Cloning And Functional Analyse Of CgATG4and CgATG8in Colletotrichum Gloeosporioide

Natural rubber(Hevea brasiliensis) is a kind of important industrial raw material and indispensable strategic materials. Rubber planting is the important pillar industry and the superiority industry in China tropics. Rubber tree anthracnose caused by Colletotrichum gloeosporioides is one of the important foliar disease of rubber tree, its occurrence and rage causing huge yield losses of rubber trees(7～45%). At present, there is still little known about the pathogenesis of C. gloeosporioides, and the molecular interactions between this pathogen and rubber tree. To further elucidate the disease occurrence law, especially molecular mechanisms of C. gloeosporioides pathogenicity, breeding resistant rubber tree clones, innovating control strategy, are being waited to addressed for sustainable development of the natural rubber industry.A high quality T-DNA insertion mutant library of C. gloeosporioides (HBCg01) by ATMT was generated in the preliminary. On the basis, this study establishs HBCg01mutant strains pathogenicity screening method and obtains pathogenic defects mutant strains CG1988. Flanking sequence of T-DNA insertion (CG1988) locus is obtained by Tail-PCR. Combining with genome database(HBCg01) and Blast programe, T-DNA marker genes:CgATG4is aisolated and cloned. At the same time, according rubber Colletotrichum glooeosporioides(HBCg01) genome database, CgATG8, related closely to the CgATG4, is cloned by the method of homologous. Gene knockout mutant strains are abtained by gene knockout technology, phenotypes of mutant are obtained by phenotype analysis for preliminary clarify the function of two autophagy related gene CgATG4and CgATG8in the process of autophagy. The main research results are as follows:We developed a screening method for pathogenicity of C. gloeosporioides transformants:in a primary screen infection assays were performed on intact, detached copper brown rubber tree leaves by inoculating agar plug cut from mycelial mats, in a secondary screen by inoculation conidial suspensions at least three times.Transformants were screened for defects in pathogenicity with a detached copper brown leaf assay.32mutants showing reproducible pathogenicity defects were obtained.Flanking sequence of T-DNA insertion (CG1988) locus is obtained by Tail-PCR. Bioinformatics analysis showed that T-DNA marker genes associated with CgATG4highly homologous, CgATG4have single copy in the genome, its ORF is1129bp, CDS is960bp, and contains3exons and2introns, contains a complete open reading frame (ORF), encoding319amino acid, and the molecular weight about35.01kD, contain a highly conserved protease domain structure(peptidase_C54cysteine).CgATG8, related closely to the CgATG4, is cloned by the method of homologous. bioinformatics analysis showed that CgATG8have single copy in the genome, its ORF is600bp, CDS is366bp, and contains3exons and2introns, contains a complete open reading frame (ORF), encoding121amino acid, and the molecular weight about14.04kD, contain a highly conserved protease domain structure(GABARAP).Two gene knockout transformation:△CgATG4and△CgATG8are obtained by constructing two gene knockout vector and homologous recombination technology. CG1988,△CgATG4and△CgATG8phenotypic analysis results showed that aerial hyphae quantity of CG1988,△CgATG4and△CgATG8decreased significantly in PDA and CZAPEK; Growth rate is similar to wild fungus; Amount of spores were significantly fewer; The spore germination and appressorium formation obviously delay; Appressorium turgor pressure significantly decreased; Sensitive to starvation; Autophagy process is blocked; Pathogenicity decreased significantly.