Functional Analysis Of CH-STE7and CH-SEC1in Colletotrichum Higginsianum

Colletotrichum higginsianum is a worldwide fungal pathogen causing serious dieases on cruciferous plants. To investigate the molecular pathogenesis of this pathogen, through the methord of gene knockout and complementation, we investigated the function of gene Ch-STE7, playing a role in C.higginsianum growth rate and pathogenic process, which is the homolog of STE7in the FUS3/KSS1MAPK pathway in Saccharomyces cerevisiae. At the same time we also did some preliminary work on the T-DNA inserted broken gene Ch-SEC1. The main resuts of this research were as follows.The full length of Ch-STE7gene is1729bp which contains4exons and3introns, encoding a protein with522aa, processing a S_TKc conserved domain. Ch-STE7knockout mutants showed a white colony, irregularly shaped edge, extremely decreased growth rate and biological yield, but aerial hyphae and hyphae top bifurcation were increased. Ch-STE7knockout mutants produced spores normally which could germinate, but just produced slender germ tube only, could not form blacken appressorium and lossed of pathogenicity on Arabidopsis thaliana. These results suggested that Ch-STE7involves in the regulation of growth rate, pigment formation, appressorium production and pathogenicity.The T-DNA inserted broken gene Ch-SEC1is645bp, just contains1exons and encodes a protein of522aa which is a hypothetic protein. The gene inserted mutant G668showed normally growth rate and spores production, but when injected on A. thaliana leaves with the spores of G668, small lesions could produced on detached leaves and substantially no visible lesions could be observed on attached leaves. Microscopic observation found that spores could germinate normally on the leaf surface and melanin appressorium, but primary hyphae production were reduced significantly, and could not produce secondary hyphae, which suggested that this gene affects the primary hyphae and secondary hyphae production. The function of this gene has not yet been reported in other fungi. To validate whether the Ch-SEC1gene was associated with the virulence of C. higginsianum, targeted gene disruption was carried out but knockout transformants verified was still not finished.