Functional Analysis Of The Gene MGSP3 Of Sublitase Family In Magnaporthe Grisea

The rice blast fungus, Magnaporthe grisea, infects many economically important cereal crops, particularly rice. The interaction of M. grisea and rice is also taken as a model for the study of fungus-plant interaction. The interaction involves a complex process of signal recognition and transduction in response to each other, the signal molecules from plant fungal pathogens which the host plant recognize are named elicitors or virulence factors. Since they have the characters of being secreted outside the cells, the secreted proteins are mostly regarded as elicitors or virulence factors. It is reported that most of the pathogenic elicitors and virulence factors of pathogens are secreted proteins.With bioinformatic tools and genome database available in the internet, 24 putative sublitases were presumed in M. grisea. It is deduced that all of them have signal peptide and can be sectreted. By oncogenomic analysis and phylogenetic analysis for sublitase , we hypothesized that the 24 putative sublitases control pathogenecity in M. grisea. In this study, the functions of MGSP3 of the 24 putative sublitases which is deduced beening sectreted second most heavily were analysed.In this study, we constructed the MGSP3 overexpressed mutant, and cultured the mutant in yeast extract medium, then normal TCA method, acetone method, and Ni-NTA agarose were applied to precipitate and separate the proteins from culture solutions after the mycelia were filtered out. The precipitated or separated proteins were analyzed by SDS-PAGE eletrophoresis . The results confirmed that the products of gene expression were secreted outside the fungal cell, then large-scale proteins were isolated and purified through chromatography, centrifugation and ultra-filtration in order to detect the activity of the fungal secreted proteins.We also constructed the MGSP knock-out mutants, the phenotype both of the knock-out mutant KOsp3-92-3 and the overexpressed mutant OEsp3-17-l were analysed, both the growing rate and pathogenicity of OEsp3-17-l decrease, but the conidiation increase obviously; both of the aerial mycelium and the conidiation of KOsp3-92-3 decrease greatly. We also observed that the pathogenicity of OEsp3-17-l decreased greatly and KOsp3-92-3 lightly to the rice CO39 inoculating them by spraying, but both to the barley Jin-chang 1316 change littlely inoculating them by dropping. In addition, for further study function of the proteins in Magnaporthe grisea, we constructed MGSP3's transient expression vector and complement vector.