and nylon membranes arrayed with subtraction libraries constructed from conidial mRNA of acr1 and its isogenic wild-type strain Guy11 were used. Results from these analyses were further confirmed by RT-PCR. The expression level of Saccharomyces cerevisiae PCL1 - like G1 cyclin was reduced in acr1. Expression of several genes related to secondary metabolism were also controlled by Acr1. Recent progress on analysis of these genes will be presented.
428. Rho1 in Ustilago maydis affects mating, cell morphology, and perhaps viability. Zhanyang Yu and Michael H. Perlin*. University of Louisville, Louisville, KY 40292, USA
The highly conserved Rho/Rac small GTPase family regulates multiple signal transduction pathways involved in cell morphogenesis, cell cycle, cell-cell communication, and mating response. In U. maydis, the pathogen of maize, at least 25 putative members of this family have been identified. Several of these have been well characterized for their roles in cell separation or cell polarity (e.g., Cdc42 and Rac1). Others have been shown to be significant participants in regulating filamentous growth, pheromone response, and pathogenicity (i.e., Ras1 and Ras 2). Less is known about the function of the Rho homologues in this organism. In the fission yeast, S. pombe, Rho1 is essential for cell viability and cell polarity. From U. maydis we cloned uro1, encoding a Rho1 homologue. The genomic version contained a 400-bp intron relative to the cDNA coding region of 950 bp. The predicted amino acid sequence had 80% identity with Rho1 of S. pombe, as well as 47.9% and 49% identity with the Cdc42 homologues from S. cerevisiae and U. maydis, respectively. This identity was particularly strong in the two highly conserved GTP binding/hydrolysis motifs. The cDNA fragment was expressed in wild-type U. maydis strains and a variety of mutant strains. Over-expression of uro1 reduced the mating efficiency on YPD-charcoal plates; the pheromone response was also reduced, especially for a2 cells, where it is normally the greatest. Attempts to disrupt the uro1 gene were unsuccessful for either a1 or a2 haploid strains. On the other hand, successful disruption of one copy of the gene was achieved in two different diploid strains. This suggests that the uro1 gene, in addition to its role in regulating mating, may also be required for viability. Moreover, disruption of a single copy of uro1 in a diploid strain produced a dominant negative phenotype, eliminating the fuz reaction on charcoal media.
429. Slave Oscillators in Neurospora's Circadian Clock: a Role for RNA-binding Proteins? Nathan Price-Lloyd & Christian Heintzen. University of Manchester, Lancashire, UK
Metronomic changes in the physical environment have led to the evolution of correspondent molecular clocks. Oscillating with a period of approximately 24 hours, circadian clocks allow pre-adaptation to daily rhythmical changes concurrent with the Earth's rotation. In 1960, a hierarchical system of ‘master' and ‘slave' oscillators was proposed by Pittendrigh, whereby a central clock can relay information via a number of subordinate cogs to a variety of outputs. This model allowed for increased plasticity in clock-generated signal pathways, and the option to evolutionarily tinker with downstream components, bypassing the central clock.
Increasing evidence suggest that Neurospora's clock functions via such a model. Importantly, some plant RNA-binding proteins (RBPs) have been shown to act as subordinate oscillators, with evidence from fruit-flies and humans also showing circadian regulation of RBPs.
We BLASTed the Neurospora on-line database with the conserved regions of the plant, fruit-fly, and human RBPs, and recognised 43 Neurospora RBPs. One is of known biochemical function, one is putatively homologous to the plant RBPs, and several others have defined functional domains. We have begun characterising transcript levels in terms of temporal expression and reaction to zeitgebers. Prime candidate RBPs have been knocked out of the genome by homologous gene replacement. Rhythmic analysis of phenotype will be presented.
430. Regulation of cuticle and cell wall-degrading enzyme expression in Fusarium oxysporum . F.J. Calero-Nieto, A.L. Martínez-Rocha, A. Di Pietro, C. Hera & M.I.G.Roncero. Departamento de Genetica, Universidad de Cordoba, Campus Rabanales Ed. C5, 14071Cordoba, Spain
Degradation of different plant polymers by fungal pathogens requires the coordinated action of numerous hydrolytic enzyme activities. Vascular pathogens such as Fusarium oxysporum, have to overcome primary physical barriers of the plant cell wall in order to reach the vascular elements and to accomplish their rapid colonization. On the other hand, leaf pathogens have to degrade cutin, an insoluble lipid polyester matrix covering the surface of all aerial parts of plants. The implication of cell wall-degrading enzymes in vascular wilt disease caused by F. oxysporum has been suggested during penetration of the different layers of the root cortex to reach the vascular system and during colonization of the host by spreading upward through the xylem vessels. Whereas the role of cutin degrading enzymes in vascular wilt disease is currently unknown. Previous studies showed that F. oxysporum secretes an array of extracellular degradative enzymes capable of hydrolyzing different components of the plant cell wall and cuticle, including polygalacturonases, pectate lyases, xylanases and cutinases. Targeted disruption of individual corresponding structural genes showed no significant reduction in virulence of F. oxysporum on tomato plants, possibly due to functional redundancy. In order to elucidate the role of xylanases and cutinases during the pathogenesis of F. oxysporum, and to establish their common regulatory mechanisms, we have identified and characterised two transcription factors, XlnR and Ctfa, known to regulate the expression of fungal xylanase and cutinase genes, respectively. Two types of mutants have been created by genetic transformation of F. oxysporum f.sp. lycopersici wild type strain 4287: loss-of-function mutants (DxlnR and Dctfa) were created by targeted gene replacement, and mutants harbouring a constitutively expressed allele of ctfa and xlnR were obtained by fusing the coding regions to the gpdA promoter. Conidiation, growth rates and virulence behaviour of these mutants on tomato plants were similar to those of the wild type strain. The expression level of xylanase and cutinase structural genes have been determined by real time quantitative PCR. Total extracellular xylanase and esterase activities were also determined for each pair of mutants.
