Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSrisks and significantly limit grain marketability. Despite extensive breeding efforts, adequate resistance to mycotoxin accumulation has not beendeveloped. Additionally, tools currently available to control mycotoxin contamination are limited in number and efficacy. Recently, host-induced genesilencing (HIGS) has emerged as a way to manipulate gene expression in fungal pathogens via expression of pathogen-specific hairpin RNA (hpRNA) inplants. The goal of this research was to generate transgenic corn inhibiting mycotoxin accumulation via HIGS. To this end, a high-throughput workflow wasdeveloped to create and validate HIGS expression vectors. First, candidate fungal genes regulating mycotoxin biosynthesis were identified through variousapproaches, including expression profiling and functional genomics. Second, a one-step cloning process was developed to simultaneously create hpRNAencodingconstructs comprised of sense and antisense orientations of target fungal genes, separated by an intron from the Magnaporthe oryzae cutinasegene, flanked by the TrpC promoter and terminator. Constructs were validated by phenotypic assessment after transformation into either A. flavus or F.verticiliioides. Finally, constructs encoding hpRNAs that significantly reduced mycotoxin accumulation were cloned into a plant expression vector andtransformed into maize. Currently, silencing vectors have been created that target the a-amylase (AMY1) and hexokinase (HXK1) genes in F. verticillioidesand the polyketide synthase (aflC) and hexokinase (kxk) genes in A. flavus, and transgenic corn plants have been created. Thus, this research will advancethe current understanding of HIGS in maize and will ultimately provide new tools to control mycotoxin contamination of corn.614. CDiT1, a novel type of proteinaceous toxin secreted by the necrotrophic pathogen of the roots of tomato Pyrenochaeta lycopersici. Pierre-HenriClergeot 1 , Herwig Schuler 2 , Ejvind Mørtz 3 , Maja Brus 1 , Simina Vintila 1 , Sophia Ekengren 1 . 1) Vaxtfysiologi, Stockholms Universitet, Stockholm, Sweden; 2)Karolinska Institutet, Stockholm, Sweden; 3) Alphalyse A/S, Odense, Denmark.During the 24th Fungal Genetics Conference in Asilomar in 2009, we reported the isolation by Fast Protein Liquid Chromatography of a putativeproteinaceous toxin of 18 kDa, secreted in liquid medium by the corky root rot pathogen of tomato, the filamentous ascomycete Pyrenochaeta lycopersici.This molecule, CDiT1, was thought to be the cause of cell death observed in tomato leaves after infiltration of culture filtrates into their apoplast. Furthercharacterization of CDiT1 revealed that it is secreted as a dimer and encoded by a single gene, whose expression peaks during tomato root infection.Infiltration into leaves of various hosts of P. lycopersici of recombinant CDiT1 purified by affinity confirmed its phytotoxic, but differential activity.Especially, currant tomato (Solanum pimpinellifolium) proved to be tolerant to a higher concentration of recombinant CDiT1 than cultivated tomato (S.lycopersicum). This correlates with the observation that roots of currant tomato are less prone to intracellular infection by a transformant of the fungusexpressing a reporter gene than those of cultivated tomato. Affinity-purified recombinant CDiT1 was also used in cut root assays to confirm lethal activityof the toxin on tomato root cells. Finally, searches made by sequence similarity in the genomes of other pathogenic Pleosporales showed that CDiT1 has aputative orthologue in the cereals pathogens Stagonospora nodorum, Pyrenophora teres f.sp. teres and Pyrenophora tritici-repentis, species known forsecreting proteinaceous toxins contributing to virulence as well. In conclusion, our data validate the experimental approach of characterizing moleculessecreted by Pyrenochaeta lycopersici and inducing disease-related symptoms after infiltration into host leaves, this in order to highlight potential geneticresistance against corky root rot in related species or varieties (see also Clergeot et al. 2012, Phytopathology, 102:878-891).615. Nonhost-specific phytotoxicity of the polyketide-derived toxin solanapyrone A produced by Ascochyta rabiei and Alternaria solani. W. Kim 1 , L.Tymon 1 , D. Johnson 1 , W. Chen 2 . 