| Project/Area Number |
11660047
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
植物保護
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| Research Institution | Yamaguchi University |
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Principal Investigator |
ITO Shin-ichi Faculty of Agriculture, Yamaguchi University, Professor, 農学部, 教授 (30243629)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | Fusarium oxysporum f. sp. cucumerinum / Fusarium oxysporum f. sp. lycopersici / mRNA / gene / gene silencing / 糸状菌 / クローニング |
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| Research Abstract |
1) Fusarium oxysporum f. sp. cucumerinum (Foc) genes expressed specifically in cucumber xylem sap were examined at mRNA and protein levels. Twenty-nine Foc cDNA clones isolated by suppression subtractive hybridization were sequenced and the resultant nucleotide sequences were subjected to homology searches. Two cDNA clones showing high homology with NIPSNAP gene and putative cation transporter gene were further examined for their expression in planta. RT-PCR analysis with specific primers designed from the two sequences revealed that their corresponding genes are actually expressed in host plants. Foc proteins accumulated in cucumber xylem sap in vitro were analyzed by two-dimensional SDS polyacrylamide gel electrophoresis. Twenty-two protein spots were extracted from the gels and subjected to peptide mass fingerprinting analysis. Sequence alignment searches showed that no proteins in databases were matched to the peptide mass fingerprintings obtained.
2) Tomatinase gene (Fotoml) of Fusarium oxysporum f. sp. lycopersici which has been known to be expressed in planta was chosen as a target gene for gene disruption. Eighteen mutants obtained showed increase in sensitivity to α-tomatine and decrease in tomatinase activity. Inoculation assay showed reduced pathogenicity of the mutants, suggesting an important role of tomatinase in pathogenicity of this fungus. Northem blot analysis showed that antisense RNA of Fotoml was expressed when one of the mutants was induced by α-tomatine, suggesting posttranscreptional gene silencing is involved in the suppression of tomatinase activity at least in this mutant strain.
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