Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSmolecular function of the genes which shown significant phenotype in these analysis.236. Penicillium purpurogenum degrades lignocellulose. What can we learn of this process by analyzing the genome, transcriptome and secretome ofthe fungus? Wladimir Mardones 1 , Eduardo Callegari 2 , Jaime Eyzaguirre 1 . 1) Department of Biology, Universidad Andres Bello, Santiago, Chile; 2) SanfordSchool of Medicine, Division of Basic Biomedical Sciences, University of South Dakota, Vermillion, SD.Penicillium purpurogenum grows on a variety of natural carbon sources and secretes to the medium numerous cellulolytic and hemicellulolytic enzymes.Although some information on the lignocellulose biodegradation process has been obtained by the study of individual enzymes, a more comprehensiveapproach has been attempted by analysis of the genome, transcriptome and secretome of the fungus. A genome sequence draft has been attained bymeans of Illumina Hi-Seq 2000 analysis followed by assembly (Allpaths-LG) and partial annotation (MAKER pipeline): 36 Mb total length, 579 scaffolds, N50238 Kbp, 8984 genes predicted. Using the same sequencing technology and the Trinity assembler, a transcriptome of the fungus grown on sugar beet pulp(50% pectin, 20% cellulose) has been obtained. It includes 7,172 ESTs with mean length of 307 bp; 5195 ESTs were significantly identified in the genome.The secretome of the sugar beet pulp culture was analyzed by shotgun mass spectrometry (2D Nano-LC MS/MS) and 53 proteins were identified byMASCOT. An analysis of the genome draft for genes related to lignocellulose biodegradation enzymes (using dbCAN) showed 347 genes of putativeCAZYmes (38 carbohydrate esterases, 245 glycosyl hydrolases, 56 glycosyl transferases, 6 polysaccharide lyases and 2 carbohydrate binding modules). Thetranscriptome data (using BLASTX) showed that 111 CAZy genes were transcribed. In addition, 46 putative CAZymes were identified in the secretome.Among the 46 recognized, 6 are cellulases and 19 are pectinases, directly related to the degradation of sugar beet pulp. This is the first Penicillium genomesequenced using next generation technology and annotated for its lignocellulose biodegradation enzyme genes. Most of the identified genes correspondto putative non-characterized enzymes. This information will be of value for a better understanding of the lignocellulose biodegradation by filamentousfungi. Support: FONDECYT 1100084; UNAB DI-61-12/R.237. Functional genomics of lignocellulose degradation in the Basidiomycete white rot Schizophyllum commune. Robin A. Ohm 1 , Martin Tegelaar 2 , HanA. B. Wösten 2 , Igor V. Grigoriev 1 , Luis G. Lugones 2 . 1) US DOE Joint Genome Institute, Walnut Creek, CA, USA; 2) Department of Microbiology and KluyverCentre for Genomics of Industrial Fermentations, Utrecht University, Utrecht, The Netherlands.White and brown rot fungi are among the most important wood decayers in nature. Although more than 50 genomes of Basidiomycete white and brownrots have been sequenced by the Joint Genome Institute, there is still a lot to learn about how these fungi degrade the tough polymers present in wood. Inparticular, very little is known about how these fungi regulate the expression of genes involved in lignocellulose degradation. In Ascomycetes, severalconserved transcription factors involved in regulation of complex carbon source degradation have been identified, but there are no homologs of these inBasidiomycetes. Few Basidiomycete white or brown rots are genetically amenable, hindering a functional genomics approach to the study of lignocellulosedegradation. A notable exception is Schizophyllum commune, for which numerous genetic tools are available. S. commune was grown on several carbonsources (glucose, cellulose, lignin or beech wood) and gene expression was analyzed. Numerous genes are strongly up-regulated on the complex carbonsources, compared to on glucose. As expected, many of these encode CAZymes (notably glycoside hydrolase family 61) and FOLymes, but also several wellconserved proteins with unknown function. Interestingly, three transcription factor genes are up-regulated during growth on complex carbon sources,suggesting they may be involved in regulating this process. These transcription factors are highly conserved in Basidiomycetes, but not in Ascomycetes.The two laccase genes