Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTShere that localization of the exocyst at the appressorium pore is septin dependent. The exocyst is furthermore involved in secretion of symplastic (hostcell-delivered) effectors but not apoplastic effectors. Targeted gene deletion of exocyst components Exo70 and Sec5 causes significant virulence defectsbecause of impaired secretion. We will present new information on the role of the exocyst during invasive growth of M. oryzae.166. Functional analysis of protein ubiquitination in the rice blast fungus Magnaporthe oryzae. Yeonyee Oh, Hayde Eng, William Franck, DavidMuddiman, Ralph Dean. Dept Plant Pathology, NCSU, Raleigh, NC.Rice blast is the most important disease of rice worldwide, and is caused by the filamentous ascomycete fungus, Magnaporthe oryzae. Proteinubiquitination, which is highly selective, regulates many important biological processes including cellular differentiation and pathogenesis in fungi. Geneexpression analysis revealed that a number of genes associated with protein ubiquitination were developmentally regulated during spore germination andappressorium formation. We identified an E3 ubiquitin ligase, MGG_13065 is induced during appressorium formation. MGG_13065 is homologous tofungal F-box proteins including Saccharomyces cerevisiae Grr1, a component of the Skp1-Cullin-F-box protein (SCFGrr1) E3 ligase complex. Targeted genedeletion of MGG_13065 resulted in pleiotropic effects on M. oryzae including abnormal conidia morphology, reduced growth and sporulation, reducedgermination and appressorium formation and the inability to cause disease. Our study suggests that MGG_13065 mediated ubiquitination of targetproteins plays an important role in nutrient assimilation, morphogenesis and pathogenicity of M. oryzae.167. The role of autophagy in Cryphonectria hypovirus 1 (CHV1) infection in Cryphonectria parasitica. M. Rossi, M. Vallino, S. Abba', M. Turina. Instituteof Plant Virology, National Research Council (CNR), Torino, Italy.The interaction between Cryphonectria parasitica, the causal agent of chestnut blight, and Cryphonectria hypovirus 1 (CHV1) results in fungalhypovirulence associated with alterations of fungal development, reduced sporulation and pigmentation, accumulation of cytosolic vesicles. The role ofthese vesicles is to support CHV1 maintenance and replication, but the origin of these compartments is still under debate. Due to the phylogeneticproximity between CHV1 and poliovirus, which induces autophagosome proliferation in infected cells, we decided to explore the involvement ofautophagy in vesicle accumulation and virus replication in CHV1-infected mycelium. We are studying the autophagy dynamic in CHV1-infectedCryphonectria expressing GFP-CpAtg8. Atg8 is the fungal orthologue of the mammalian LC3, an essential protein for autophagosome formation which isconsidered a reliable autophagosome marker. In CHV1-free hyphae, GFP-CpAtg8 distribution was mostly cytosolic, but in presence of CHV1 we observed apunctate distribution of fluorescence which is compatible with the binding of GFP-CpAtg8 with autophagosome membranes. The induction of autophagy isalso supported by the observed increase of accumulation of GFP-CpAtg8 in presence of CHV1 compared with virus-free mycelium which could be due to anactivation of gene transcription and/or to protein stabilization. Overall our results seem to confirm the activation of autophagy by CHV1. We are nowtesting through various approaches if CHV1 is able to induce autophagosomes proliferation to support its own replication or if this is an effect of fungaldefense against hypovirus infection.168. Neurospora crassa protein arginine methyl transferases are involved in growth and development and interact with the NDR kinase COT1. D.Feldman, C. Ziv, M. Efrat, O. Yarden. Dept of Plant Pathology and Microbiology, Faculty of Agricutlure, The Hebrew University of Jerusalem, Rehovot, Israel.The protein arginine methyltransferaseas (PRMTs) family is conserved from yeast to human, and regulates stability, localization and activity of proteins.We have characterized deletion strains corresponding to genes encoding for PRMT1/3/5 (designated prm-1, prm-3 and skb-1, respectively) in N. crassa.Deletion of PRMT-encoding genes conferred reduced growth rates and altered Arg-methylated protein profiles (as determined immunologically). Dprm-1exhibited reduced