Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS104. Maltose permease-encoding mRNA is cleaved under induction condition of amylolytic gene expression in Aspergillus oryzae. Mizuki Tanaka,Takahiro Shintani, Katsuya Gomi. Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan.Eukaryotic mRNA is degraded by two degradation pathways: the 5' to 3' degradation pathway by Xrn1 and the 3' to 5' degradation pathway by exosome-Ski complex. To investigate the mRNA degradation mechanism in filamentous fungi, we generated the disruptions of orthologous genes encoding mRNAdegradation machinery in Aspergillus oryzae. Interestingly, the disruptants of ski2 and ski3, which encode the components of Ski complex, showed theremarkable growth defect on minimal medium containing maltose or starch as a sole carbon source, whereas they normally grew on the medium withglucose or fructose as a sole carbon source. Northern blot analysis showed that the 3'-truncated fragment of mRNA encoding maltose permease (malP)was accumulated in Ski complex deficient mutants. Circularized RT-PCR analysis revealed that the malP mRNA was cleaved at a large stem-loop structuresituated within the coding region. These results suggested that the malP mRNA is cleaved by endonuclease and the resultant 3'-truncated malP mRNA isdegraded rapidly by 3' to 5' degradation pathway. In higher eukaryotes, it has been reported that the mRNAs encoding secreted and membrane proteinswere cleaved by endoplasmic-reticulum (ER) endonuclease Ire1 during ER stress. Since A. oryzae produces copious amounts of amylolytic enzymes in thepresence of maltose, we presumed that malP mRNA is cleaved by Ire1 with the induction of amylolytic gene expression. Therefore, we generated thedouble deficient mutant for Ski complex and AmyR, the regulator of amylolytic genes expression. The resultant double mutant showed normal growth onmaltose medium, and 3'-truncated fragment of malP mRNA was not detected by Northern blot analysis. This result clearly indicated that malP mRNA iscleaved under induction condition of amylolytic gene expression in A. oryzae.105. Functional characterisation of Rac GTPase in Botrytis cinerea reveals impact on polarity, cell cycle and pathogenicity. Anna Minz-Dub 1 , LeonieKokkelink 2 , Paul Tudzynski 3 , Amir Sharon 1 . 1) Department of Plant Sciences, Britannia 536, Tel-Aviv University, Tel-Aviv 69978; 2) Universität zu Köln,Biozentrum, Institut für Botanik, Zülpicher Str. 47 b, 50674 Koeln, Germany; 3) Institut für Biologie und Biotechnologie der Pflanzen ,WestfaelischeWilhelms-Universitaet Muenster, Schlossplatz 8, D-48143 Muenster, Germany.Small GTPases of the Ras superfamily are involved in regulation of different cellular mechanisms including cell cycle and differentiation. Furthermore,small GTPase proteins are interconnected with many different signalling pathways. In this study we describe functional characterization of a Rho-typeGTPase BcRac from the necrotrophic plant pathogen Botrytis cinerea. Role of this protein in cell cycle, development and pathogenicity is described.Expression of a constitutively active (CA) version of the BcRac protein, or deletion of the gene had a severe impact on fungal growth and differentiation.The mutant strains have polarity defects, they do not produce conidia, disease symptoms on plants are delayed, and they produce and accumulateincreased amounts of ROS in culture. In addition, nuclear content and actin localization were altered in the CA-BcRac strain as compared to wild type. Aneffect of Rac-specific inhibitor NSC23766 on spore germination of wild type strain indicated that BcRac might be necessary for spore germination duringG2/M phase. Based on our observations, BcRac is an important regulator of development in B. cinerea, and alteration of its activity disrupts themorphogenetic program and influences fungal infection.106. Light matters: The transcription factor LTF1 regulates virulence and light responses in the necrotrophic plant pathogen Botrytis cinerea. JuliaSchumacher 1 , Adeline Simon 2 , Kim Cohrs 1 , Muriel Viaud 2 , Paul Tudzynski 1 . 