Functional Analysis Of A Pathogen-Associated Molecular Pattern FoEG1 From Fusarium Oxysporum In Plant Immunity Induction And Pathogenicity

Fusarium oxysporum is a typical soil-borne plant pathogenic fungus with an extremely wide host range,which can cause crop wilt disease by infecting the vascular system of plant,and seriously threaten the production of many important economic and horticultural crops.Many plant pathogens can secrete a large number of cell wall degrading enzymes during the process of infecting plants,while some cell wall degrading enzymes not only play the role of virulence factors,but also may have the function of PAMPs,which can trigger plant immune response and enhance the resistance of plants to the subsequent infection of pathogens.However,there are relatively few research reports on F.oxysporum in cell wall degrading enzymes and plant immunity.In this study,a pathogen-associated molecular pattern(PAMP)FoEG1 was identified from F.oxysporum,and its function in plant immunity induction and pathogenicity was analyzed.The results obtained in this study are as follows:1.Identification of a PAMP FoEG1 from F.oxysporum.The secreted proteins in the Glycoside hydrolase 12(GH12)protein family of F.oxysporum were analyzed,and a protein elicitor FoEG1 was identified,which can induce cell death in Nicotiana benthamiana.The ability of FoEG1 protein to cause plant cell death depended on its signal peptide,indicating that FoEG1 acted as a PAMP to target at the apoplast of plants.In addition,FoEG1 can also induce cell death in other different plants,such as common tobacco,tomato,and cotton,indicating that its activity had a wide range of effects.FoEG1 was a cellulose degrading enzyme,but the mutants of FoEG1 which lost the activity of hydrolase can still cause the cell death in N.benthamiana,indicating that the ability of FoEG1 to induce cell death of plants did not depend on its hydrolase activity.Moreover,FoEG1 could not trigger cell death in BAK1-and SOBIR1-silenced N.benthamiana,which indicated that the ability of FoEG1 to induce plant cell death was mediated by BAK1 and SOBIR1.2.FoEG1 induced plant disease resistance.FoEG1 protein can induce ROS accumulation and callose deposition in N.benthamiana,and it can also induce the up-regulated expression of defense related genes in N.benthamiana by q RT-PCR,which indicated that FoEG1 can trigger PTI immune response of plants.After pretreatment with FoEG1 protein,the resistance of N.benthamiana to Botrytis cinerea was significantly enhanced,while the resistance of cotton and tomato to F.oxysporum also increased significantly.3.Functional fragment analysis of FoEG1 induced plant immunity.Through site-directed mutation,it was found that the mutants of cysteine residues did not affect the ability of FoEG1 to induce plant cell death,so FoEG1 may have a functional fragment of cell death-inducing ability.Different types of truncated mutants were expressed by transient expression system in N.benthamiana,and an 86 amino acid residues fragment from amino acid position 144 to 229 was identified,which can still cause cell death in N.benthamiana.In addition,the recombinant protein in this fragment can also induce cell death and ROS accumulation,and enhance the resistance of N.benthamiana to B.cinerea.4.The role of FoEG1 in the pathogenesis of F.oxysporum.In the early stage of F.oxysporum infection in cotton and tomato,FoEG1 gene was significantly induced expression 24-48 h after inoculation,which indicated that FoEG1 may be involved in the infection process of F.oxysporum.The deletion and complementation mutants of FoEG1 in F.oxysporum were generated,but the phenotypic characteristics of the mutants were not significantly different compared with that of the wild type.The pathogenicity assays results showed that the virulence of the deletion mutants were significantly reduced compared with that of the wild type and complementation mutants,indicating that FoEG1 might be involved in the pathogenic process of F.oxysporum as a virulence factor.