Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSThe dermatophyte Trichophyton rubrum is worldwide spread and is the most prevalent causative agent of clinical cases of skin and nail mycoses inhumans. Adhesion, invasion and colonization of keratinized host tissues are crucial for the success of the infection process and depend on the modulationof genetic responses during host-pathogen interactions. The APSES transcription factors are exclusive of the fungi kingdom and have been reported to playimportant roles in cell growth, differentiation, pathogenicity and virulence in several fungi species. In this work, in silico analyses of T. rubrum genomerevealed the presence of genes encoding distinct proteins containing the APSES domain, suggesting that these proteins may play different roles in the cell.A high number of genes potentially regulated by the APSES regulators were identified by in silico analyses of 1000nt upstream of the annotated ORFs of T.rubrum. Transcriptional profiles of the APSES genes were analyzed during growth of T. rubrum on human nail or keratin, as the sole source of nutrients. Invitro infection of human nail was also evaluated by light microscopy. The results revealed that the transcription levels of the APSES genes are modulatedduring the human nail infection process and keratin degradation. Taken together, our findings suggest that the APSES genes of T. rubrum may beimplicated in host-pathogen interactions. Financial support: FAPESP, CAPES, CNPq, FAEPA.618. Carbohydrate binding proteins of two Leptosphaeria pathogens of Brassica napus. Rohan G T Lowe 1 , Bethany Clark 1 , Angela Van de Wouw 1 , AndrewCassin 1 , Jonathan Grandaubert 2 , Thierry Rouxel 2 , Barbara Howlett 2 . 1) School of Botany, University of Melbourne, Melbourne, Victoria, Australia; 2) INRA-Bioger, Campus AgroParisTech, Thiverval-Grignon, France.Effectors include small secreted proteins (SSPs) produced by pathogens to modify or subvert defence responses of the host organism. Leptosphaeriamaculans “brassicae”, the foremost pathogen of Brassica napus (canola), has 651 genes predicted to encode SSPs. The related species L. biglobosa"canadensis" more aggressively infects Brassica cotyledons, but causes fewer stem cankers. This difference in symptomology may be due to a differentresponse from the host innate immune system. Compared to the L. maculans “brassicae” v23.1.3 reference genome, L. biglobosa has a relatively compactgenome (30 Mbp) lacking the characteristic AT-rich, gene-poor repeats of L. maculans. We are using comparative transcriptomics to identify genesinvolved in early stages of infection. RNAseq analysis revealed that >300 L. maculans “brassicae” genes are highly upregulated (>100-fold) 7 days afterinfection compared to in vitro growth. These genes are enriched for SSPs, which comprise 25 of the top 100, but only 5% of the total gene complement. Amajor class of SSPs are carbohydrate active enzymes (CAZY), some of which play a role in evasion of chitin-triggered immunity in plants. L. maculans“brassicae”and L. biglobosa "canadensis" have different complements of the chitin-associated CAZY domains, CBM18 and CBM50 (aka LysM). Both ofthese domains bind chitin or peptidoglycan and may be found with chitinase domains in the same protein. The genomes of both Leptosphaeria speciesencode a similar number of CBM18 domains (27 and 29, respectively). L. maculans “brassicae” has predominantly multi-domain CBM18 proteins that arenot highly upregulated 7 days after infection, whilst L. biglobosa "canadensis" has more abundant single domain proteins, two of which are highlyupregulated (>100-fold) at 7 days post inoculation. Homologs of the well-characterised CBM50/LysM protein, ECP6, from Cladosporium fulvum are presentin both species, and a triplication of the N terminal LysM domain has occurred in L. maculans “brassicae”. The composition and regulation of CBM18 andCBM50-containing genes in Leptosphaeria may be involved in determining the degree of chitin-triggered immunity in the host canola, and concomitantly,the success of the pathogen. Silencing of key CAZY genes in L. maculans “brassicae” is underway, with a focus on members of the CBM50/LysM family.619. Domains for plant uptake of Ustilago maydis secreted effectors. Anupama Ghosh, Armin Djamei, Shigeyuki Tanaka, Regine Kahmann. Max PIanckInstitute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-Von-Frisch-Strasse 10, D-35043 Marburg, Germany.The genome of the corn