431. The calpain-like protease PalB from A. nidulans is required for retarding the proteolytic processing of transcription factor PacC. Carlos J. Ono, Mônica S. Ferreira-Nozawa, Nilce M. Martinez-Rossi and Antonio Rossi. Universidade de São Paulo, FMRP, Ribeirão Preto, Brazil.
The conserved PacC signal transduction pathway mediates many metabolic events involved in ambient pH sensing in A. nidulans, and it is widely accepted that it governs only the response to neutral-to-alkaline pHs. The pacC gene codes for a Zn-finger transcription factor whose transcription is itself induced under alkaline growth conditions. The pal genes (palA, B, C, F, H, and I) are putative members of a signaling cascade, whose function is presumed to promote the proteolytic activation of PacC. Thus, the current model states that the full-length version of PacC (PacC72) is activated at alkaline pH by two sequential proteolytic steps that remove the C-terminal negatively acting domain. The conversion of PacC72 to PacC53 is PalB- and pH-dependent, whereas the conversion of PacC53 to PacC27 is pH-independent. It is assumed that the PacC27 processed form is perhaps the sole functional form of PacC at alkaline pH. This model implies that inactivation of any of the pal genes should lead to an abundant expression of PacC72 irrespective of the extracellular pH. To test this hypothesis we purified PacC as briefly described here: the truncated PacC protein, which contains the three zinc-fingers of gene pacC, was expressed in E. coli, purified by affinity chromatography, and polyclonal antibodies were raised in albino male rabbits. The anti-PacC antibodies was purified, chemically cross-linked to hydrazide-Sepharose resin, and used for purification of PacC by immunoaffinity chromatography. We present evidence that proteolytic processing of PacC72 occurs earlier in the palB7 mutant grown under acidic conditions. Furthermore, proteolysis of PacC is extensively detected in the palB7 mutant grown under alkaline conditions, where PacC72 should be abundant. Thus, the full-length version of PacC was abundantly detectable only in strains in which palB is functional.
Financial support: FAPESP, CNPq, FAEPA and CAPES.
432. Aspergillus oryzae atfB encodes a transcription factor, which is required for stress tolerance of conidia. Kazutoshi Sakamoto. NRIB, Higashi-Hiroshi, Japan
In the solid-state culture, Aspergillus oryzae exhibits phenotypes such as the high production of enzymes, conidiophore development, and the production of various kinds of secondary metabolites. Though these characteristics should involve various gene expressions, the only a few regulatory systems have been understood. From the EST database of A. oryzae, we found two genes encoding transcription factors that show high homology to atf1 of Shizosaccharomyces pombe and named atfA and atfB respectively. The atfB gene was upregulated at the late phase of solid-state culture, while atfA gene was constitutively expressed. We tried to identify atfB target genes by microarray analyses. In the atfB-overexpression strain, about 30 genes including catalase gene (catA) were upregulated. Almost all of these genes were upregulated at the late phase of solid-state culture in wild type strain. Furthermore the expression of these genes were downregulated in the atfB-disrupted strain. The disruptant strain grew normaly but its conidia lost several stress tolerances.
433. Characterization of lentinan degrading exo-glucanase-encoding gene exg2 from Lentinula edodes. Y. Sakamoto1, K. Minato2, S. Kawakami2, M. Mizuno2, T. Irie1 and T. Sato1 1Iwate Biotechnology Research Center, Iwate, Japan. 2Department of Agriculture, Kobe University, Hyogo, Japan
Lentinan, which is a beta-1, 3-linked-D-glucan with beta-1, 6 branches isolated as anti-tumor active-substrate from Lentinula edodes, is purified from fresh fruit-bodies and marketed for clinical use. However, it is known that lentinan content decreases during post-harvest preservation. Lentinan contents were measured by ELISA using anti-lentinan anti-bodies, and it was revealed that lentinan degradation is mainly occurred in gill. We also revealed that beta-glucanase activity increased during post-harvest preservation. Then we cloned two exo-glucanase encoding genes, exg1 and exg2 from L. edodes. The exg1 gene had 1.2 kbp cDNA length, and calculated molecular weight and pI value of the encoded protein was 44 kDa and 4.36, respectively. Putative amino acid sequence of the exg1 displayed 67% identity to AbEXG1 of Agaricus bisporus. Two conserved glutamic acids that are within the catalytic active site in yeast exo-beta-1, 3-glucanase-encoding genes were conserved in exg1 of L. edodes. The exg2 gene had 2.4 kbp cDNA length, and calculated molecular weight and pI value of the encoded protein was 79 kDa and 4.66, respectively. Putative amino acid sequence of the exg2 displayed 40% identity to exgS of Aspergillus saitoi. The exg2 gene had two imperfect copies of motif that are conserved in ascomycetous exo-glucanases. Transcription levels of the exg1 and exg2 genes were higher in stipe than that in pileus of young fruiting bodies. Transcription level of the exg1 gene decreased, but the exg2 gene increased after harvest. Western blot analysis showed that EXG2 protein expression increased after harvest. Purified EXG2 protein degraded lentinan, therefore, we concluded that the exg2 gene is a lentinan degrading exo-glucanase encoding gene.
