Studies on morphogenesis of infection structure and signal network in plant pathogenic fungi - regulatory mechanisms of environmental response and gene expression

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1) Isolation and characterization of genes involved in morphogenesis of infection structure and signal networkThe C. lagenarium GMK1 gene, a homologue of the Saccharomyces cerevisiae FUS3/KSS1 MAP kinase genes, was shown to regulate conidial germination, appressorium formation, and invasive growth. In S. cerevisiae, Ste12p is known to be a transcriptional factor downstream of Fus3p/Kss1p MAP kinases. In this research, to evaluate the CMK1 MAP kinase pathway, the Ste12 homologue CST1 gene from C. lagenarium was isolated and characterized. cst1Δ strains were nonpathogenic on intact host leaves, but could form lesions when inoculated on wounded leaves. Conidia of the cst1Δ strains could germinate and form melanized appressoria on both host leaf surface and artificial cellulose membrane, but could not produce infectious hyphae from appressoria, suggesting that CST1 is essential for appressorium penetration in C. lagenarium. In addition, matured appressoria of the cst1Δ strains contained ex … More tremely low level of lipid droplets, compared with that of the wild type strain. Lipid droplets were abundant in conidia of the cst1Δ strains, but rapidly disappeared during appressorium formation. This misscheduled lipid degradation might be related to the failure of appressorium penetration in the cst1Δ strain.2) Establishment of Agrobacterium tumefaciens-mediated transformation as an experimental tool for random insertional mutagenesis in Colletotrichum lagenariumIn this research, Agrobacterium tumefociens-mediated transformation (AtMT) was applied to Colletotrichum lagenarium for random insertional mutagenesis. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2, a binary vector carrying hygromycin-resistant gene cassette, introduced A. tumefaciens C58C1 led to good production of hygromycin-resistant transformants. The fungal genomic DNA segments flanking T-DNA could be identified from several pathogenicity or morphogenesis deficient mutants by thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR). Sequence from one of the mutants was identical with melanin biosynthesis gene PKS1 of C. lagenarium that we have previously characterized. These results indicated that AtMT could be an effective tool for tagging genes relevant to pathogenicity and morphogenesis in the plant pathogenic fungus C. lagenarium. Less