The Functional Study Of Plant Innate Immunity Regulated By Xanthomonas Campestris Pv.Campestris 8004 Effector Proteins

Plants survival in the complicated and changeable environment and have natural barriers to defense against pathogens,such as the rigid cell wall,low pH in apoplast,and enzymes or antibacterial compounds secreted by plants.They also have evolved highly sophisticated immune systems to cope with the invading pathogens:Pathogen-associated molecular patterns(PAMPs)triggered innate immunity(PTI)and effector-triggered innate immunity(ETI).PTI and ETI,although two independent layer of plant immune,have a close relationship in some ways that they share downstream signaling mechanism and the receptors involved in ETI or PTI often form complex.The type III secretion system is the most important tool of bacteria to deliver effectors into plant cells,which possess extraordinary abilities to control host cell functions and cause devastating diseases.Xanthomonas campestris pv.campestris(Xcc)is a gram-negative bacterium that causes black rot disease of cruciferous plants including important vegetables(broccoli,cabbage,mustard,etc.),ornamental plants and the model plant Arabidopsis thaliana.As the genome of 8004 strain(Xcc 8004)has been sequenced,Arabidopsis-Xcc model pathosystem become an effective tool to study the interactions between plant and bacteria.In this study,we generated different gene deletion mutants and analyzed there pathogenicity on cruciferous plants.In addition,we investigated the pathogenic mechanism of XopLXcc8004 and XopQXcc8004 protein in Arabidopsis,aiming to clarify the biological function of the candidate effectors,XopLXcc8004 and XopQXcc8004.The results were outlined as follows:1.Several gene deletion mutant strains were generated by triparental conjugation,including Xcc 8004?xopQ,Xcc 8004?xopL,Xcc 8004?avrBS1,Xcc 8004?avrAC and the multiple-gene deletion mutant strains.The pathogenicity of each mutant was analyzed on cruciferous plants.The results showed that all single gene deletion mutants caused decreased disease symptoms on different cabbage cultivar comparing to the wild type strain Xcc 8004,while the multiple-gene deletion mutants displayed different disease symptoms.No obvious difference was observed betweent these mutants and Xcc 8004 when they were inoculated on Arabidopsis Col-0.These results indicated that these genes are required for the full virulence of Xcc 8004 on cabbage,and interplay might exist between effectors.Gene deletion and pathogenicity test can provide clues for investigating function of effector genes.2.Bioinformatics analysis indicated that XC₄273 encodes XopLXcc8004 protein which contains leucine-rich repeats(LRR)domain,and XC₃177 encodes XopQXcc8004 that contains inosine-uridine-nucleoside hydrolase(IU_NHs)domain.They are both conserved proteins in plant pathogenic bacteria.3.The expression and regulation of XC₄273 and XC₃177 were analyzed using qRT-PCR.The results showed that transcription of these genes was regulated by the type? regulator HrpG and HrpX,and the coding proteins were secreted in HrcV-dependent way,indicating that XopLXcc8004 and XopQXcc8004 are the type ? effectors of Xcc 8004.4.The expression of PTI marker gene FRK1(Flagellin receptor kinase 1)was measured by dual luciferase reporter system.Results showed both XopLXcc8004 and XopQXcc8004 in Arabidopsis protoplasts inhibited flg22-induced FRK1::LUC transcription,implying that both XopLXcc8004 and XopQXcc8004 can inhibit PTI signaling pathway.5.XopLXcc8004 and XopQXc8004 transgenic plants of Arabidopsis were obtained via Agrobacterium-mediated inflorescence dipping methods.Then,Xcc8004 was inoculated on these transgenic plants and virulence analysis indicated that both transgenic plants could promote the virulence of Xcc 8004 comparing to wild type Col-0.Over-expression of xopLXcc8004 in Col-0 would lead to Arabidopsis seedlings developmental defect,while XopQXcc8004-expressed Agrobacterium could lead to HR on Nicotiana benthamiana.Col-0 expressing XopQXcc8004 showed HR reaction after inoculating with Xcc 8004.These results suggests both proteins display virulence function on Arabidopsis and XopQXcc8004 may induce ETI signaling pathway.6.XopLXcc8004 and XopQXcc8004 suppressed flg22 induced reactive oxygen species(ROS)and callose deposition,and the expression of plant pathogenic mechanism/defense related gene including PR1.Moreover,XopQXcc8004 inhibited flg22-induced MAPKs activity while XopLXcc8004 could not.These results suggest that both XopLXcc8004 and XopQXcc8004 can inhibit PTI signaling pathway,and the activity of XopQXcc8004 is dependent on the MAPKs7.The confocal microscopy revealed that XopQXcc8004-GFP fusion proteins were localized at the plant cell plasma membrane in Arabidopsis protoplasts and epidermal cells of N.benthamiana,while XopLXcc8004-GFP localized randomly in cytoplasm,cell membrane and nucleus.8.The proteins which may interaction with XopQXcc8004 protein were determined by the Co-Immunoprecipitation and yeast two-hybrid screening using Mate&plate Libraries.In conclusion,this study provide a possible clue for the mechanism of effector proteins-triggered plant innate immunity,which will make us know more about the mechanisms of bacterial virulence and plant resistance.