434. Protein Production and Unfolded Protein Response in Fermentations of Trichoderma reesei and its Transformant Expressing Endoglucanase I with a Hydrophobic Tag. Anna Collén1, Michael Bailey, Markku Saloheimo, Merja Penttilä and Tiina Pakula. VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland. 1Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden. Present address: AstraZeneca R&D Mölndal, S-431 83 Mölndal, Sweden
The effect of induction of protein production was studied in bioreactor cultures of the T. reesei strain Rut-C30 and its transformant expressing endoglucanase I (EGI, Cel7B) fused with a hydrophobic tag. The peptide tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The first phase of the bioreactor cultivations was carried out on glucose containing minimal medium. At the stage when glucose was nearly depleted, the medium was supplemented with rich medium containing lactose as a carbon source to induce production of cellulases. The transformant produced somewhat less secreted protein and cellobiohydrolase I (CBHI, Cel7A) activity than the parental strain. Western analysis of intracellular proteins showed that the fusion protein EGICORE-P5(WP)4 accumulated inside the cell, indicating impaired secretion of the protein. Two-dimensional gel analysis suggested that the fusion protein was possibly trapped early in the secretory pathway. The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdi1, and the level of the activated hac1 transcript encoding the UPR transcription factor, increased at the same time with an increase in the transcript levels of cellulase genes, suggesting UPR activation in response to cellulase induction. However, only a minor increase in pdi1 and bip1 transcript level was observed in the transformant expressing the fusion protein compared to its parental strain. In addition, slightly lower CBHI production and cbh1 mRNA levels were measured in the transformant as compared to the parental strain, indicating activation of the novel repression mechanism of genes encoding secreted proteins in response to secretion stress, RESS (repression under secretion stress).
435. ENVOY, a novel PAS/LOV domain protein, regulates cellulase gene transcription dependent on light and connects carbon source signaling to light response in Hypocrea jecorina (anamorph Trichoderma reesei). Monika Schmoll1, Lisa Franchi2, and Christian P. Kubicek1. 1Institute of Chemical Engineering, Vienna University of Technology, Getreidemarkt 9/1665, A-1060 Wien, Austria. 2Universita' di Roma "La Sapienzia", Policlinico Umberto I, Viale Regina Elena 32, 00161 Roma, Italy
Envoy, a PAS/LOV domain protein with high similarity to the Neurospora light regulator VIVID and links cellulase induction by cellulose to light signaling. An env1PAS- mutant grows slower in the presence of light but remains unaffected in darkness as compared to the wild-type strain QM9414. env1 transcription rapidly responds to a light pulse, this response being different upon growth on glucose or glycerol, and it encodes a regulator essential for H. jecorina light tolerance. Despite their similarity, env1 could not compensate for the lack of vvd function. Induction of cellulase formation in H. jecorina by cellulose is enhanced by light in the wild-type strain as compared to constant darkness, whereas a delayed induction in light and an initially stronger induction of cellulases followed by a shut down of expression was observed in the env1PAS- mutant in constant darkness. Light does not lead to cellulase expression in the absence of an inducer.
Envoy thus connects light response to carbon source signaling and consequently light must be considered as an additional external factor influencing gene expression in this fungus.
436. New components of nitrogen regulation in Fusarium fujikuroi: the role of NPR1-Gf und CPC1-Gf. Birgit Schönig, Bettina Tudzynski. Westfälische Wilhelms-Universität Münster, Schlossgarten 3, 48149 Münster, Germany
The rice pathogen Fusarium fujikuroi produces gibberellins (GAs), a group of economically important phytohormones. The production of GAs is subject to nitrogen catabolite repression: the central regulator of nitrogen repression, AREA-Gf, had been shown to directly activate the transcription of 6 out of 7 biosynthetic genes under nitrogen limitation conditions. Besides homologues to other key elements of nitrogen regulation in Saccharomyces cerevisiae, we are interested in homologues of the NPR1 protein and the effector of cross pathway control in filamentous fungi, CPC1 and their influence on secondary metabolism.
The NPR1 protein kinase regulates sorting and stability of nitrogen permeases and, as shown recently, also the transport of the AREA homologue GLN3 into the nucleus. In order to show if the NPR1 homologue in Fusarium fujikuroi is involved in nitrogen regulation and GA production, we cloned and deleted the corresponding gene, npr1-Gf.
The central effector of cross pathway control in filamentous fungi, CPC1, has been shown not only to affect the availability of amino acids, but also secondary metabolism, stress response and pathogenicity. To examine if the orthologue in F. fujikuroi is involved in general secondary metabolism control under amino acid starvation, we cloned and deleted the genomic copy, cpc1-Gf. Macroarray experiments revealed several target genes of cpc1 not described in filamentous fungi before. The effect of gene replacement on GA production is under investigation.
437. Siderophore biosynthesis is negatively regulated by SreA in Aspergillus fumigatus. Markus Schrettl, Claudia Kragl, and Hubertus Haas. Department of Molecular Biology, Innsbruck Medical University, Austria.
Virtually all organisms require iron for their growth, because this metal is indispensable for various cofactors, e.g. heme moieties and iron-sulfur clusters. Induced by iron starvation, most fungi excrete siderophores - low molecular-mass ferric iron chelators - in order to mobilize extracellular iron. The filamentous ascomycete A. fumigatus produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin as an intracellular iron storage compound. Recently it was shown, that the siderophor system is absolutely required for virulence of this fungus and therefore regulation of this iron uptake system is of great interest. Inactivation of the GATA-type transcription factor SreA caused derepression of the reductive iron uptake system, of extracellular siderophore biosynthesis and accumulation of intracellular siderophores during iron-replete condition. Such mutants displayed increased sensitivity to the redox cycler menadione. Moreover, an increase of extracellular iron availability caused a decrease of radial growth in these mutants. These data suggest that deregulation of the iron uptake system leads to increased oxidative stress, presumably via Fenton/Haber-Weiss-chemistry. Taken together, the data show that SreA is a key regulator of iron uptake in A. fumigatus and gene profiling of the mutant will help to identify genes involved in iron metabolism.
This work was supported by Austrian Science Foundation.
