Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS272. Functional analysis of catalase-peroxidase encoding genes in the fungal wheat pathogen Zymoseptoria tritici. A. Mirzadi Gohari 1,3 , R. Mehrabi 1,2 , P.J.G.M. de Wit 2 , G. H.J. Kema 1 . 1) Wageningen University and Research Centre, Plant Research International, P.O. Box, 16, 6700 AA, Wageningen, TheNetherlands; 2) Wageningen University, Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands; 3) Department ofPlant Protection, College of Agriculture, University of Tehran, Plant Pathology Building, Karaj, Iran.Zymoseptoria tritici is the new name of the foliar wheat pathogen cereal Mycosphaerella graminicola (teleomorph)/Septoria tritici (anamorph) thatcauses septoria tritici blotch particularly in regions with high rainfall and moderate temperatures during the production season. Similar to many plantpathogens, Z. tritici possesses three catalase-peroxidase genes that are known to detoxify H2O2 accumulated in the foliage during colonization. In thecurrent study, we functionally analysed these three catalase-peroxidase genes and found that MgCatD-1, encoding a secreted catalase-peroxidase, playsan important role in the pathogenicity of Z. tritici. MgDCat-1 mutants hardly induced any disease symptoms and expression analysis of MgDCat-1 in plantarevealed that it is up-regulated during pathogenesis, particularly at 8 dpi (days post inoculation). This coincides with an important switch from a biotrophicto a necrotrophic lifestyle during pathogenesis suggesting that this gene is likely required to overcome H2O2-dependant defence responses duringcolonization. Furthermore, the MgDCat-1 strain is hypersensitive to H2O2 as the spore germination dropped to 50% at 4mM H2O2 and to completeinhibition at 6 mM H2O2 compared to the WT IPO323 strain. These results show that secreted catalase-peroxidase is an important pathogenicity factor forsuccessful pathogenesis of Z. tritici.273. Efficient recycling of selective marker genes with the Cre-loxP recombination system via anastomosis in Cryphonectria parasitica. Dong-Xiu Zhang,Donald Nuss. Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD.Reverse genetic analysis has played a significant role in advancing fungal biology, but is also limited by the number of available selectable marker genes(SMGs). The Cre-loxP recombination system has been adapted for use in filamentous fungi to overcome this limitation. Expression of the Cre recombinaseresults in excision of an integrated SMG that is flanked by loxP sites allowing subsequent rounds of transformation with the same SMG. However, currentprotocols for regulated expression or presentation of Cre require multiple time consuming steps. During efforts to disrupt four independent RNAdependentRNA polymerase genes in a single strain of the chestnut blight fungus Cryphonectria parasitica, we tested whether Cre could successfully exciseloxP-flanked SMGs when provided in trans via anastomosis. Stable Cre-producing donor strains were constructed by transformation of wild-type C.parasitica strain EP155 with the Cre coding domain under the control of a constitutive promoter. Excision of multiple loxP-flanked SMGs was efficientlyachieved by simply pairing the Cre donor strain and the loxP-flanked SMGs transformed recipient strain and recovering mycelia from the recipient colonyjust above the anastomosis zone. This method was shown to be as efficient as and much less time consuming than excision by transformation-mediatedexpression of Cre and should be applicable for optimizing reverse genetics analysis in a broad range of filamentous fungi.274. RNA-seq reveals the pleiotropic regulating functions of the transcription factor XYR1 in Trichoderma reesei. Liang Ma 1 , Lei Zhang 1 , Ling Chen 1 , GenZou 1 , Chengshu Wang 2 , Zhihua Zhou 1 . 1) Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for BiologicalSciences, Chinese Academy of Sciences, Shanghai, China; 2) Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiologyand Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.Trichoderma reesei is a well-known cellulase producer and widely applied in enzyme industry. XYR1 has been demonstrated to be the main transcriptionactivator of cellulase and hemicellulase gene expression in T. reesei. To comprehensively investigate the genes regulated by XYR1, RNA-seq was used todetermine the transcriptional profile of T. reesei Rut-C30 and the Dxyr1 strain cultured on either cellulose or glucose. A total of 467 distinct genes wereclassified as differentially expressed comparing parent strain with the xyr1-deleted strain when induced by cellulose, among which were mainly thoseinvolved in carbohydrate metabolism, lipid metabolism, protein fate and solute transport. Almost all the functional cellulase genes were found to bedownregulated in the Dxyr1 strain while all the differentially expressed protease genes were upregulated. Furthermore, the expression of 84 genesidentified as cellulose-induced were significantly impaired when xyr1 was deleted. In contrast, 281 genes showed expression difference in the Dxyr1 strainwhen cultured on glucose as carbon source, and most of these were involved in solute transport, lipid metabolism, secondary metabolism and amino acidmetabolism. In addition, we also found that the functional restoration of XYR1 in the Dxyr1 strain required the homologous insertion of xyr1 at its originallocus. Our study provides the global insight into the genes regulated by XYR1 beyond the hydrolase-encoding genes in T. reesei and might aid futurestudies to improve its cellulase production.275. Tyrosinase an important enzyme for melanin production in the oomycete Saprolegnia parasitica. Marcia Saraiva 1* , Irene de Bruijn 2 , Laura Grenville-Briggs 3 , Debra McLaggan 1 , Vincent Bulone 3 , Pieter van West 1 . 1) School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom; 2)Laboratory of Phytopathology, Wageningen University, 6709 PD Wageningen, Netherlands; 3) KTH - Royal Institute of Technology, School of BiotechnologyAlbaNova University Center SE-106 91 Stockholm Sweden.Filamentous fungi posses a pigment that often is considered an indirect virulence factor giving the fungi leverage. The pigment, melanin, acts as aprotective agent against several threats and hazards that these organisms may encounter such as oxidants, killing by macrophages, UV and alsoantimicrobial compounds. In the biosynthetic pathway of melanin Tyrosinase is the first enzyme involved. Tyrosinases are widely distributed in nature,where they have been found in prokaryotes, eukaryotic microorganisms, invertebrates, plants and mammals. Here we describe the first functionalcharacterisation of a tyrosinase (SpTyr) from the fish pathogenic oomycete Saprolegnia parasitica. This aquatic water mould infect a wide range of fish,amphibians and crustaceans that are relevant to the aquaculture industry and aquatic ecosystems, causing a disease called Saprolegniosis. This disease isresponsible for millions of losses in aquacultures. Previously we found that SpTyr is highly expressed in sporulating mycelium. After developing andimplementing a transient gene silencing method (RNAi), based on the delivery of in vitro synthesized dsRNA into protoplasts of this water mould, weobtained SpTyr-silenced lines with a significantly decreased tyrosinase activity of 40-60% compared to control lines. The tyrosinase activity correlateddirectly with the level of SpTyr-silencing in the transient lines, which ranged from 68.7-37.5%. Furthermore, the melanin content was measuredspectrophotometrically in the SpTyr-silenced lines and found to be significantly reduced to 2-70%. Microscopic observations of SpTyr-silenced linesresulted in aberrant zoosporangium formation, with less pigment and abnormal morphology. Moreover, our results demonstrate that transient genesilencing can be successfully used to functionally characterise genes in S. parasitica and can provide a high-throughput tool for S. parasitica functionalgenomics.276. Comparative transcriptomics of Cordyceps bassiana to understand expression levels of its NRPS related genes. B. Shrestha 1 , J. Oh 2 , M.-W. Hyun 3 , J.-G. Han 1 , H.-W. Kwon 3 , S.-H. Hyun 2 , S. H. Kim 3 , H.-K. Choi 2 , G.-H. Sung 1 . 1) Mushroom Research Division, Rural Development Administration, Suwon 441-707,Republic of Korea; 2) College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea; 3) Department of Microbiology and Institute of Basic188