Screening And Resistance Identification Of Ralstonia Solanacearum Effector RipQ Transgenic Arabidopsis Thaliana

Bacterial wilt caused by Ralstonia solanacearum is a devastating soil-borne disease that spreads widely in the world and causes serious harm.The Type Ⅲ secretion system(T3SS)and the Type Ⅲ effectors(T3Es)are important pathogenic factors of R.solanacearum.The complex species can secrete more than 100 effector proteins,which can cause hypersensitive response(HR)of resistant host plants and pathogenicity of susceptible hosts in the long-term coevolution process with host plants.Rip Q is an effector protein widely present in the phylotypes Ⅰ,II and Ⅲ of R.solanacearum.Transient expression of Rip Q in tobacco induced the expression of HR marker gene and defense gene PR1 a,suggesting that Rip Q activates the resistance response of tobacco.This study heterologously expressed Rip Q in Arabidopsis thaliana,and investigated the effects on plant phenotype,disease resistance,and its mediated disease resistance pathway.The contents include:1.24 h after inoculated with GMI1000,rip Q deletion mutant(△rip Q)and overexpression mutant(GMI1000::rip Q)in the leaves of A.thaliana,a large amount of hydrogen peroxide was accumulated in the injection area of GMI1000::rip Q.The expression levels of HR marker gene hin1,Salicylic acid(SA)signal pathway marker gene PR1 a and Jasmonic acid(JA)signal pathway marker gene PDF1.2 were all upregulated at the sites inoculated with GMI1000::rip Q.Furthermore,there was no difference in the expression of Ethylene(ET)signaling pathway marker gene EIN3 with wild-type and deletion mutants;The results of pathogenicity test in A.thaliana showed that the pathogenicity of the over-expression mutant GMI1000::rip Q was significantly lower than that of the wild-type and △rip Q.When GMI1000::rip Q was inoculated with SA,JA and ET signal pathway mutants Sid2-1,Coi-1,Ein2-5 and wild-type Col-0 by root irrigation respectively,it was found that Sid2-1 and Coi-1 mutants showed significiently reduced resistance to GMI1000::rip Q compared with wild-type Col-0,while Ein2-5 mutants showed a little difference compared with wild-type.These results suggest that Rip Q can activate SA and JA mediated signaling pathways and improve disease resistance in A.thaliana.2.RipQ transgenic Arabidopsis plants with 35 S constitutive promoter and WRKY18-induced promoter were obtained with Agrobacterium-mediated flower dipping.Four Lines of T2 generation p35S:rip Q transgenic A.thaliana and three Lines of p W18:rip Q transgenic A.thaliana were verified by PCR,indicating that they were all successful transgenic A.thaliana;The morphological indexes of the transgenic A.thaliana wereanalyzed in terms of number of division,number of rosettes,time of moss extraction,time of flowering,time of pod setting,plant height and pod length,suggested that these phenotypes were similar to those of wild-type Col-0 and empty vector transgenic A.thaliana;The wild-type and p35S:rip Q transgenic A.thaliana were inoculated with R.solanacearum GMI1000,Pseudomonas syringae DC3000 and Xanthomonas Campestris respectively,and the results showed that the resistance of transgenic A.thaliana to the three pathogens was significantly enhanced,among which Line 3 was the most resistant.These results suggest that Rip Q can improve the disease resistance of plants by activating SA and JA mediated signaling pathways.Rip Q transgenic A.thaliana showed enhanced resistance to multiple pathogenic bacteria,indicating that rip Q gene has potential application value in crop breeding.