438. Alternative initiation of translation and light-specific phosphorylation yield two forms of the essential light and clock protein White Collar-2. Carsten Schwerdtfeger, Jennifer J. Loros, and Jay C. Dunlap, Dartmouth Medical School, Genetics, Hanover, NH, USA
Blue light and temperature are important environmental cues and also play an important role in the Neurospora crassa circadian clock. To date, the core components of the circadian clock in N. crassa are WHITE COLLAR-1 (WC-1), WHITE COLLAR-2 (WC-2), and FREQUENCY (FRQ) proteins with several kinases, phosphatases, and a ubiquitin ligase also identified. WC-1 and WC-2 are both GATA-type zinc finger, DNA binding transcription factors with a nuclear localization signal and PAS (Per, Arnt, Sim) dimerization domains (Ballario, et al. 1996, Taylor and Zhulin, 1999).
Here we demonstrate the detection of a short form of WC-2 (sWC-2), which emerges from the same transcript as long WC-2 (lWC-2), arising via alternative initiation. sWC-2 is missing ~15kDa from its N-terminus and migrates at around 45kDa. WC-2 antibodies show sWC-2 to be predominantly a nuclear protein that undergoes a hyperphosporylation in response to blue light. By generating a mutant that makes sWC-2 only we find the light-driven transcriptional responses of the albino genes and frq to be at normal levels. However, the circadian oscillation is abolished. Furthermore, the lack of lWC-2 leads to a change in light adaptation and the high light response similar to the vvd (vivid) mutant. Our results suggest different roles for lWC-2 and sWC-2 in clock and blue light signal transduction in Neurospora crassa.
439. The effect of viral infection on gene silencing in Cryphonectria parasitica. Gert C. Segers and Donald L. Nuss, UMBI-CBR, University of Maryland, College Park, MD 20742.
The C. parasitica / hypovirus system provides unique opportunities to study the effect of virus infection on gene silencing as well as antiviral responses in fungi. To induce gene silencing in C. parasitica, a vector (pGS) that allows expression of a hairpin construct was developed. pGS contains multiple cloning sites on either side of an intron from the C. parasitica GPD gene, the A. nidulans GPD promoter to drive expression and the C. parasitica GPD terminator to terminate transcription. Transformation of pGS containing EGFP (pGS-EGFP) into an EGFP expressing C. parasitica strain resulted in transformants of which app. 50% were silenced, as judged by monitoring EFGP fluorescence. EGFP-silenced strains showed severe reduction in EGFP transcript and protein accumulation. Subsequent infection of EGFP silenced strains with a hypovirus resulted in a moderate increase in EGFP transcript levels, suggesting a negative effect of virus infection on gene silencing.
Hypoviral protein P29 shares similarity with HC-Pro of potyviruses, a known suppressor of silencing in plants. Transformation of a C. parasitica strain expressing both EGFP and a p29 transgene with pGS-EGFP failed to silence EGFP. This suggests that P29 can preventively suppress gene silencing. Experiments are underway to determine the mechanism of P29 suppression of silencing.
Genes encoding two Dicer homologues (DCL1 and DCL2) and one Argonaute homologue (AGL1) have been isolated, and generation of corresponding null mutants is in progress. The effect of AGL1 gene deletion on viral accumulation will be determined.
440. Structural and functional analysis of Cryptococcus neoformans hybrid histidine kinase gene CnNIK1. Kiminori Shimizu, Antra Drivinya, Akira Yoshimi1), Chihiro Tanaka1), Susumu Kawamoto. Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Japan, 1)Graduate School of Agriculture, Kyoto University, Japan.
Hybrid histidine kinases (HHKs) are known to be involved in drug resistance and osmosensitivity in filamentous fungi. We cloned a gene CnNIK1 encoding an HHK from a human pathogen Cryptococcus neoformans. Comparison of genomic DNA and cDNA sequences revealed that the gene consists of 4149-bp open reading frame coding for 1383 amino acid residues interrupted by 13 introns, one of which is present in the 5f untranslated region. Homology searches against GenBank database were carried out to identify conserved sequences such as HAMP domains, a phosphoacceptor histidine residue, an ATP binding domain and a response regulator domain, which are characteristics of HHK genes belonging to NIK1 family found in other filamentous fungi including Neurospora crassa NIK1 and Cochliobolus heterostrophus Dic1. A CnNIK1 disruption strain (delta CnNIK1) was created in order to study its biological function in C. neoformans. The MIC of delta CnNIK1 strain against a fungicide fludioxionil was higher than 48 ug/ml, whereas that of wild type strain was 3 ug/ml, suggesting that CnNIK1 is responsible for this drug sensitivity. However, delta CnNIK1 strain did not show any growth defect in high osmotic conditions, which phenotype is seen in many fungal species lacking genes of NIK1 family, suggesting that the function of CnNIK1 differs from those in other filamentous fungi. Further characterization of the strain is underway.
441. Genetic Regulation of Conidiation in Trichoderma hamatum. Johanna Steyaert1, Margaret Carpenter1, Alison Stewart1, Travis Glare2 & Hayley Ridgway1. 1National Centre for Advanced Bio-Protection Technologies, PO Box 84, Lincoln University. 2AgResearch Ltd., PO Box 60, Lincoln
Trichoderma spp. are ubiquitous soilborne ascomycetous fungi with superior biocontrol capabilities towards fungal phytopathogens. This capability has led to massive exploitation and currently Trichoderma spp. represent one third of all commercial fungal biocontrol agents sold globally. Commercial fungal biocontrol products involve bulk preparations of conidia, however considerable variability in conidiation rates exist between biocontrol agents. This variation can restrict the suitability of a particular strain for production. The majority of studies on Trichoderma conidiation have focused on the species T. viride and T. atroviride. These species form conidia in response to blue and near-UV light and/or nutrient deprivation. Conidiation proceeds in a highly co-ordinated fashion, however relatively little is known about the genetic basis of Trichoderma conidiation. In this study, conidiation in the lesser known biocontrol species T. hamatum is being investigated using a combined morphological and molecular approach. A selection of genes implicated in sporulation and the blue-light responses are currently being isolated and characterised from T. hamatum. Two genes, phr1 and cmp1, which were isolated previously from T. atroviride will be used as early and late markers of gene expression during the photoresponse in T. hamatum. Their expression will be used to define time points for harvesting comparable stage-specific RNA from T. hamatum and T. atroviride. Using degenerate PCR putative sporulation gene orthologues, rcoT, blr1 and blr2, have been identified in T. hamatum. Gene disruption studies on the orthologues are currently underway. Results of these studies, in addition to sequence and expression analysis, will be presented and discussed in relation to the current knowledge of the molecular basis of conidiation in Trichoderma and other filamentous fungi.