1) IBBP, WWU Muenster, Schlossplatz 8, 48143 Muenster, Germany; 2) INRA,BIOGER, Avenue Lucien Brétignières, 78850 Grignon, France.The lifecycle of Botrytis cinerea/ Botryotinia fuckeliana includes the formation of white mycelia generating pigmented conidiophores with macroconidiafor propagation, pigmented sclerotia for over-wintering and sexual reproduction, microconidia for spermatization of the sclerotia, and the formation ofapothecia as fruiting bodies on spermatized sclerotia. Full-spectrum light induces the differentiation of conidia and apothecia, while sclerotia areexclusively formed during incubation in constant darkness. The relevance of light for virulence of the fungus is not that clear, however, infections areobserved under natural illumination conditions as well as in constant darkness. By a T-DNA insertional mutagenesis approach, we identified a novelvirulence-related gene encoding a GATA-type transcription factor (TF) with homologues in A. nidulans (NsdD) and N. crassa (SUB-1). As transcription isinduced by light (2.5-fold), it is called BcLTF1 for ‘Light-regulated TF 1’. By deletion and over-expression of BcLTF1, we confirmed the predicted role of theTF in virulence, and discovered furthermore its extraordinary functions in regulating light-dependent differentiation processes (growth defect of Dbcltf1 inlight, loss of sclerotia formation in darkness), the equilibrium between production and scavenging of reactive oxygen species (ROS), and secondarymetabolism. Hence, microarray analyses (WT, Dbcltf1; dark vs. exposure to light for 1h) revealed that the expression levels of 206 out of 313 lightdependentgenes are modulated by BcLTF1, including the genes of the putative carotenoid gene cluster and six out of eleven genes encoding TFs. Inaddition, the mutation of bcltf1 affects the expression of 1,616 genes irrespective of the light conditions, including the over-expression of known and so faruncharacterized secondary metabolism gene clusters. The over-expression of the gene encoding the alternative oxidase (AOX) and the under-expression ofgenes involved in oxidative stress responses are in accordance with the observed phenotypes of the deletion mutant, i.e. the hypersensitivity toexogenously applied oxidative stress even in the absence of light and the restoration of growth rates in continuous light by offering antioxidants, indicatingthat BcLTF1 is required to cope with oxidative stress that is caused by the exposure to light.107. Functional analysis of genes in the mating type locus of Botrytis cinerea. Razak Bin Terhem, Joost Stassen, Jan van Kan. Laboratory ofPhytopathology, Wageningen University, Wageningen, The Netherlands.Botrytis cinerea is a heterothallic ascomycete with two mating types, MAT1-1 and MAT1-2, each containing two genes. Besides the archetypal genesencoding the MAT1-1-1 (alpha-domain) protein and the MAT1-2-1 (HMG-box) protein, each idiomorph contains one additional gene, designated MAT1-1-5and MAT1-2-4, respectively. Homologs of these genes are only found in closely related taxa, and their function is as yet unknown. Knockout mutants weregenerated in all four genes in the B. cinerea MAT locus, either in the MAT1-1 strain SAS56 or in the MAT1-2 strain SAS405. Mutants were crossed with astrain of the opposite mating type, either the wild type or a knockout mutant, in all possible combinations. Knockout mutants in the MAT1-1-1 gene andthe MAT1-2-1 gene fail to show any sign of primordial outgrowth and are entirely sterile. This confirms the essential role of the alpha-domain protein andthe HMG-box protein in the mating process. By contrast, mutants in the MAT1-1-5 gene and the MAT1-2-4 gene do produce stipes, but these fail todevelop further into an apothecial disk. The MAT1-1-5 and MAT1-2-4 mutants show identical phenotypes, suggesting that these two genes jointly controlthe transition from stipe to disk development. RNA-seq data were obtained from a cross between two wild type strains and from a cross involving a MAT1-1-5 knockout mutant, from tissue at the stage of transition from stipe to disk. Differential gene expression analysis was performed to identify genes thatare possibly involved in development of the apothecial disk.27th Fungal Genetics Conference | 147