Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSchromosomes, which may be consistent with their being dispensable for asexual plant infection.632. A genomic analysis of the infection strategies employed by Phoma medicaginis a necrotrophic fungal pathogen of alfalfa and the model legumeMedicago truncatula. Angela H. Williams 1,4 , James K. Hane 2 , Robert D. Trengove 3 , Karam B. Singh 2 , Richard P. Oliver 4 , Judith Lichtenzveig 4 . 1) MurdochUniversity, Perth, Australia; 2) CSIRO Plant Industry, Perth, Australia; 3) Separation Science and Metabolomics Laboratory, Murdoch University, Perth,Australia; 4) Department of Environment and Agriculture and the Australian Centre for Necrotrophic Fungal Pathogens, Curtin University, Perth, Australia.Phoma medicaginis is a necrotrophic plant pathogen that causes black spot of alfalfa (Medicago sativa) and the closely related model legume Medicagotruncatula. It is a member of the Didymellaceae family, a distinct clade within the order Pleosporales which includes some of the most importantpathogens of legume crops. We present here the first genome assembly of P. medicaginis and the results of investigations into the host-pathogeninteraction, focusing on identification of necrotrophic effectors (NEs) using a combination of proteogenomic and transcriptomic analyses. A draft genomeassembly was constructed using Illumina paired-end reads, de novo assembled into 952 nuclear scaffolds totaling 31.4 Mbp, with ~27 x coverage andencoding ~10,500 predicted proteins (>50 amino acids) . Of these, ~1,000 are predicted to be secreted. Peptide sequencing via mass spectrometry wasconducted in order to validate the gene set and characterise the protein content of intracellular and necrosis-inducing secreted fractions. This enabled theconfirmation of 554 predicted genes and identified 162 proteins in the necrosis-inducing secreted fraction. To further validate the predicted gene set andexamine differences in gene expression, the transcriptome was sequenced via RNA-seq at four important lifestyle phases. These included: 1) 1-5 days postinfection of M. truncatula; 2) vegetative growth in vitro; 3) sporulation in vitro and 4) during growth in media where the culture filtrate produces necrosisand chlorosis when infiltrated into the plant. Close to 10,000 genes were expressed under one or more of these conditions with ~ 3,000 showingdifferential expression between the in planta and in vitro samples. The combination of proteogenomic and transcriptomic analyses has enabled thevalidation and fine-tuning of the majority of de novo predicted gene models. Several novel genes were identified via manual annotation of RNA-seq data.We have previously demonstrated that the genome is manipulable via Agrobacterium-mediated transformation which means that the functions ofpotential effector genes can be readily investigated. Collectively these data form a valuable resource from which a short list of effector candidates wasderived and genes involved in the pathogenicity mechanisms of Didymellaceae fungi against their legume hosts were predicted.633. Two G protein-coupled receptors, GprC and GprD, regulate density-dependent development in Aspergillus flavus. Katharyn J. Affeldt, Nancy P.Keller. University of Wisconsin-Madison, Madison, WI.Aspergillus flavus is an opportunistic pathogen of several plant hosts, including maize. This interaction is mediated in part by oxygenatedpolyunsaturated acids, or oxylipins, that are produced by both the fungus and the plant host. Although much has been learned about the synthesis ofthese oxylipins, how the fungus perceives them remains unknown. We hypothesize that G protein-coupled receptors (GPCR) are responsible for receivingand transducing oxylipin signals in A. flavus. We have deleted and overexpressed two GPCRs, gprC and gprD, and found that they are important inregulating density-dependent development, which is thought to involve oxylipin signaling. Specifically, depletion of both gprC and gprD locks the fungusinto a low-density state, even when grown at high density. Furthermore, this mutant is unable to respond to spent medium of a wild type high-densityculture. Inoculation of these mutants on corn kernels will ask whether GprC and GprD are important for pathogenicity, and heterologous expression ofGprC and GprD in Saccharomyces cerevisiae is being used to address questions concerning direct ligand-receptor activation.634. Characterization of genes encoding putative secreted proteins during pathogenesis in Magnaporthe oryzae. Seongbeom Kim, Kaeun Kim, Sook-Young Park, Jaeyoung Choi, Junhyun Jeon, Yong-Hwan Lee. Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea.The repertoire of secreted proteins defines the nature of interactions between microbe and host at the molecular level. Thus, cataloging andcharacterizing the list of secreted proteins from a given pathogen is a pivotal step in understanding molecular mechanisms of pathogenesis. Unlikebacterial and Oomycete pathogens, however, only a limited number of secreted proteins has been identified and analyzed in plant pathogenic fungi. Herewe set out to identify and characterize new secreted proteins in the rice blast fungus. SingalP program predicted a total 1,885 genes encoding secretedproteins in M. oryzae. We prioritized 15 genes, MoSPE1 to MoSPE15, with T-DNA mutants available for in-depth analysis. To reveal their roles inpathogenicity, gene deletion mutants were generated and characterized their functionality. Deletion of MoSPE1 rendered the fungus non-pathogenic,while deletion of MoSPE3, MoSPE6, and MoSPE15 resulted in reduced virulence. Rice sheath inoculation of DMospe1 and DMospe15 showed that defectsin pathogenicity could be attributed to the inability to grow inside plant tissues, suggesting their implication in interaction with rice. In addition, the twogenes were indeed up-regulated during invasive growth in rice. Proteins encoded by MoSPE1, MoSPE6 and MoSPE15 were capable of being secreted inyeast secretion trap system. We believe that our work would reveal novel function of secreted proteins, providing new insight into fungal pathogenesis.635. The biosynthesis of oxalate is entirely dependent on oxaloacetate acetylhydrolase in Sclerotinia sclerotiorum . X. Liang 1 , D. Liberti 2 , M. Li 3 , Y.-T.Kim 4 , R. Wilson 1 , J. Rollins 1 . 1) Plant Pathology Department, University of Florida, 1453 Fifield Hall, Gainesville, FL, 32611-0608; 2) Nunhems NetherlandsBV, PO Box 4005, Haelen 6080 AA, Netherlands; 3) Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL; 4)Environmental Biotechnology Research Centre, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea.Sclerotinia sclerotiorum (Lib.) de Bary is one of the most devastating necrotrophic fungal plant pathogens worldwide and its successful infection involvesthe accumulation of oxalate (up to 10 mM) in plant tissues. Oxaloacetate acetylhydrolase (EC 3.7.1.1), catalyzing the hydrolytic cleavage of oxaloacetate toform acetate and oxalate, has been shown to be the key enzyme catalyzing oxalate biogenesis in Aspergillus niger, Botrytis cinerea and Cryphonectriaparasitica. To dissect the genetic regulation of oxalate biogenesis and pathogenesis of S. sclerotiorum, the S. sclerotiorum oxaloacetate acetylhydrolasegene Ss-oah1 was functionally characterized. Previously we demonstrated that oxalate accumulation in S. sclerotiorum is under strong alkaline induction.Strikingly Ss-oah1 gene expression is regulated in the same manner; neutral pH strongly induces the accumulation of Ss-oah1 transcripts and this pHinduction is completely suppressed in the Ss-pac1 knock out mutant. Ss-oah1 knock out mutants fail to accumulate oxalate in culture and during plantinfection and these phenotypes are restored by complementation with the wild type gene. These data demonstrate that Ss-Oah1-catalyzed oxaloacetatehydrolysis is solely responsible for oxalate production in S. sclerotiorum. On all tested host plants, Ss-oah1 knock out mutants are dramatically reduced invirulence and induce a strong host defense response. On leaves, Ss-oah1 knock out mutants produce limited dark brown-green lesions compared with thespreading, necrotic, light brown lesions produced by the wild type. Host tissue bordering the lesion is clearly defined with a thin, dark zone and while theuninfected leaf tissue becomes yellow and senescent the colonized area often retains chlorophyll reminiscent of “green islands”. In sum, our experimentaldata establish the key function of oxaloacetate acetylhydrolase in oxalate biogenesis and pathogenesis in S. sclerotiorum and indicate that the oah1oxalate minus mutant retains some aspects of virulence but cannot suppress host defense.27th Fungal Genetics Conference | 277