442. Regulation of gluconeogenesis in Aspergillus nidulans. Yumi Suzuki1, Edyta Szewczyk2, Meryl A. Davis1 and Michael J. Hynes1. 1Department of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia. 2Department of Molecular Genetics, Ohio State University, Columbus, OH 43210, USA.
Gluconeogenesis is required for the utilization of carbon sources metabolized via the TCA cycle. Gluconeogenesis requires the enzymes fructose-1,6-bisphosphatase and PEP-carboxykinase that mediate irreversible steps in glycolysis. There is at present little known about the mechanisms underlying the regulation of gluconeogenic genes in A. nidulans. The expression level of genes encoding these enzymes is significantly lower in strains with mutations of the acuK and acuM genes. These genes encode Zn(II)2Cys6 binuclear cluster proteins (Sealy-Lewis, personal communication) and are likely to be involved in transcriptional regulation of gluconeogenic genes. A double deletion strain of acuK and acuM shows a phenotype equivalent to that of the single deletion strains. It can therefore be concluded that these two proteins are involved in a single regulatory mechanism. Expression studies have been conducted on maeA, which encodes an NADP-dependent malic enzyme. Malic enzyme is required for growth on carbon sources metabolized via 2-oxoglutarate, such as proline. It converts malate to pyruvate, which is used to produce acetyl-CoA, and also provides NADPH. Mutations in acuK and acuM genes significantly reduce maeA expression supporting their role as regulatory proteins. However, the study showed that maeA had a different regulatory pattern from the gluconeogenic genes studied previously. One significant difference is that the high induction level brought about by proline is abolished in the presence of acetate. Acetate does not need to be utilized for this effect.
443. Differential gene expression during sclerotium formation in Sclerotium rolfsii. Johanna E. Takach and Scott E. Gold. Department of Plant Pathology, University of Georgia, Athens.
Sclerotium rolfsii, the causal agent of southern blight or white mold of peanut, survives poor environmental conditions by differentiating into sclerotia. Resistant sclerotia are critical to the persistence of disease potential in infested fields. Environmental factors, including variation in light, temperature, and oxygen are known to trigger sclerotium development, yet little is known about the genetic basis of this differentiation. To further understand this process, we have begun to identify and characterize genes up- and down-regulated during sclerotium formation. We are evaluating gene expression in a S. rolfsii isolate that consistently produces sclerotia in a highly programmed manner. With this strain we are using suppressive subtractive hybridization PCR (SSHP) to identify genes differentially expressed during sclerotium formation. Characterization of these genes will yield insight into the process and regulation of sclerotium differentiation. Preliminary results of this study will be presented.
444. Genetic and molecular basis of the dominant RIP suppressor of the Adiopopdoume strain of Neurospora crassa. Ranjan Tamuli and Durgadas P. Kasbekar. Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
An assay for RIP was developed that makes use of a tagged duplication that targets RIP to the erg-3 gene. Using this assay seven dominant RIP suppressor strains of Neurospora were identified out of 446 strain screened. One of the seven suppressor strains is the Adiopodoume strain, which contains an active transposon, Tad. Interestingly, within this region I have identified a region that differs between mat A and mat a versions of the Oak Ridge background. I have also observed de novo DNA methylation, within this region. Understanding the molecular basis of dominant RIP suppression in Adiopopdoume might explain how Tad has survived in this strain.
447. Transcriptional control of dimorphic switching in Penicillium marneffei. Kaeling Tan and Alex Andrianopoulos. Department of Genetics, University of Melbourne, Melbourne, Victoria, 3010, Australia.
Many fungi are dimorphic; able to switch between yeast and filamentous form. Dimorphism is believed to be a crucial determinant for fungal pathogenicity in both plants and animals. Penicillium marneffei is an opportunistic pathogen of increasing medical importance due to the increase in immuno-compromised individuals especially in Southeast Asia. Previous studies demonstrated that there is differential expression of abaA in the different cell types of P. marneffei. Loss of abaA leads to asexual developmental abnormalities at 25ºC, similar to A. nidulans, such as the production of uninucleate, swollen chains of phialides (abacus structure) that fails to switch from aceropetal to basipetal division, and are unable conidiate. This implies that abaA may be involved in both yeast growths and in its conserved role in asexual development. The multi-nucleate yeast cells and arthroconidiating filamentous cells at 37ºC, compared to the uninucleate wildtype cells, suggests a role of AbaA in cell cycle control. These lead to the interest in investigating the regulation of abaA and its role in cell cycle regulation. The intimate coordination abaA expression involved in morphogenesis and cell cycle control is by studying the cyclin-dependent kinase (CDK) activity in P. marneffei (NimA and NimXCdc2 homologue) during growth and development in the different cell types.
The abaA expression is investigated by assaying the reporter gene expression and by complementating the abaA promoter deletion strains to identify the promoter region required for AbaA regulation in yeast and asexual morphologies. DNA footprinting studies and mobility shift studies are performed in parallel to identify the specific regulatory sequences necessary for protein binding.