smut fungus Ustilago maydis codes for a large repertoire of secreted effectors. Some of them play crucial roles for virulence andestablishment of the biotrophic phase. The chorismate mutase Cmu1 is one such secreted translocated effector of U. maydis. cmu1 deletion strains areattenuated in virulence that is attributed to higher salicylate levels in plants infected with the mutant strain, most likely through alterations in thechanneling of chorismate from the plastids to the cytosol. Here we identify the motif in Cmu1 that is necessary for the translocation of the protein acrossthe plant plasma membrane and present a mutational analysis of this region. To test for uptake we assayed the ability of mutant proteins to complement acmu1 mutant strain as well as the retained ability to complement the growth defect of a Daro7 strain of S. cerevisiae in minimal medium. By deletionanalysis a region of 20 amino acids adjacent to the signal peptide was shown to be essential for the translocation. Microscopic analysis of maize tissueinfected with U. maydis strains expressing Cmu1-mcherry fusion proteins with or without the probable uptake motif revealed that the 20 amino acid motifallows binding of the protein to an as yet unknown plant plasma membrane component. We hypothesize that the translocation of Cmu1 across the plantplasma membrane is a two step process; initiated by binding followed by translocation across the membrane. In addition, we present results where the 20amino acid motif is substituted by motifs from other effectors.620. Lipid metabolism influences virulence in Ustilago maydis. Scott Lambie, Matthias Kretschmer, Jim Kronstad. Michael Smith Laboratories, Universityof British Columbia, Vancouver, BC, Canada.Plant tissues and surfaces are a source of lipids which act as a potential carbon source and signals for the pathogenic development of the biotrophic smutfungus Ustilago maydis. This pathogen is an excellent model for the molecular genetic analysis of lipid use during disease and responds to lipids with amorphologic transition from budding to filamentous growth. In addition, the fungus possesses both peroxisomal and mitochondrial b-oxidation pathways,and numerous putative phospholipases (PLs), to exploit lipid carbon sources for nutritional and signalling purposes. We have shown that bothmitochondrial and peroxisomal b-oxidation is important for the utilization of fatty acids and the pathogenic development of U. maydis and may thereforerepresent a potential target to combat crop disease caused by fungal pathogens. In U. maydis, deletion of components of these pathways influencedmating, lead to a decrease in virulence, caused a defect in fatty acid metabolism, a loss of acetate metabolism and the accumulation of toxicintermediates. To further explore the role of the b-oxidation pathway during morphogenesis and pathogenic development, we have investigated theeffects of several non-steroidal anti-inflammatory drugs (NSAIDs), which are known to interfere with b-oxidation functions at various stages. Diclofenacinhibited the usage of fatty acids of different chain length and saturation state as sole carbon sources and had an influence on the filamentation efficiencyof those fatty acids. Further it showed fungicidal activity by inducing apoptosis, and influenced mating and pathogenic development. In an attempt tofurther elucidate the role of lipid utilization during infection we are investigating the role of PLs as a potential mechanism by which host-derived lipidsignals or fatty acids for subsequent b-oxidation are generated. We present an analysis of 17 candidate PL genes identified by genome mining, as well as apreliminary functional characterization of these genes during mating and infection. Overall, our work demonstrates the utilization of host-lipids by U.maydis as an important nutritional and signalling source that is required for pathogenic development. Furthermore, we have begun to elucidate theunderlying mechanisms involved which represent a potential target to combat crop diseases caused by fungi.621. Functional characterization of the putative cell surface receptor for hydrophobicity, Msb2, in Ustilago maydis. Marino Moretti, Daniel Lanver, IrinaL. Schmidt, Regine Kahmann. MPI for Terrestrial Microbiology, Marburg, Germany.Msb2 is a transmembrane mucin protein involved in plant surface sensing in U. maydis. Msb2 deletion mutants are defective in sensing the hydrophobic27th Fungal Genetics Conference | 273