The Functional Analysis Of Inducing Plant Immunity And Pathogenicity Of A Pectate Lyase VdPEL1 From Verticillim Dahliae

Verticillium dahliae is a wide-host-range fungal pathogen that causes Verticillium wilt.V.dahliae can secrete a large number of cell wall degrading enzymes in infection process,which destroy the cell wall and promote the infection.Meanwhile,plants can specifically recognize cell wall degrading enzymes to activate plant immune response and resist the invasion of pathogens.The study of cell wall degrading enzymes of V.dahliae is helpful to understand its pathogenicity and the mechanism of inducing plant resistance,thus providing insights and ideas for the prevention and control of verticillium wilt.In this study,a pectate lyase VdPEL1 was isolated and identified from V.dahliae.VdPEL1 contributed to V.dahliae virulence and induced plant defense responses.The following results were obtained in this study:1.VdPEL1 played an important role in the infection process by V.dahliaeBy referring to the early secretory proteomics data of V.dahliae Vd991 infected cotton,a pectate lyase was identified and named VdPEL1.The results of qPCR showed that VdPEL1 gene was significantly induced during the early stage of infection of cotton and tobacco plants by Vd991,indicating that VdPEL1 may be involved in the infection process.We generated two independent VdPEL1 deletion lines?VdPEL1-1 and?VdPEL1-2.?VdPEL1-1 and?VdPEL1-2 showed normal growth phenotypes as the wild-type stain.The results of the pathogenicity test showed that VdPEL1 deletion strains displayed significantly reduced virulence on plants compared with the wild-type stain.EC-1 and EC-2 recovered the high virulence phenotypes.2.Eukaryotic expression and enzyme activity detection of VdPEL1 proteinThe eukaryotic VdPEL1 expression vector pPICZaA-VdPEL1 was successfully constructed and transformed into the competent cells of P.pastoris,and the recombinant proteins with high purity and concentration were obtained after induced expression and purification.SDS-PAGE indicated that the size of recombinant VdPEL1protein was 30 kDa.We found that purified VdPEL1 had a hydrolase activity,which was affected by the temperature,Ca²⁺concentration and pH.3.VdPEL1 induced plant cell deathVdPEL1 induced cell necrosis in tobacco at a minimum concentration of 0.3?M.Meanwhile,VdPEL1 could induce cell death in several plants,including tomato,soybean and cotton plants.Transient expression of VdPEL1 could also trigger cell necrosis in plants,and cell death-inducing activity of VdPEL1 in plants required its signal peptide.The enzymatic activity of VdPEL1 was required for cell death-inducing activity,and VdPEL1^REC?VdPEL1 without pectate lyase activity?abolished the ability to produce cell necrosis in plants.4.BcXyl1 conferred plants disease resistanceVdPEL1 activated PTI responses in plants,including ROS accumulation,leakage of ion electrolytes,expression of plant defense genes,and callose deposition.In addition,VdPEL1 could confer plants disease resistance.VdPEL1-treated tobacco plants enhanced disease resistance against B.cinerea and V.dahliae.Cotton plants pretreatment with VdPEL1 had more resistance to V.dahliae,and significantly fewer verticillium wilt symptoms and fungal biomass compared with the plants treated with VdPEL1^recec or PEVC controls...