Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSmating types can be isolated. After germination of U. bromivora spores (kindly provided by T. Marcel, INRA-AgroParisTech, France) all haploid progenieswere of the a1 mating type. This suggests a mating type bias where the a2 mating type might be linked to a deleterious recessive allele making theisolation of strains harboring this mating type under laboratory conditions impossible. Hence, the genomes of several strains harboring the a1 mating typeand of diploid spore material containing both mating types were sequenced via an Illumina-Next-Generation Sequencing approach. Based on thesesequences we plan to reconstruct the a2 mating type and to generate a strain containing this mating type as well as a solopathogenic haploid strain.495. T-DNA mediated insertional mutagenesis: evidence of a new gene implied in the early phase of pathogenic development of Botrytis cinerea.Nathalie Poussereau 1* , Eytham Souibguy 1 , Marie-Pascale Latorse 2 , Geneviève Billon-Grand 1 , Cindy Dieryck 1 , Vincent Girard 1 , Adeline Simon 3 , Muriel Viaud 3 ,Julia Schumacher 4 , Paul Tudzinsky 4 . 1) Unité Mixte CNRS BayerSAS, Université LYON I, 14 impasse Pierre Baizet, BP 99163, 69263, Lyon cedex09, France; 2)Centre de Recherche Bayer SAS 14 impasse Pierre Baizet, BP 99163, 69263, Lyon cedex09, France; 3) BIOGER, INRA Versailles, route de Saint Cyr, 78026,Versailles France; 4) Institut fûr Biologie and Biotechnolgie der Pflanzen, West.Wihelms-universitât, Hindenburgplatz 55, 48143 Mûnster, Germany.A collection of mutants of the grey mould fungus Botrytis cinerea has been constructed in order to provide the support for the identification of bothfungal functions that are essential for the pathogenic development and/or plant defence traits raised in the host plant. A random insertional mutagenesisstrategy based on the Agrobacterium tumefaciens-mediated transformation (ATMT) is used to enlarge an existing mutant library (2367 lines, Giesbert etal. 2011). 2144 additional T-DNA integrated transformants have been generated. The insertion sites of the T-DNA are being determined using TAIL-PCR andcapacity to infect the host plant is assayed. These data are organized into a genome-orientated database of tagged genes and will be soon available for thescientific community. One exploitation of this mutant library focuses on the characterization of mutants whose parasitic development in planta ishampered. We present here an example of the study of a new gene encoding a DnaJ domain protein. The T-DNA mutant exhibited a drastic alteration ofthe infectious process on bean leaves. Deletion of the studied gene confirmed this phenotype and revealed that colonization process was also altered ondifferent host plants. A defect in penetration and an abnormal infection cushion formation were registered. A dramatic reduced conidiation and anabnormal hyphal morphology were also observed. Resistance/sensitivity to ROS, formation of ROS, organic acids and cell wall degrading enzymes secretionwere investigated. Finally, proteomic analyses are currently developed in order to attribute a function to this gene.496. The NADPH Oxidase Complexes in Botrytis cinerea. Ulrike Siegmund, Jens Heller, Sabine Giesbert, Paul Tudzynski. IBBP, WWU Muenster, Muenster,Germany.Reactive oxygen species (ROS) are generated in all aerobic environments and therefore play a major role for many organisms depending on oxygen. Forexample they act as messenger molecules for intercellular signaling or play a role during defense mechanisms against pathogens (Takemoto et al., 2007).One good example is the oxidative burst; plants rapidly produce large amounts of ROS as the first defense reaction towards pathogen attacks. NADPHoxidases (Nox) are the most common enzymatic system to produce these ROS. Nox are enzyme complexes, which transport electrons through biologicalmembranes and therewith reduce oxygen to superoxide. In fungi they are shown to be involved in differentiation processes and pathogenicity and aretherewith in our focus to gain insights into plant - fungi interactions. In the phytopathogenic fungus Botrytis cinerea two NADPH oxidases (BcNoxA andBcNoxB) as well as their putative regulator (BcNoxR) were previously identified (Segmueller et al., 2008). Besides their involvement in pathogenicity andsclerotia production, deletion studies have revealed that BcNoxA and BcNoxR are also involved in