448. The multiple 3'-5' DNA helicases of Neurospora crassa cooperate in the mei-3 -mediated homologous recombination repair. K. Suzuki, A. Kato, Y. Sakuraba , S. Tanaka, and H.Inoue* (Dept. Regulation-Biol., Fac. Sci., Saitama Univ., Saitama, Japan)
Homologous recombination repair and post-replication repair are important for restart of stalled and collapsed replication forks. The SRS2 gene of Saccharomyces cerevisiae encodes 3'-5' DNA helicase which functions in both of homologous recombination repair and post-replication repair. Here we identified the SRS2-homolog gene in the filamentous fungus Neurospora crassa and disrupted it by RIP. As the mutant showed significant sensitivity to several DNA-damaging agents, it was named mus-50. A series of epistasis analyses indicated that mus-50 belongs to the homologous recombination repair group, in which mei-3 (RAD51 homolog), mus-11 (RAD52 homolog), mus-48 (RAD55 homolog) and mus-49 (RAD57 homolog) are included, but mus-50 was not involved in post-replication repair. Also, double-repair deficient mutant carrying mus-25 (RAD54 homolog) and mus-50 mutations was not lethal, though combination of rad54 and srs2 mutations in S.cerevisiae is lethal. Tetrad analysis revealed that triple-repair deficient mutant carrying 3 mutations of mus-50 and two RecQ homologs, qde-3 and recQ2, is lethal. This lethality was suppressed by mei-3, mus-11 or mus-25 mutation. We also demonstrate that camptothecin-induced collapse of replication fork is repaired by two independent pathways: one is a QDE3- and MUS50-dependent pathway and another is a MUS25- and RECQ2-dependent pathway.
449. Promoter motifs needed for inducing Phytophthora infestans genes during zoosporogenesis in response to cold and increased membrane rigidity. Shuji Tani and Howard S. Judelson, Department of Plant Pathology, University of California, Riverside, CA 92521 USA
Zoospores, important components of the late blight disease caused by Phytophthora infestans, are released from sporangia in cold water (usually PinifC genes are regulated by a cold-induced inositol trisphosphate-mediated calcium signaling pathway. Using GUS reporter fusions, analyses of truncated, chimeric, and mutated PinifC3 promoters revealed that a 7-bp sequence between positions -139 and -133 was sufficient for cold-induced transcription. This “cold box” was also detected in promoters of PinifC1, PinifC2, and orthologs from P. sojae. Protein(s) binding the cold box were detected by EMSA, and are being purified from nuclear extracts for sequence analysis. Furthermore, zoospore release and cold box-regulated transcription were induced by the membrane rigidifizer DMSO (mimicking a cold treatment), but inhibited by the membrane fluidizer benzyl alcohol. Our data delineate a cold signaling pathway in which sporangia perceive reduced temperatures via increased membrane rigidity, which triggers oscillations in cytosolic calcium, zoosporogenesis, and the induction of genes containing the cold box.
450. The ascospore discharge mechanism of Gibberella zeae. LUIS VELASQUEZ, Y. LeTourneau, C. Platt, H. Hallen and F. Trail. Department of Plant Biology, Michigan State University. East Lansing, MI. 48824
Gibberella zeae causes Fusarium headblight (FHB), one of the most destructive plant diseases in the world. The fungus overwinters in infested crop residues and is the main source of primary inoculum for next year's crop. Asci inside newly formed perithecia forcibly discharge sexual spores (ascospores) which are dispersed by wind and water to flowering hosts. The mechanism of spore discharge is the main focus of this research project. We have randomly mutated and subsequently screened mutants for a discharge minus colony. We have identified a mutated gene from a discharge minus colony that codes for a protein related to DNA binding. Additionally, we have characterized the ascus fluid discharged with the spores and determined that the major sugar component is mannitol. We have begun the targeted mutation of the genes involved in the mannitol biosynthesis of G. zeae. The two main enzymes in this pathway, Mannitol dehydrogenase (MtDH) and mannitol 1-phosphate dehydrogenase (M1PD) have been mutated. Additionally generation of a double mutant by mating is under way. Moreover, a series of candidate genes which could be involved in the ascospore discharge mechanism have been targeted for knockouts. We report, in this work, the progress of these approaches.
451. RPDA, a Class-i Histone Deacetylase, Is Essential for Viability of Aspergillus nidulans. Martin Tribus and Graessle Stefan. Innsbruck Medical University
During the past years it has become clear that chromatin represents an important regulatory element that affects nuclear processes such as DNA replication, recombination, DNA repair and transcription by tuning the accessibility of DNA for various regulatory factors. Cells have elaborated a specific machinery to modify nucleosomes for specific processes occuring in chromatin. Acetylation and methylation of the N-terminal tails of the core histones is the most prominent modification. Enzymes responsible for the dynamic equilibrium of histone acetylation are histone acetyltransferases (HATs) and histone deacetylases (HDACs). The latter form highly conserved protein among eukaryots. HDACs are categorized according to the yeast RPD3-like (class 1), HDA1-like (class 2), SIR2-like (class 3) and the plant specific HD2-like enzymes.
Recently, we have deleted representatives of each HDAC class in A. nidulans. Since we were not able to generate deletion mutants of RpdA, an RPD3-related class I HDAC, we put the rpdA coding sequence under the control of the inducible/repressible promoters alcA of A. nidulans and xylP of Penicillium chrysogenum. Repression of RpdA led to a drastic deficiency in viability of knock down strains. The data presented demonstrate that RpdA of A. nidulans is the first HDAC analyzed so far, which seems to be essential for survival of a eukaryotic organism.
This work was supported by the Austrian Science Foundation Grant FWF-P15439 to S.G.