1) Plant Pathology, Washington State University, Pullman, WA; 2) USDA-ARS, Grain Legume Genetic and PhysiologyResearch Unit, Pullman, WA.Solanapyrone A is a polyketide-derived metabolite produced by Ascochyta rabiei and Alternaria solani, which are the most destructive necrotrophicpathogens of chickpea and potato/tomato, respectively. They belong to the Order Pleosporales within the Class Dothideomycetes, but are phylogeneticallydistantly-related. All isolates of the two fungi tested so far are capable of producing solanapyrone A in synthetic media, which may imply that it isindispensable for their life cycle. However, very little is known about the genetics of solanapyrone A production and its role in pathogenesis and their lifecycle. Recently, solanapyrone biosynthesis gene cluster was identified in Al. solani. Six genes (Sol1 - Sol6) form the gene cluster, spaning about 20 kb of thegenome. Among them, Sol5 gene encodes a Diel-Alderase which catalyzes the final step of solanapyrone biosynthesis pathway. Knockout of the Sol5 genein both A. rabiei and Al.solani resulted in the production of three compounds (presumably solanapyrone precursors), instead of solanapyrone A. Colony ofsol5 mutants showed expansive growth in agar medium until covering the entire plates, in contrast to restricted growth of colony of their correspondingwild-type progenitors. The restricted growth of the wild type strains is likely due to solanapyrone toxicity. Phytotoxicity of solanapyrone A was examinedwith various plant species including their natural host plants. Solanapyrone A produced similar size of necrotic lesions on all plant species tested. On theother hand, one of the putative solanapyrone precursors with the same molecular weight of solanapyrone A caused much smaller lesion only around thewounds of application sites. These results indicate that solanapyrone A is a nonhost-specific phytotoxin because it caused similar degree of lesions on hostand nonhost species.616. Crosstalk of the unfolded protein response and regulatory pathways controlling pathogenic development in Ustilago maydis. Kai Heimel 1 , JohannesFreitag 2 , Martin Hampel 1 , Julia Ast 2 , Michael Bölker 2 , Jörg Kämper 3 . 1) Department of Molecular Microbiology and Genetics, Georg-August-University,Göttingen, Germany; 2) Genetics Department, Philipps-University, Marburg, Germany; 3) Genetics Department, Karlsruhe Institute of Technology (KIT),Karlsruhe, Germany.Development of eukaryotic pathogens is accompanied by dramatic changes in morphology, lifestyle, nutrient acquisition and growth behavior.Consequently, pathogens require robust control systems to adapt to changing host environments and to maintain cellular physiology and homeostasis.The unfolded protein response (UPR) is a conserved eukaryotic signaling pathway counteracting endoplasmic reticulum (ER) stress during situations ofincreased demands on the secretory pathway. We identified and characterized the homologs of the central UPR regulators, Hac1 and Ire1 in the biotrophicfungus U. maydis. The UPR is tightly interlinked with the b mating-type-dependent signaling pathway that regulates pathogenic development. Exact timingof UPR is required for virulence and premature activation interferes with the b-dependent switch from budding to filamentous growth. A smut-specific C-terminal extension of the U. maydis Hac1 homolog, Cib1, mediates direct interaction with Clp1, an essential component of the b-mediated signalingcascade. This interaction leads to stabilization of Clp1, increased ER stress resistance and thus prevents deleterious hyperactivation of the UPR duringbiotrophic growth of U. maydis. Since Clp1 expression is decisive for cell cycle release and fungal proliferation in planta we suggest that UPR activationserves as a checkpoint to time developmental progression and secretion of effector molecules, which promote the establishment of the biotrophicinteraction.617. Transcriptional profiling of the APSES genes in Trichophyton rubrum during growth on human nail. Elza A. S. Lang 1 , Nalu T. A. Peres 1 , Maíra P.Martins 1 , Tiago R. Jacob 1 , Pablo R. Sanches 1 , Larissa G. Silva 1 , Antonio Rossi 2 , Nilce M. Martinez-Rossi 1 . 1) Department of Genetics, Ribeirao Preto School ofMedicine, University of Sao Paulo, Brazil; 2) Department of Biochemistry and Immunology, School of Medicine of Ribeirao Preto, University of Sao Paulo,Brazil.272