hyphal elongation rates (70% of wild type) and increased susceptibility to the ergosterol biosynthesis inhibitor voriconazole. In Dprm-3,distances between branches were significantly longer than the wild type, suggesting this gene is required for proper regulation of hyphal branching.Deletion of skb-1 resulted in hyper conidiation (2-fold of the wt) and increased tolerance to the chitin synthase inhibitor polyoxin D. Inactivation of twoPRMTs responsible for asymmetric dimethylation (Dprm-1;Dprm-3) conferred changes in both asymmetric as well as symmetric protein methylationprofiles, suggesting either common substrates or cross-regulation of different PRMTs. Taken together, all N. crassa PRMTs are involved in fungal growth,hyphal cell integrity and affect asexual (but not sexual) reproduction. The PRMTs in N. crassa apparently share cellular pathways which were previouslyreported to be regulated by the NDR (Nuclear DBF2-related) kinase COT1, whose dysfunction leads to a pleiotropic change in hyphal morphology. Usingco-immunpercipitation experiments, we have shown that SKB1 and COT1 can physically interact. To date, two isoforms of COT1 (67 and 73KDa) have beenidentified and studied. We have now identified a third, 70kDa, isoform of COT1, whose abundance was increased in a Dskb-1 background. This isoform, aswell as the two others, are Arg-methylated, as determined on the basis of immunological detection and results indicate that the methylation observedinvolves the activity of more than one PRMT enzyme. The fact that environmental suppression of the cot-1 phenotype is more pronounced in prm-3 andskb-1 backgrounds links these PRMTs to the environmental response associated with COT1 function. Based on the highly conserved structure of the PRMTsand the NDR kinases in eukaryotes, it is likely that these proteins undergo similar interactions in other organisms.169. Role of tea1 and tea4 homologs in cell morphogenesis in Ustilago maydis. Flora Banuett, Woraratanadharm Tad, Lu Ching-yu, Valinluck Michael.Biological Sciences, California State University, Long Beach, CA.We are interested in understanding the molecular mechanisms that govern cell morphogenesis in Ustilago maydis. This fungus is a member of theBasidiomycota and exhibits a yeast-like and a filamentous form. The latter induces tumor formation in maize (Zea mays) and teosinte (Zea mays subsp.parviglumis and subsp. mexicana). We used a genetic screen to isolate mutants with altered cell morphology and defects in nuclear position. One of themutants led to identification of tea4. Tea4 was first identified in Schizosaccharomyces pombe, where it interacts with Tea1 and other proteins thatdetermine the axis of polarized growth. Tea4 recruits a formin (For3), which nucleates actin cables towards the site of growth, and thus, polarizessecretion (Martin et al., 2005). Tea1 and Tea4 have been characterized in Aspergillus nidulans and Magnaporthe oryzae (Higashitsuji et al., 2009; Patkar etal., 2010; Takeshita et al., 2008; Yasin et al., 2012). Here we report the characterization for the first time of the Tea4 and Tea1 homologs in theBasidiomycota. The U. maydis tea4 ORF has coding information for a protein of 1684 amino acid residues that contains a Src homology (SH3) domain, aRAS-associating domain, a phosphatase binding domain, a putative NLS, and a conserved domain of unknown function. All Tea4 homologs in theBasidiomycota contain a RA domain. This domain is absent in Tea4 homologs in the Ascomycota, suggesting that Tea4 performs additional functions in theBasidiomycota. We also identified the Umtea1 homolog, which codes for a putative protein of 1698 amino acid residues. It contains three Kelch repeats.The Tea1 homologs in the Ascomycota and Basidiomycota contain variable numbers of Kelch repeats. The Kelch repeat is a protein domain involved inprotein-protein interactions. The tea1 gene was first identified in S. pombe and is a key determinant of directionality of polarized growth (Mata and Nurse,1997). To understand the function of tea1 and tea4 in several cellular processes in U. maydis, we generated null mutations. We demonstrate that tea4 andtea1 are necessary for the axis of polarized growth, cell polarity, normal septum positioning, and organization of the microbutubule cytoskeleton. We alsodetermined the subcellular localization of Tea1::GFP and Tea4::GFP in the yeast-like and filamentous forms.162