Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSleaf surface which is a prerequisite for the differentiation of infection structures. Consequently, msb2 mutants are attenuated in virulence (Lanver et al.,2010). The molecular mechanism leading to an activation of Msb2 and the downstream MAP kinase cascade is so far unknown. In yeast Msb2p isprocessed by the aspartyl protease Yps1p leading to an active cell-associated form and a secreted glycosylated part which has an inhibitory function in thefull length protein. In U. maydis Msb2 is also processed, but so far there is no evidence that this leads to an activation of surface sensing. Using a yeastmutant lacking five aspartyl proteases we could demonstrate that yeast Yps1p is able to cleave U. maydis Msb2. In addition, by using this heterologoussystem, two U. maydis aspartyl proteases were identified that were weakly able to cleave Msb2. The respective genes were deleted in the solopathogenicstrain SG200 and its Dmsb2 derivative expressing Msb2-HA-GFP. Possible phenotypic alterations in virulence as well as in Msb2 processing will bemonitored. In addition, a synthetic codon-adapted YSP1 gene has been introduced in the above-mentioned U. maydis strains to analyze the effects of anincrement in Msb2 cleavage on biological activity of the protein. Finally, the extracellular domain of Msb2 was subjected to a mutational analysis toidentify regions with a presumed positive regulatory function. Lanver, D., Mendoza-Mendoza, A., Brachmann, A. and Kahmann, R. (2010). Sho1 and Msb2-Related Proteins Regulate Appressorium Development in the Smut Fungus Ustilago maydis. The Plant Cell 22, 2085-2101.622. The U. maydis effector Pit2 inhibits maize cysteine proteases to suppress host defense. Andre Mueller 1 , Sebastian Ziemann 1 , Steffi Treitschke 2 ,Daniela Abmann 1 , Gunther Doehlemann 1 . 1) MPI for Terrestrial Microbiology, Karl-von-Frisch-Strabe 10, 35043 Marburg, Germany; 2) Fraunhofer ITEM-R,Biopark I, Josef-Engert-Strabe 9, 93053 Regensburg, Germany.The basidiomycete Ustilago maydis is the causal agent of smut disease in maize. Infected plants show tumor formation in all infected aerial parts asprominent symptoms. As a biotroph pathogen, U. maydis depends on living plant tissue and hence efficient suppression of plant immunity is required.Therefore, infectious hyphae secrete effector proteins that interfere with specific components of the plant immune system. One such secreted effectorproteinis Pit2 (Protein important for tumor-formation 2), which, in a previous study, was found to be essential for tumor formation in infected plants [1].Instead of tumors, necroses can be observed at infection sites indicating that plant defense and cell death reactions are triggered in Dpit2 infections [1].Using a combination of yeast-two-hybrid- and protease activity assays, we could show that Pit2 acts as an inhibitor of apoplastic plant cysteine proteaseswhose activity is directly linked with salicylic acid (SA)-associated plant defenses. Sequence comparisons with Pit2 orthologs from related smut fungiidentified a conserved 14 amino acid motif. Mutation of this motif leads to a loss-of-function of Pit2 and consequently to avirulence of U. maydis,suggesting that the protease inhibition by Pit2 is essential for plant infection. Moreover, synthetic peptides of the conserved motif show full activity asprotease inhibitor. Interestingly, expression of only this motif in U. maydis partially restores virulence of the Dpit2-mutant, substantiating the importantrole of this novel protease inhibitor in suppression of host immunity. [1] Doehlemann et al. 2011. Mol Micobiol 81: 751-766.623. The Ustilago maydis MAP Kinase signaling pathway: Identification of direct MAP kinase targets by phospho-peptide enrichment. Vikram Naik 1 ,Gerold J.M. Beckers 2 , Wolfgang Hoehenwarter 3 , Regine Kahmann 1 . 1) Max Planck Institute for Terrestrial Microbiology, Marburg, Germany; 2) PlantBiochemistry and Molecular Biology Group, RWTH-Aachen University, Aachen; Germany; 3) Department for Molecular Systems Biology, Faculty of LifeSciences, University of Vienna, Vienna, Austria.In the plant pathogenic fungus Ustilago maydis three MAP kinase modules have been identified mostly via their homology to genes in Saccharomycescerevisiae. The module consisting of the MAP kinase kpp2, the MAP kinase kinase fuz7 and the MAP kinase kinase kinase kpp4 controls pheromonesignalling and plays an essential role in mating and pathogenicity. Kpp2 is involved in filamentation and appressorium development