Functional Analysis Of Gene VdSch9 In Verticillium Dahliae

Verticillium dahliae is a soilborne fungus that causes Verticillum wilt in a broad range of hosts and result in huge economic losses.The mechanism of Verticillum wilt is complicated,and many genes associated with growth and pathogenicity involve in this process.However,the molecular mechanisms of infection are poorly understood.Tor pathway is highly conserved in fungals.The downstream gene of Tor pathway Sch9 is critical in response to extracelluar signal and protein synthesis machinery.Since this gene has not been studied,we explored the function of gene Vd Sch9?encodes a serine/threonine protein kinase?in growth and pathogenicity by knocking out Vd Sch9.The main results wew exhibited as follows.1.Based on the genome database of Verticillium dahlia strain Vd Ls.17,we cloned and analyzed the sequence of VDAG₀4427.Gene Vd Sch9 has a full length of 2768 bp,it encodes a protein kinase of 904 amino acids.Knockout plasmid p OSCAR-Vd Sch9 was achieved successfully by one step method for construction of vector.2.Gene Vd Sch9 was knocked out using Agrobacterium tumefaciens mediated transformation method.We genetated 24 deletion transformant strains through resistance screening of hygromycin.Through PCR detection with the hygromycin primers and target gene primers,3 deletion mutant strains named VS-1,VS-2 and VS-3 were obtained.Using polyethylene glycol?PEG?mediated protoplast transformation approach,we genetated 3 complementary transformation strains.The results of G418 resistance screening and PCR amplification detection revealed that we obtained 2 complementary mutant strains named VS-C1 and VS-C2.VS-C1 was chosen for subsequent research.3.The VS-1,VS-2 and VS-3 strains significantly reduced in growth rate,the mutant was also defective in aerial hyphal growth and hyphal branching.Pathogenicity assay showed,the disease index of deletion mutant is 56.6 that significantly lesser than wild type and complement mutant which are 90.5 and 82.8 inoculated on cotton.The same result was acquired when the pathogenicity was tested by inoculating on eggplant.The disease index of deletion mutant is 65.9,significantly lower than 91.1 and 89.8 in wild type and complement mutant.Deletion mutants was more sensitive to oxidative,hyperosmotic,cell wall,and membrane stresses.These results suggest that Vd Sch9 is important for growth,stress response and pathogenesis in V.dahliae.