Cloning And Functional Analysis Of MgS11, MgATG5 And MgATG8 Gene In Magnaporthe Grisea

Magnaporthe grisea is a well-known ascomycete that causes rice blast. Understanding of the molecular basis of this disease is not only beneficial for rice blast control, but also can serve as a model for revealing other fungal pathogen-plant interactions. Identification of pathogenicity-relative genes is a key step towards this goal. Mutation analysis of pathogen is a powerful strategy in study of the parasitic interaction between the pathogenic fungus and its plant host, which might make the identification of pathogenicity-relative genes possible. In this study, MgS11, MgATG5 and MgATG8 gene from M. grisea were cloned. The expression pattern of MgS11 and MgATG5 gene was analyzed by eGFP fusion and the role of MgS11, MgATG5 and MgATG8 in pathogenicity was analyzed by targeted gene replacement. The results are showed as follows:1. MgS11 was found from a library of mature appresorium cDNA and its cDNA clones were obtained by PCR strategy, while the sequence homologues were found by using the yeast amino acid sequence of ATG5 and ATG8 to search from M. grisea genome databases, assigned as MgATG5 and MgATG8. The genomic DNA clones covered the full length of ORF and cDNA clones were obtained by PCR strategy and the nucleotide sequences of the genomic DNA and cDNA were analyzed.2. Hygromycin-resistance transformants were obtained by MgS11, MgATG5 and MgATG8 gene replacement vector construction and fungal transformation. Knockout mutants were identified by PCR and Southern blot. Single copy was detected by genomic Southern blot in the genome of Guy11 for each gene.3. The colony modality of AMgSll was different from Guy11. Compared to Guy11, the growth rate of the mutants on CM, CM-C, CM-N and CM with 1M NaCl had no obvious change. Conidia germination and appressoria formation of mutant AMgSll was different from that of wild type. Hydrophobic property test for AMgSll and Guy11 shows that AMgSll was more wettable than Guy11.4. Several eGFP fusion expressing vectors, including PHNS11E, PHNES11 and PHS11PE were constructed. MgS11-eGFP fusion analysis showed that MgSll gene was expressed in hyphaetips, conidia and mature appressoria at different levels, especially in conidia and appressoria.5. The colony modality of AMgATG5 was different from Guyll. Compared to Guyll, the growth rate of the mutants on CM, CM-C, CM-N and CM with 1M NaCl had no obvious change. Conidia germination and appressoria formation of mutant AMgATG5 was suppressed, compared to wild type. The pathogenicity of'AMgATGS mutant was partially reduced on barley.6. High expression of MgATG5 in cell was indicated by prokaryotic expression experiment.7. Conidiation of mutant AMgATGS was declined.