Studies on the involvement of transposons in the pathogenicity variations of the rice blast fungus

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1. Deletion analysis of the MAGGYLTR promoterSeveral constructs having a deletion in the MAGGY promoter were made and examined for the promoter activity upon stress using the GUS gene as a reporter. The deletion constructs with the GUS gene were introduced into M.grisea isolate Br 48. Total protein was extracted from the M.grisea transformants after beat shock treatment (42C, 1hr), and the GUS activity was measured using MUG as a substrate. The construct that lacks up to 60 bp at 5' terminus of LTR retained the promoter activity and responded to heat shock whereas one lacking 90 bp at 5' terminus lost the promoter activity. The results indicated that some critical sepuence required for both promoter activity and stress response occur in the 60 to 90 region of MAGGYLTR.2. Analysis of the promoter activity of MAGGY during infection processes using the GFP geneTo investigate the promoter activity of MAGGY during infection processes, GFP was expressed in a M.grisea transformant under the control of the MAGGY promoter. Infection test was performed with the GUS-expressing transformants. However, no obvious upregulation of the MAGGY promoter was found during infection processes on either resistant or susceptible cultivar of rice.3. Isolation of a new LTR-retrotransposn from M.grisea.As a new candidate for a stress responsive transposable element, an LTR-retrotransposon, Pyret was isolated from M.greisea. Pyret containg gag and pol genes as do ordinary gypsy-class retrotransposons. However, this element is unique since it carries an extra domain in the gag gene, which is designated as WCCH domain according to a characteristic amino acid sequence. The WCCH domain was found only in four gypsy-class retrotransposons identified in fungal genomes so far. Therefore, the functional role of the WCCH domain is unknown at this moment.