452. Microarray analysis of the Aspergillus niger transcriptome reveals that XlnR plays an important role in the regulation of different pathways. Luisa Trindade and Leo de Graaff. Wageningen University, Fungal Genomics, Wageningen, The Netherlands
The XlnR protein was initially identified as a transcription activator of different genes encoding cellulolytic and xylanolytic enzymes in Aspergillus niger (van Peij et al., 1998). A number of experiments have been performed to elucidate the regulation mechanism of XlnR and the results suggest that the transcription of the xlnR gene is induced by the presence of D-xylose in the culturing media and repressed by D-glucose. As the mRNA levels of xlnR gene are very low, and they can hardly be detected by Northern blot analysis, further research is necessary to confirm these results. To study the XlnR regulon, XlnR mutants were grown under inducing (D-xylose and xylan), repressing (D-glucose) and neutral (sorbitol) culturing conditions. The whole transcriptome was examined by microarray analysis. The XlnR mutants used in these experiments are an xlnR knock out mutant, a mutant where XlnR is constitutively expressed, and the wild type phenotype. Comparison of the transcriptome of different XlnR strains under inducing and repressing conditions showed that XlnR regulates several genes that are involved in different pathways. Among these are genes encoding proteins involved in signal transduction, in the regulation of transcription, in sugar transport but also genes encoding enzymes.
Previous work of Graaff et al. (1994) suggested that the CreA transcription factor might play a role in the transcriptional regulation of the XlnR regulon. This has been shown for the enzyme encoding genes of the regulon, but whether CreA regulates the transcription of the xlnR gene still was uncertain. In order to investigate this the transcriptome of different single and double CreA derepressed and XlnR mutants were compared using both microarray and qPCR analysis. The results of these experiments lead to a new model for the transcription regulation of XlnR.
Graaff LH, de Broek HC, van den Ooijen AJJ, Visser J. 1994. Regulation of the xylanase-encoding xlnA gene of Aspergillus tubingensis. Mol. Microbiol. 12: 479-490.
van Peij NN, Gielkens MM, de Vries RP, Visser J, de Graaff LH. 1998. The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger. Appl Environ Microbiol. 64 (10): 3615-3619.
The authors thank DSM for giving us access to the Aspergillus niger genome and microarrays.
453. Transcriptional Profiling During Pathogenic Development of Ustilago maydis. M. Vranes, M. Scherer and J. Kaemper. Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany.
Little is known about the change of gene expression on planta and the role of the b-mating type during morphogenesis and pathogenicity of Ustilago maydis on planta. We have performed first experiments using DNA-Array analysis to address the gene expression in late tumour tissue. We identified 487 genes that were differentially expressed in planta when compared to haploid sporidia grown in liquid culture, among others genes encoding proteins with putative functions in plant cell wall degradation, iron uptake, lignin degradation and transport.
To address earlier stages of development we have employed a method to isolate fungal cells from the leaf surface. To identify genes differentially expressed on planta prior to plant penetration, we have performed DNA-Arrays with a solopathogenic strain containing an active bE/bW-heterodimer, mutant strains which are not able to penetrate and non-pathogenic wild type haploid sporidia. Our aim is to identify genes important for pathogenesis. Among those genes, which are upregulated in the solopathogenic strain, but are not expressed in the mutants, we have identified riz1, a gene encoding for a C2H2 zinc-finger-protein, as an important regulator of pathogenicity. Currently we are investigating the role of riz1 during pathogenic development of U. maydis.
454. Rbf1 is a zinc finger transcription factor required for regulation of pathogenic development in Ustilago maydis. M. Scherer and J. Kaemper. Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Str., 35043 Marburg, Germany.
The phytopathogenic fungus Ustilago maydis has a dimorphic life cycle. For successful infection of its host plant maize two compatible haploid sporidia have to fuse on a leaf surface and form a filamentous dikaryon, which is able to penetrate the plant cuticula and subsequently leads to the fungal proliferation in planta. Pathogenic development is controlled by the multiallelic b-mating type locus encoding the homeodomain proteins bE and bW. bE and bW proteins expressed from different alleles can form a heterodimer, which is thought to regulate the b-dependent processes via its function as a transcriptional regulator. By expression profiling of b-mediated gene regulation we previously found 246 genes to be b-responsive.
We identified a gene encoding a C2H2 type zinc finger transcription factor as a very early induced b-target. Since deletion mutants are unable to form a b-dependent filament and to penetrate the plant surface, we named the gene rbf1 (regulator of b-filament). Induced expression of rbf1 leads to filamentous growth even in the absence of an active b-heterodimer. Microarray analysis revealed that Rbf1 is necessary as well as sufficient for the activation of a large subset of b-responsive genes. Thus, Rbf1 is an essential part of a regulatory cascade acting downstream of the b-heterodimer.
Current work is focussing on the identification of additional members of the regulatory cascade triggering pathogenic development.
455. Molecular aspects of dominant RIP suppression by large duplications and the associated barrenness in Neurospora. Meenal Vyas, Ravindran Chinnarajan and Durgadas P. Kasbekar. CCMB, Hyderabad, India
Previous studies in our lab have shown that large duplications can act as dominant suppressors of RIP in Neurospora possibly by titrating out the RIP machinery. Apart from this, seven wild isolated strains were identified as dominant suppressors of RIP in a screen in our lab. One of the suppressor strains, Sugar town, appears to harbor a large duplication based on its barren phenotype. We present data to map and molecularly characterize the Sugartown duplication and compare its size with that of synthetic large duplications like Dp (AR17), Dp (OY329) etc. that were shown to act as dominant RIP suppressors.
The role of RIP and RNAi – based mechanisms in rendering crosses involving duplication bearing strains barren, is also being studied in our lab. We report an intriguing observation where in crosses that are homozygous for Dp(AR17) and also defective for meiotic silencing and RIP, are fertile whereas a similar cross that is proficient for either RIP or meiotic silencing, is barren.
