Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSon other fungi, disruption mutants had a slow radial growth rate in culture, and colonies were highly compact relative to controls. Furthermore, thehyphae were convoluted and hyper-branched suggesting that apical dominance had been disrupted. Nitro blue tetrazolium straining of hyphae showedthat cAMP disruption mutants were impaired in their ability to synthesise superoxide indicating that cAMP signalling is important for the production ofROS in culture in this species. This defect was reversed by re-insertion of a functional wild type acyA gene into mutant strains. Despite significant defects inhyphal growth and ROS production in culture, E. festucae DacyA mutants were infectious and capable of forming symbiotic associations with grasses,albeit at a lesser infection frequency than wild type. Plants infected with E. festucae DacyA mutants were indistinguishable from controls. However, as inculture, microscopic evidence showed that the mutant strains within the host were hyper-branched, and host tissues heavily colonised, indicating that thetight regulation over hyphal growth normally observed in developing and mature host tissues requires a functional cAMP signalling cascade. Furtherresearch is currently underway to understand how cAMP affects the hyphal growth transitions undertaken during host colonisation, particularly at thelevel of the cell cytoskeleton and hyphal cell wall synthesis.570. Role of VCP1 and SCP1 proteases in the mutitrophic behaviour of the nematophagous fungus Pochonia chlamydosporia. Nuria Escudero 1 ,Christopher R. Thornton 2 , Luis Vicente Lopez-Llorca 1 . 1) Laboratory of Plant Pathology, Multidisciplinary Institute for Environment Studies (MIES) RamónMargalef. University of Alicante, Alicante, SPAIN; 2) Food Security and Sustainable Agriculture, Biosciences, College of Life & Environmental Sciences,University of Exeter, Exeter. UK.Pochonia chlamydosporia (Goddard) Zare and Gams is a fungal parasite of female nematodes and eggs, which has been widely studied as a biologicalcontrol agent of cyst and root-knot nematode egg-shells. The nematode egg-shell is formed by several layers, including a chitinous layer composed of aprotein matrix embedding chitin microfibrils. Extracellular enzymes, such as serine porteases (e.g. VCP1), secreted by egg-parasitic nematophagous fungiare known to play an important role in egg infection. SCP1, a recently reported serine carboxypeptidase from P. chlamydosporia was found during plantroot endophytic colonisation by the fungus, its role in eggs parasitisim is unknown. We have investigated the role of VCP1 and SCP1 proteases in themutitrophic behaviour of the nematophagous fungus Pochonia chlamydosporia using immunological approaches using antiVCP1 and SCP1 polyclonalantibodies, these were raised against synthetic peptides of both proteases. ELISA and immunofluorescence have confirmed the production of bothproteases when Meloidogyne javanica eggs were used as inducer. P. chlamydosporia under starvation condition (water) also expressed both proteases. Itseems that the signal of SCP1 was more intense than of VCP1 under most conditions tested (eggs, protein substrate and starvation). Using proteomic,chitosan was previously found in our lab to induce VCP1 in P. chlamydosporia liquid cultures. Consequently, we have also evaluated the amount of VCP1and SCP1 in media with chitosan, to quantify the production of these proteases under multitrophic conditions. This study is casting light into the molecularaspects of the multitrophic behaviour of P. chlamydosporia. This will help to understand the biocontrol potential of the fungus and open newbiotechnological applications.571. Cellular development integrating primary and induced secondary metabolism in the filamentous fungus Fusarium graminearum. Jon Menke 1 ,Jakob Weber 2 , Karen Broz 3 , H. Corby Kistler 1,3* . 