while the MAP kinase,Kpp6, which also acts downstream of Fuz7, is required for appressorial penetration of plant epidermal cells. Our goal is to identify crucial virulence factorswhich act directly downstream of the MAP kinases Kpp2 and Kpp6. For this we generated a strain in which MAP kinase signaling can be induced byexpressing a constitutively active version of the MAPKK Fuz7 (Fuz7DD) under an inducible promoter in the presence or absence of kpp2 and kpp6. We thenused a two-step chromatographic procedure combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography (MOAC),followed by tryptic digest of enriched phosphoproteins, and TiO2-based MOAC for phosphopeptide enrichment. This enabled detection of low abundantphosphorylated peptides using LC-MS/MS and allowed direct identification and site-specific quantification of phosphorylated peptides that differentiallyaccumulated after MAP kinase activation in wild type and mutant cells. LC-MS/MS analysis of the phosphopeptide fraction obtained after the two-stepMOAC yielded 111 putative substrates of Kpp2 and Kpp6 MAP kinases in three replicate experiments. Of these 20 differentially phosphorylated proteinswere chosen for subsequent functional analyses. We are presently generating deletion mutants of these genes in compatible U. maydis strains that carrydifferent a and b alleles and in a solopathogenic strain. In addition, we are analysing the expression pattern of the chosen genes during the differentdevelopmental stages of U. maydis. Results on the role of these U.maydis genes on signaling and pathogenicity will be presented.624. See1 : A novel organ specific effector in the Ustilago maydis - maize interaction. Amey Redkar 1 , Christoph Hemetsberger 1 , Ziba Ajami-Rashidi 1 ,Virginia Walbot 2 , Gunther Doehlemann 1 . 1) Max Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, Karl von FrischStrasse 10, Marburg, 35043 Germany; 2) Department of Biology, Stanford University, Stanford, California. 94305-5020 USA.Ustilago maydis is a biotrophic smut fungus which infects all aerial organs of its host plant maize. The disease progression and development of infectionis by reprogramming of the plant tissue which ultimately results in formation of tumors. This tumor induction is likely being triggered by small secretedproteins by the fungus, so called effectors. Given the fundamental differences between the different maize organs that are colonized by U. maydis, wehypothesized that the fungus deploys organ specific effectors to manipulate physiology and development of specific host tissues (1). To further investigatethe role of individual organ specific effectors in modulating biotrophy, we in the present study identified a novel secreted protein, termed See1 (Seedlingefficient effector 1) that is strongly induced in seedling leaves but only weakly expressed in tassels and ears. U. maydis deletion mutants for see1 show astrong reduction of tumor formation in maize seedlings but not in floral tissues. Laser scanning confocal microscopy shows that the mutant hyphaesuccessfully enter the leaf tissue but might be blocked during pre proliferation stages in the mesophyll tissue of the leaf. Moreover, by labeling replicatingDNA by 5-ethynyl-2’-deoxyuridine (EdU) we observed that maize seedling colonized by Dsee1 do not show mitotic activity during infection, while celldivision in leaves is specifically induced in wildtype infected host cells. In contrast, the Dsee1 mutant induces normal tumor formation in tassels and alsoshows the stable cell division rate in colonized anthers. Overexpression of see1 causes a hypervirulent phenotype only in the vegetative parts of the tassel,which are not transformed to tumors in wild type infections. To localize See1 during the disease progression we are applying confocal microscopy with livecell imaging using mCherry-tagged See1 protein. Most importantly, we are aiming for the identification of see1 interaction partners to link the observedphenotypes with its molecular function to understand its organ-specific function for U. maydis virulence. (1) Skibbe D*, Doehlemann G*, Fernandes J,Walbot V. (2010) Maize tumors caused by Ustilago maydis require organ-specific genes in host and pathogen. Science 328:89-92.625. Investigation of unconventionally secreted proteins in Ustilago maydis. Stefanie Reissmann 1 , Sina Krombach 1 , Florian Bochen 1 , Till Ringel 1 , SaskiaKreibich 1 , Thomas Brefort 1 , Kerstin Schipper 1,2 , Matthias Mann 3 , Regine Kahmann 1 . 1) Organismic Interactions, Max Planck Institute for Terrestrial274