Cloning And Functional Characterization Of Mgvam7 Gene In Magnaporthe Grisea

Magnaporthe grisea is an important pathogenetic fungus which leads to a lot of rice production loss worldwidely every year.Due to its social and economic significance M. grisea has been a model system for the study of phytopathogenetic fungi.During the infection of plant,M.grisea secreted a lot of proteins suggesting the significance of vesicle transport components to the biology of M.grisea.SNARE proteins play a central role during the protein veiscle transport and membrane fusion,upto present time the functions of SNARE in phytopathogenetic fungi was limitedly reported.Here we present the cloning and functional analysis of a putative SNARE coding gene Mgvam7 in M.grisea.The fungal macrocyclic lactone brefeldin A(BFA) has proved to be of great value as an inhibitor of protein trafficking in the research of endomembrane system in eukaryotic cell.BFA treatment repressed the growth of fungal hyphae,blocked the germination of conidia and decreased the development of appresorium in M.grisea.SNARE proteins are class of proteins that are conserved among all eukaryotic organisms.A gene coding for a SNARE protein were cloned from M.grisea disignated as Mgvam7.Southern blotting revealed that there was a unique copy of gene in the genome of M.grisea.The phylogenetic analysis revealed Mgvam 7 was much divergent to yeast protein VAM7 though both of them have a similar domain organization.yeast functional complementation assays showed that Mgvam 7 could rescued the CFW sensitivity and homeotypic vesicle fusion defect of vam 7 gene deleted mutant in S.cerevisiae suggesting a conservatism of the function in this gene. The analysis of the fluorescence of eGFP which was under the control of promoter of Mgvam7 suggested the Mgvam7 expressed during the vegetative growth,conidiation and germination of spore and the development of appresorium.Deletion of Mgvam7 resulted in many defects in the growth and development of M.grisea.The Mgvam 7 deleted mutants displayed more sensitive responses to the stresses which related to the function of vacuole including metal ionic stress,nutrient stress and hyperosmic stress.Treatment of various cell wall and cell membrane stressor indicated a cell wall lintegrity defect in the mutants and CFW staining revealed a change in the distribution of newly synthesed cell wall components.Staining by specific endocytosical dye FM4-64 revealed a defect in the maintance of intact The Spitzenk(o|¨)rper and endocytosis, BFA was able to partly rescued the endocytosic defect in mutants suggesting that the Mgvam 7 might be function on the same pathway with BFA.Reactive oxygen species(ROS) play a crucial role in the development and differentiation of fungi.Staining ROS in vivo revealed that the mutants reduced the accumulation of ROS in the apex of fungal hypha. The mutants produced few conidia and were defective in the infection of plant.Our results suggested that one hand like its countpart in S.cerevisiae the Mgvam7 had a conserved role in assemble and maintance of vacuole;on the other hand the Mgvam 7 had developed some new functions related endocytosis and the regulation of ROS in vivo.In our conclution,the Mgvam7was a multifunctional protein and had a essential role in the growth,development and pathogenicity of M.grisea.