hyphal germling fusions (Roca and Weichert et al., 2011).Recent analyses show a localization of the catalytical subunits BcNoxA and BcNoxB to the ER and partly to the plasma membrane of hyphae, while theregulator BcNoxR is localized in vesicles and at the hyphal tips. Nox are multi-enzyme complexes, whose regulatory process and the participating proteinsare well described in mammals. However, in fungi not all components have been identified, yet. For B. cinerea interaction studies with potentialcandidates identified the small GTPase Rac, the GEF BcCdc24, the scaffold protein BcBem1 and the PAKs BcCla4 and BcSte20 as interacting proteins withinthe BcNox complex. Roca M.G. and Weichert M. et al., (2012) Fungal Biol 116(3): 379-387. Segmueller N. et al., (2008) Mol Plant Microbe Interact 21: 808-808-819. Takemoto D. et al., (2007) Fungal Genet Biol 44(11): 1065-1076.497. A putative function of small RNAs in the plant pathogen Botrytis cinerea. Arne Weiberg, Ming Wang, Hailing Jin. Plat Pathology & Microbiology, UCRiverside, Riverside, CA.Small RNAs (sRNAs) are a class of non-coding transcripts that regulate gene expression. sRNA-directed gene regulation is a common phenomenon ineukaryotes, and in fungal systems function in differentiation, genome defense, and heterochromatin formation has been described. However, it isunknown in any systems whether sRNAs play an important role in fungal pathogenicity. To study sRNAs in the plant pathogen Botrytis cinerea we arecurrently undertaken a dual approach: I) sRNA deep sequencing was performed analyzing RNA profiles of fungal in vitro culture samples and Botrytisinfectedplant tissues using two host systems, the model plant Arabidopsis thaliana and tomato (Solanum lycopersicum). The goal is to identify infectionspecificB. cinerea-derived sRNAs (Bc-sRNAs). II) Genetic analysis of important sRNA biogenesis factors in B. cinerea is currently piloted. B. cinereapossesses all relevant RNAi components including two Dicer-like genes (Bc-DCL1 and Bc-DCL2) and two Argonaute-like genes (Bc-AGO1 and Bc-AGO2).Targeted gene disruption by homologous recombination of Bc-DCL1 and Bc-DCL2 led to growth retardation on artificial media and to delay of massiveconidiospore production. In planta, no reduction in virulence was observed. However, a dcl1dcl2 double mutant was strongly impaired in virulence andwas unable to produce a set of Bc-sRNAs. Taken our observations together, it is proposed that B. cinerea expresses Bc-sRNAs during infection in order toregulate important processes to facilitate pathogenesis.498. The Role of Quorum-sensing Molecules in Interactions between Candida albicans and its Host. Jessica C. Hargarten 1 , Thomas M. Petro 2 , Kenneth W.Nickerson 1 , Audrey L. Atkin 1 . 1) School of Biological Sciences, University of Nebraska, Lincoln, Lincoln, NE; 2) Department of Oral Biology, University ofNebraska Medical Center, Lincoln, NE.Candida albicans is a polymorphic fungus that is capable of causing the life threatening disease Candidiasis once it reaches the bloodstream of asusceptible host. The capability to switch between morphologies, and its ability to synthesize and secrete the quorum sensing molecule (QSM) farnesol areknown virulence factor. Previously, we showed that C. albicans mutants that produced less farnesol are less pathogenic to mice than their parental strainin a tail vein assay. Also, oral administration of farnesol to the mice prior to infection increased mortality. In contrast, farnesol blocks the yeast to myceliatransition in vitro, which should have a protective effect. These observations pose the dilemma of finding a mechanism whereby a molecule which blocksthe yeast to mycelia transition can also act as a virulence factor. We hypothesize that farnesol functions as a virulence factor by modulating the hostinnate immune response. Distinct Candida morphologies elicit different host immune responses. Both white and opaque cells stimulate leukocytemovement, but only white cells secrete a small molecular weight chemoattractant that draws the leukocyte directly towards the white cell and stimulatesengulfment by mouse macrophages. The white cells are also less susceptible to killing by human macrophages and neutrophils than opaque cells, possiblydue to their increased capabilities of escape once phagocytosed. The chemical identity of this chemoattractant is currently unknown, but the reason27th Fungal Genetics Conference | 243