| Research Abstract |
Action mechanisms of inosine and cyclicAMP were analyzed in order to clarify the differentiation mechanism of multicellular appressoria, the infection organ of gray mold fungus (Botrytis cinerea). The inosine induced the formation of the appressoria in the some starins, and promoted infection to cucumber plants. Though the induction effect by the cAMP was lower than the inosine, its effect increased with the progress in the time. The inosine was suggested to be an inducer of the appressorium differentiation, because cyclicAMP was metabolized into inosine in the fungal mycelia. By the combination of SDS-PAGE and interaction analyzer (Iasys), 19kD protein, which peculiarly combined with the inosine, was got in the fractionation of the fungal cell membrane protein. In the N-terminal sequence, this protein showed the high homology for the amino acid sequence of the GTP-binding protein.
Next, the isolation of the gene, which peculiarly appeared in the differentiation of the multicultural appresoria, was tried by Differential Display method. The result showed that there was a difference between both samples in the band pattern of PCR amplified product with the polyacrylamide electrophoresis, when several primer was used. The base sequences of the bands peculiarly detected in the appressorium differentiation were decided. The amino acid sequence of which the one band was anticipated showed the high homology for the arrangement of aspartic proteinase. Aspartic proteinase is known as an enzyme peculiarly secreted in the infection in opportunistic infection fungi such as Aspergillus fumigatus and Candida albicans, and it is suggested to be deeply concerned in the pathogenicity. At present, the roles in the formation of the appressorium and the infection are analyzed by disruption of the aspartic proteinase gene.
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