1) Department of Plant Pathology, University of Minnesota, St. Paul, USA; 2) Molekulare Phytopathologie,Universität Hamburg, Germany; 3) USDA ARS Cereal Disease Laboratory, St. Paul, MN, USA.Several species of the filamentous fungus Fusarium colonize plants and produce toxic small molecules that contaminate agricultural products, renderingthem unsuitable for consumption. Among the most destructive of these species is F. graminearum, which causes disease in wheat and barley and oftencontaminates the grain with harmful trichothecene mycotoxins. Induction of these secondary metabolites occurs during plant infection or in culture inresponse to chemical signals. Here we report that trichothecene biosynthesis involves a complex developmental process that includes dynamic changes incell morphology and the biogenesis of novel subcellular structures. Two cytochrome P-450 oxygenases (Tri4p and Tri1p) involved in early and late steps intrichothecene biosynthesis were tagged with fluorescent proteins and shown to co-localize to vesicles we call “toxisomes.” Toxisomes, the inferred site oftrichothecene biosynthesis, dynamically interact with motile vesicles containing a predicted major facilitator superfamily protein (Tri12p) previouslyimplicated in trichothecene export and tolerance. The immediate isoprenoid precursor of trichothecenes is the primary metabolite farnesylpyrophosphate. When cultures are shifted from non-inducing to trichothecene inducing conditions, changes occur in the localization of the isoprenoidbiosynthetic enzyme HMG CoA reductase. Initially localized in the cellular endomembrane system, HMG CoA reductase increasingly is targeted totoxisomes. Metabolic pathways of primary and secondary metabolism thus may be coordinated and co-localized under conditions when trichothecenesynthesis occurs.572. DNA double-strand breaks generated by yeast endonuclease I-Sce I induce ectopic homologous recombination and targeted gene replacement inMagnaporthe oryzae. T. Arazoe 1 , T. Younomaru 1 , S. Ohsato 1 , T. Arie 2 , S. Kuwata 1 . 1) Meiji University, Kanagawa, Japan; 2) Tokyo University of Agricultureand Technology, Tokyo, Japan.The filamentous fungus Magnaporthe oryzae causes the rice blast disease that is one of the most destructive fungal diseases of cultivated rice plants. Tocontrol this fungal disease, many resistant genes have been introduced into cultivated rice germplasm, however, breakdowns of the resistance often occurwithin several years by rapid evolution of the fungus. Therefore, studies on the evolutionary mechanisms of the fungus are important for elucidation of therapid evolution. We set out a novel detection/selection system of DNA double-strand breaks (DSBs)-mediated ectopic homologous recombination (HR)that is one of the evolutionary mechanisms. The system consists of two nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusiongenes as a donor and a recipient, and a yeast endonuclease I-Sce I gene as a DSB-inducer. In this system, ectopic HR can be detected and selected byrestorations of YFP fluorescence and blasticidin S (BS)-resistance at a single cell level. These donor and recipient genes were simultaneously integrated intothe M. oryzae genome and transformed lines were isolated. In the absence of the DSB-inducer, transformed lines showed relatively low frequencies of HRevents (>2.1%). On the other hand, by integration of the DSB-inducer gene into transformed lines, we could observe the frequencies of DSB-mediated HRraising up to ~40%. This result clearly showed that DSB into a certain gene induce ectopic HR events between the gene and its homologs. Accordingly, wefurther applied I-Sce I mediated DSB for TGR in M. oryzae. To detect TGR, we constructed simple system using donor and recipient genes. The recipientgene was integrated into the M. oryzae genome and transformed lines were isolated. To recipient gene integrated lines, the donor gene was introducedand restorations of YFP fluorescence and BS-resistance were evaluated. As we expected, the TGR frequencies were increased at least 37-folds by I-Sce I cotransformationas compared with those obtained without I-Sce I. This result provides a new method using DSB for improving the TGR frequency in M.oryzae. Taken together, it is strongly suggested that DSBs can drive genomic rearrangement and accelerate pathogenic variability in M. oryzae through theectopic HR between homologous sequences such as transposable elements and avirulence genes.573. Investigation of the Magnaporthe oryzae proteome and phosphoproteome during appressorium formation. William L. Franck 1 , Emine Gokce 2 ,Yeonyee Oh 1 , David C. Muddiman 2 , Ralph A. Dean 1 . 1) Plant pathology, NC State University, Raleigh, NC; 2) W.M. Keck FT-ICR Mass Spectrometry27th Fungal Genetics Conference | 261