456. DNA methylation in the dimorphic fungus Candida albicans. Tithira Wimalasena, Heather Lee, Dolores Montiel, David Archer. School of Biology. University of Nottingham, University Park. Nottingham. NG7 2RD. UK.
Candida albicans is a dimorphic fungal pathogen of humans. It grows in yeast or mycelia forms depending on environmental conditions and, in the mycelial form, it is capable of invasive infection in humans. The dimorphic switch is associated with changes in gene expression patterns (Brown et al., 1999) and some of these may be associated with DNA methylation. We know from chemical analysis of DNA extracted from C. albicans that there is low level DNA methylation and that the level is different in the two morphotypes (Russell et al., 1987). We are investigating the presence of DNA methylation in C. albicans and its role in dimorphism. We have used methylation-sensitive restriction endonuclease isoschizomers (HpaII, MspI and Dpn II, Sau3AI) to analyse the methylation status of CCGG and GATC sequences. Amplified fragment length polymorphisms (AFLPs) were detected when DNA was digested with the isoschizomers suggesting that DNA methylation patterns are different in the yeast and mycelial forms. One of the AFLP bands has been cloned and sequenced and we will report on further analysis of methylation in that sequence. Furthermore, we have used the nucleotide analogue 5-azacytidine (Pancaldi et al., 1988) to examine its impact on morphogenesis and to determine also the effect of azacytidine on DNA methylation using AFLP and Southern hybridisation analyses.
Brown, A. J. P., Gow, N. A. R. 1999. Regulatory networks controlling Candida albicans morphogenesis Trends in Microbiology 7(8): 333-338.
Pancaldi, S., Del Senno, L, Fasulo, M. P. Poli, F. Vannini, G. L 1988. 5-Azacytidine accelerates yeast mycelium conversion in Candida albicans. Cell Biology International Reports 12(1): 35-40.
Russell, P. J., J. Welsch, E. M. Rachlin, and J. A. McCloskey. 1987. Different levels of DNA methylation in yeast and mycelial forms of Candida albicans. Journal of Bacteriology 169: 4393-4395.
457. Identification of Novel Genes Expressed During Conidiogenesis in Beauveria bassiana Using Suppression Subtractive Hybridisation. Jiang Wu1, Hayley Ridgway1, Drion Boucias3 and Travis Glare2. 1National Centre for Advanced Bio-Protection Technologies, PO Box 84, Lincoln University. 2AgResearch Ltd, PO Box 60, Lincoln. 3Department of Entomology and Nematology, University of Florida, Florida, U.S.A
The fungus Beauveria bassiana is an aggressive pathogen of a wide range of insect pests economically important to the agricultural industry. Although this fungus has considerable potential for development as a biopesticide, bulk production of the infective stage of the fungus (conidia) is inconsistent, restricting the choice of strain for commercial production. In this research we aim to enhance the development of B. bassiana fungal-based biopesticides by identifying and subsequently modifying the expression of sporulation genes to increase both consistency in in vitro spore production and resistance to the detrimental effects of UV light. Suppression subtractive hybridization (SSH) was used to isolate genes differentially expressed during conidiation of B. bassiana isolate B17. This was achieved by extracting RNA from both mature mycelium with conidia (tester mRNA population) and submerged synchronised mycelium without conidia (driver mRNA population). cDNA were synthesised for both the tester and driver populations. The tester and driver cDNAs were then hybridised and hybrid sequences removed, the remaining un-hybridised cDNA represent genes that are expressed during sporulation in the tester. The cDNAs were amplified with primary and secondary PCR and cloned into pGEM-T. A total of 91 clones were generated from transformation and PCR showed that most of clones contained 200-600 bp inserts. So far, twenty-three cDNA clone inserts have been sequenced and 17 fragments identified that were not redundant. The DNA sequence of each of the 17 cDNA fragments was used to search GenBank with both nucleotide and translated Blast searches and the sequences resulted in no notable similarity to sporulation genes from Aspergillus or Neurospora. Four of the cDNA fragments were used as probes for northern blot analysis. These fragments were strongly expressed in the lane containing RNA from the sporulating culture. Currently, the full DNA sequence of two novel genes are being isolated from a genomic library.
458. Cryptococcus neoformans senses CO2 during growth, mating, and infection. Bahn Yong-Sun and Joseph Heitman. Duke University
Carbon dioxide (CO2) and bicarbonate (HCO3-) sensing and transport are essential cellular processes in diverse organisms from fungi to plants and mammals. The enzymatic interconversion between CO2 and HCO3- is mediated by a ubiquitous protein, carbonic anhydrase (CA). In pathogenic fungi, including Cryptococcus neoformans, which can survive and proliferate in both environments and animal hosts, it will be crucial to understand the mechanisms of CO2 sensing and its conversion to HCO3- since cells must accommodate dramatic changes in CO2 concentrations during infection. Here we identified and characterized genes encoding CA in C. neoformans to investigate the role of CO2 sensing in the human pathogenic fungus. C. neoformans contains two ?-CA proteins, Can1 and Can2, which are homologous to CA in other fungi but distinct from ?-CA in mammals. Can2, but not Can1, was found to be required for normal growth in ambient conditions. The can2? mutant was able to resume growth in the presence of high CO2 concentrations (5%). Both Can1 and Can2 are dispensable for capsule and melanin production and stress responses with or without high CO2 pressure. Most interestingly, we found that CO2 completely blocks mating between wild-type MAT? and a strains by inhibiting cell fusion, but not filamentation. This CO2-dependent inhibition of mating is reversed by can2, but not by can1 mutations. Regardless of their growth defect in ambient conditions, can2? mutants were found to survive better than the wild-type in the rabbit model of systemic cryptococcosis. In conclusion, the carbonic anhydrase Can2 catalyzes CO2-HCO3- conversion that modulates in vitro growth, mating, and virulence of C. neoformans