Function Alanalysis Of Virulence Genes In Verticillium Dahliae From Cotton

Verticillium wilt is a serious disease of cotton,which is mainly caused by the pathogen Verticillium dahlia Kleb?V.dahlia?in China.The pathogen can stay in the soil in the form of sclerotium for a long time and when conditions are favorable,spore start to germinate.Plant roots are infected by the growth of mycelia through the vascular bundle.LRR protein contain leucine-rich repeats sequence and it has been reported in yeast,Drosophila and human.LRR had been proved playing an important role in regulating the relationship between proteins.The function and pathogenic mechanism of LRR in verticillium dahlia is currently unclear.We acquired ten genes that contain the LRR structure after screening the verticillium dahlia genomic data.Eight genes were selected as the candidate gene after the analysis of sequences.All the genes didn't contain the classic signal peptide at their N terminal.Secretome anlysis found that VDAG₀9119T0,VDAG₀1074T0 and VDAG₀7522T0 may encode non-classical secretory proteins.These LRR proteins belong to different categories and the RI class is very conservative.We cloned the conserved VDGA9119 for the further analysis and research.In the process of infection,the expression level of VDAG09119 was down regulated from 24h to 96h.The knockout and complementation vectors were constructed for the Knockout mutant strains ?LRR1-7,?LRR1-10 and complementation mutant strain Ect? LRR-B?Ect? LRR-D;The Knockout mutant strains didn't have influence on the growth rate and colonial morphology.The results showed that the wild strains and complementation mutant strain lead more serious disease than the Knockout mutant strains after cotton and tobacco infection.Knockout mutant strains could increase callose deposition and improve the expression of defense genes when the cotton roots were infected.So the preliminary judgment was that the VDAG09119 of Verticillium dahliae affect the pathogenicity and plant immune reaction.Because the isochorismate can be resolved by hydrolysis,and the isochorismate is the raw material for the synthesis of SA.In order to explore the gene of isochorismatase hydrolase in verticillium dahlia,we analyzed the biological information and gene identification to find the Vdlsc1 gene which code isochorismate hydrolase in Verticillium dahlia.Contrast the known sequence of isochorismate hydrolase and find that Vdlscl gene contain a highly conserved amino acid residues,the two stage structure and the catalytic residues are conserved.In order to explore the Vdlscl gene whether affects the activity of isochorismate hydrolase,we measured the absorbance of 340nm of VdIsc1 and VdIsc1A3,and the content of DDHB.The result showed that VdIsc1 gene improved the activity of isochorismate hydrolase,and the catalytic residues of Vdlscl plays a key role in the influence of isochorismate hydrolase.In order to further verify toxic effect of Vdlscl gene in Verticillium dahlia,we inoculation the ?1-12,?1-12VdIsc1,?1-12VdIsc1?Nt of cotton.In conclusion,Vdlscl gene enhances Verticillium dahlia infection of cotton,and the N terminal of Vdlscl gene is very important in transportation of pathogens in the process.VdIsc1 and VdIscl?Nt expression in plants,and inoculation with Phytophthora capsici showed that the VdIsc1 inhibited the immune response of plant.At the same time we measured the SA and the expression of PR1 gene,and the result is the Vdlscl gene inhibited the salicylic acid synthesis of plant and the expression of PR1 gene,therefore inhibit the immune responses of plant.