of S. commune are very lowly expressed on complex carbon sources, suggesting that their function in lignocellulose degradation islimited. A promoter analysis of up-regulated genes reveals a conserved putative transcription factor binding site, which is also present in related fungi.Experiments to validate these findings, as well as a proteomics analysis during growth on complex carbon sources, are currently in progress.238. Functional characterization of genes expressed in early infection stages by the phytopathogenic fungus Botrytis cinerea. J. Espino, N. Temme, A.Viefhues, B. Oeser, P. Tudzynski. Institut of Plant Biology and Biotechnology, Westf. Wilhelms University, Schlossplatz 8, 48143 Muenster, Germany.Botrytis cinerea is a phytopathogenic fungus that causes important economic losses in the agricultural field, due to its aggressiveness and ability toproduce the “grey mould disease” in more than 200 plant species. Nowadays, the main strategy of control consists in the use of fungicides, although somestrains are becoming resistant to these chemicals. Therefore, the knowledge of the molecular mechanisms during host-plant interaction could be a usefultool to develop new effective treatments against this organism. In microarray studies, we have identified more than 150 genes which are expressed duringthe early stages of infection, but not in conidia, suggesting an important role during fungal germination and penetration. Most of these genes codify forproteins with unknown function. By means of bioinformatic analyses, transmembrane domains and signal peptides were identified in some of theseproteins, suggesting a possible role in signaling pathways or as effectors in the interaction with the plant. We are currently focusing on 18 of these genes,and we validated their expression by real time PCR. In all cases the expression pattern observed in the microarrays studies could be confirmed byquantitative PCR results. Some of them showed an expression at 12 hours post inoculation even 10,000-fold compared to the expression in conidia. Inorder to elucidate the possible role of these genes, we have generated knock-out mutants of 9 single genes and 9 genes located in clusters. Pathogenicitystudies as well as further characterization of the different deletion mutants are now in progress.239. Regulation of biofilm formation in Candida parapsilosis. Linda Holland, Leona Connolly, Denise Lynch, Geraldine Butler. School of Biomolecular andBiomedical Science, Conway Institute, University College Dublin, Dublin, Ireland.Candida parapsilosis is a major cause of infection in premature neonates, particularly because of its tendency to grow as biofilms on indwelling medicaldevices. The biofilm architecture of C. parapsilosis biofilms is substantially different to that of Candida albicans, in particular because C. parapsilosis doesnot make true hyphae, suggesting that the regulation of biofilm formation may also be very different. To address this question we have adapted a fusionPCR method originally developed for C. albicans to construct gene deletions in the type strain C. parapsilosis CLIB214 (1). To date, we have generated 100homozygous deletion strains. We selected predicted protein kinase genes, transcription factors and also genes that are known to be important for biofilmformation in either C. albicans or C. parapsilosis. The collection was assayed for changes in biofilm formation using 24-well Nunc polystyrene plates and bymeasurement of the dry weight of mature biofilm. Eight deletion strains, efg1, czf1, mkc1, gzf3, ume6, ace2, cph2 and bcr1 have a defect in biofilmdevelopment. Only efg1 and bcr1 deletions of C. albicans have similar defects. C. albicans and C. parapsilosis therefore share some key regulators ofbiofilm formation, but there are also substantial differences. References: (1) Noble SM, French S, Kohn LA, Chev V, Johnson AD. Nat Genet. 2010Jul;42(7):590-8. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity.240. Functional analysis of the Mps1 MAP kinase pathway in the rice blast fungus Magnaporthe oryzae. E. Grund 1 , M.-J. Gagey 1 , V. Toquin 2 , R. Beffa 3 , N.Poussereau 1 , M.-H. Lebrun 1,4 . 1) MAP CNRS-UCB-INSA-Bayer CropScience, Lyon, France; 2) Biochemistry Dept, Bayer CropScience, Lyon, France; 3) BayerCropScience AG, Frankfurt/Main, Germany; 4) BIOGER INRA, Thiverval-Grignon, France.27th Fungal Genetics Conference | 179