|Budget Amount *help
¥14,900,000 (Direct Cost: ¥14,900,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 2002: ¥7,600,000 (Direct Cost: ¥7,600,000)
Gα subunit genes, bcg1 and bcg2, of the gray mold fungus Botrytis cinerea have been previously cloned and characterized. We have isolated a novel Gα subunit gene (bcg3) and a Gβ subunit gene (bcgb1), only one copy each was present in the genome. The deduced amino acid sequences of both Bcg3 and Bcgb1 show the typical conserved motifs present in Gα or Gβ proteins from other filamentous fungi. Bcg3 is a member of the Gas class (III), which also includes MagA of Magnaporthe grisea and Gpa3 of Ustilag maydis. Bcgb1 displays a high degree of sequence homology (approximately 90%) to Gβ subunits Fgb1, Cpgb1, and Mgb1 from Fusarium oxysporum, Cryphonectria parasitica, and M.grisea, respectively. Reverse-transcription (RT)-PCR experiments show clearly that both genes are expressed at very early stages of the infection process. Analysis of transcript levels indicated that bcg3 expression levels are extremely low, whereas bcgb1 expression levels are similar to bcg1 during the infection process. Next, the isolation of the gene, which peculiarly appeared in the differentiation of the multicellular appressoria, 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 sevaral 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 fumigates 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.