Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSdisruption mutant of AoatrR. Similarly, the expression of same ergosterol biosynthetic pathway genes was also markedly down-regulated in the disruptionmutant of AosrbA, but the expression of ABC transporter genes was not affected. In contrast, the expression of ergosterol biosynthetic pathway genes wasnot up-regulated in an overexpression strain of AoatrR. These results suggest that AtrR and SrbA coordinately regulate ergosterol biosynthetic pathwaygenes in aspergilli. On the other hand, the AoatrR disruptant was more hypersensitive to azole drugs compared to the AosrbA disruptant, suggesting thathypersensitivity of the atrR disruptant to azole drugs is attributed not only to lowered ergosterol levels due to down-regulation of ergosterol biosyntheticpathway genes, but also to reduced efflux transport of the drugs due to down-regulation of ABC transporter genes.428. Effects of intron deletions in production of a heterologous protease in Trichoderma reesei. Marja Paloheimo. Roal Oy, Rajamäki, Finland.A protease gene originating from a filamentous fungus Malbranchea cinnamomea was expressed in Trichoderma reesei using the strong T. reesei cbh1(cel7A) promoter. The heterologous protease was produced at relatively high yields. However, when the protease cDNA was included in the (otherwiseidentical) expression cassette the amount of protease in the culture supernatants of the transformants was very low. The native M. cinnamomea proteasegene contains three introns. To further study the effects of the removal of introns in the production of protease, single-copy transformants wereconstructed that contained expression cassettes in which each of the three introns was separately removed and in which two of the introns (allcombinations) were deleted at a time from the genomic protease gene. The expression cassettes were targeted to the native cbh1 locus. Three single copytransformants from each transformation were chosen for further studies. These strains were cultivated in shake flasks and the amount of protease wasanalysed from the culture supernatants. A clear decrease in the protease activity was detected when any of the introns was removed. However, dependingon the intron removed there were differences in the relative decreases of protease activity. Removal of two introns at a time had a cumulative decreasingeffect on production of protease. The results obtained are shown and discussed.429. Novel core promoter elements in the oomycete Phytophthora infestans and their influence on expression pattern detected by genome-wideanalysis. Laetitia Poidevin, Sourav Roy, Howard S. Judelson. Department of Plant Pathology & Microbiology, University of California Riverside, USA.The core promoter is the region flanking the transcription start site (TSS) that directs pre-initiation complex formation. While core promoters have beenstudied intensively in mammals and yeast, little is known about more diverse eukaryotes including oomycetes. Prior studies of a small collection of clonedoomycete genes proposed that its core promoters contain a 19-nt block bearing both an Initiator-like sequence (INR) and a novel 3' sequence named FPR,but this has not been extended to whole-genome analysis. To learn more about oomycete core promoters, we used expectation maximization to find overrepresentedmotifs near TSSs of Phytophthora infestans, the potato blight pathogen. The motifs corresponded to INR, FPR, and a new element found 25-ntdownstream of the TSS called DPEP. TATA boxes were not detected. Assays of DPEP function by mutagenesis were consistent with its role as a core motif.Genome-wide searches found a well-conserved combined INR+FPR in only 13% of genes after correcting for false discovery, contradicting prior reportsthat INR and FPR are adjacent to each other in most genes. INR or FPR were found alone near TSSs in 18% and 7% of genes, respectively. Promoters lackingthe motifs had pyrimidine-rich regions near the TSS. The combined INR+FPR motif was linked to higher than average mRNA levels, developmentallyregulatedtranscription, and functions related to plant infection, while DPEP and FPR were over-represented in constitutive housekeeping genes. Themotifs were all detected in other oomycetes including Hyaloperonospora arabidopsidis, Phytophthora sojae, Pythium ultimum, and Saprolegnia parasitica,but only INR seemed present in a non-oomycete stramenopile. The absence of a TATA box and presence of novel motifs show that oomycete corepromoters have diverged from that of previously-studied model systems, and likely explains failures in prior heterologous expression studies. Theassociation of the INR+FPR with developmentally-regulated genes shows that oomycete core elements influence stage-specific transcription in addition toregulating pre-initiation complex formation.430. New insights into the phosphate-sensing network in Neurospora crassa. Antonio Rossi 1 , Gabriela F Persinoti 2 , Nalu T A Peres 2 , Diana E Gras 2 , Nilce MMartinez-Rossi 2 . 1) Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto, SP, Brazil; 2) Genética, FMRP-USP, Ribeirão Preto, SP, Brazil.The filamentous fungus Neurospora crassa is an excellent model system for examining molecular responses to ambient signals in eukaryoticmicroorganisms. Inorganic phosphate is an essential growth-limiting nutrient in nature and is crucial for the synthesis of nucleic acids and the flow ofgenetic information. Numerous ambient signals activate the recruitment of mitogen-activated protein kinase (MAPK) cascades. Thus, to identify genesinvolved in metabolic responses to exogenous phosphate sensing and the functioning of an MAPK, MAK-2, we performed microarray experiments using amak-2 knockout strain (Dmak-2) grown under phosphate-shortage conditions by comparing its transcription profile to that of a control strain grown in lowandhigh-phosphate cultures. These experiments revealed 912 unique differentially expressed genes involved in a number of physiological processesrelated to phosphate transport, metabolism, and regulation as well as posttranslational modification of proteins, and MAPK signaling pathways.Quantitative real-time-PCR gene expression analysis using independent RNA samples of 10 arbitrarily chosen genes validated our microarray results. A highPearson correlation between microarray and quantitative real-time-PCR data was observed. The analysis of these differentially expressed genes in theDmak-2 strain provide evidence that the mak-2 gene is a component of the hierarchical phosphate-signaling pathway in N. crassa in addition to itsindependent involvement in other metabolic routes such as the isoprenylation pathway. In this extended model, MAK-2 is functional regardingtranscription of Pi-repressible phosphatases when N. crassa is cultured under Pi shortage and is non-functional under abundant Pi conditions, thusrevealing novel aspects of the N. crassa phosphorus-sensing network. Financial support: FAPESP, CNPq, CAPES, FAEPA.431. Carbon source and light dependent regulation of gene clusters in Trichoderma reesei (Hypocrea jecorina). Doris Tisch 2 , Monika Schmoll 1 . 1) Healthand Environment, Bioresources, Austrian Institute of Technology AIT, Tulln, Austria; 2) Vienna University of Technology, Institute of Chemical Engineering,Vienna, Austria.Trichoderma reesei (anamorph of Hypocrea jecorina) is one of the most prolific producers of plant cell wall degrading enzymes. Regulation of the genesencoding these enzymes occurs in response to the nutrient sources available in the environment and many of them are responsive to light as well.Cellulose as the natural substrate induces the most complete enzyme set, while induction of cellulases also occurs on sophorose and lactose. In contrast,no cellulases are induced on glycerol and the respective genes are repressed on glucose. We therefore investigated the transcriptome on these five carbonsources in light and darkness and aimed to identify genes specifically expressed under cellulase inducing conditions. These conditions are characterized bya significant enrichment of genes involved in C-compound and carbohydrate degradation and transport among the upregulated gene set. Genes downregulatedunder inducing conditions show a significant enrichment in amino acid metabolism and energy metabolism. We were further interested whetherlight dependent regulation is clustered in the genome and if the carbon source is relevant for activation of light dependent clusters. We found that lightdependent clustering predominantly occurs upon growth on cellulose, with the most significant regulation in a gene cluster comprising env1. This clusterappears on glucose as well, but is not down regulated in mutants of blr1 or blr2. Also cbh2, the arabinofuranosidase gene abf2 and the histoneacetyltransferase gene gcn5 are part of light dependent clusters. Hierarchical clustering of gene expression patterns was performed to reveal functional226