Research On Pathogenicity Mechanism Of The Type Ⅲ Effector XopXccR1 Of Xanthomonas Campestris Pv.Campestris

Plants have evolved a set of effective defense mechanisms in the long-term competition with pathogens,including PTI(PAMP-triggered immunity)and ETI(effector-triggered immunity).In order to provide a beneficial environment for bacterial multiplication,phytopathogenic bacteria utilize a type Ⅲ secretion system(T3SS)to inject type Ⅲ secreted effectors(T3SEs)directly into plant cells thereby suppressing plant defenses.At present,the interaction between most effector proteins and plants is still unclear,so the study of the function of different effector proteins will help reveal the interaction between effector proteins and plants.Xanthomonas campestris pv.campestris(Xcc)is an important microbial pathogen that can cause black rot disease in cruciferous plants worldwide,bringing serious agricultural production losses and economic losses.Seventeen type Ⅲ effector proteins have been identified in Xcc 8004,of which XopXccR1 is the largest effector protein and contributes to pathogenicity significantly.Therefore,the pathogenic mechanism of XopXccR1 should be investigated.The main results were as follows:1.Two conserved motifs AvrE(155-615 aa)and AT(1884-1955 aa)were found by CD-Search in NCBI.Based on this,we generated deletion mutants D155-615,D1884-1955 and the corresponding in trans complementary strains C155-615,C1884-1955.Then pathogenicity was tested on Raphanus sativus cv.Manshenghong via leaf cutting.Compared with the wild-type Xcc 8004,both D155-615 and D1884-1955 mutants can partially reduce pathogenicity,but the complementation of conserved domain could not restore to the wild-type level.It indicates that the deletion of the conserved motifs contributes to the pathogenicity of XopXccR1 effector protein.2.For further understanding the pathogenic function of XopXccR1 effector protein,we inoculated Xcc 8004,Δ17,and Δ17/pJ3160 on non-host Nicotiana benthamiana by infiltration.After 3 days,Δ17/pJ3160 caused necrotic lesions at infection region similar to wild-type Xcc 8004,while Δ17 could not elicit any necrotic lesion,indicating that XopXccR1 effector protein could promote cell death in non-host N.benthamiana.3.In order to investigate the location of XopXccR1 effector protein in plants,subcellular localization analysis was performed on the full-length XopXccR1EGFP,XopXccR1N1-1220aa-EGFP and XopXccR1C1221-2031aa-EGFP fusion proteins.The result showed that full-length XopXccR1-EGFP and its N-terminus are localized on the cell membrane,while the C-terminus is in the cytoplasm.The different subcellular localization of N-terminus and C-terminus in N.benthamiana indicates that there might be localization signals at the N-terminus and C-terminus of XopXccR1 respectively,while the N-terminal signal dominates the final localization of the full-length XopXccR1 effector protein.4.Since most macromolecular proteins have a self-cleaving function,we generated a constitutively expressed 6×His-XopXccR1-3×FLAG fusion protein via homologous recombination technology,then detected the size of the XopXccR1 effector protein by western blotting.The results showed that there is no change in size of XopXccR1 effector protein in NYG medium,indicating that XopXccR1 did not cleave in the tested condition.5.In order to study the interaction between the effector protein XopXccR1 and the host plant targets,the transgenic Arabidopsis plants of xopXccR1 were obtained via Agrobacterium-mediated inflorescence dipping methods.The results of Kan resistance screening showed that the T3 generation lines had been obtained.6.In order to find potential target proteins of the effector protein XopXccR1 in plants,we tried to screen the target proteins that interact with XopXccR1 effector protein in Arabidopsis thaliana through the yeast two-hybrid system.Six recombination bait plasmids with different sizes were generated based on bioinformatics analysis results.Finally,three bait strains which can be used for yeast hybridization,i.e.,Y2HGold/pGBKT7-B,Y2HGold/pGBKT7-E,and Y2HGold/pGBKT7-F,were obtained after checking by toxicity assay and autoactivation test.Overall,the conserved domains AvrE and AT contributes to the pathogenicity of the XopXccR1 effector protein;XopXccR1 could promote cell death in nonhost N.benthamiana;N-terminus and C-terminus of the effector protein XopXccR1 might have a localization signal respectively,while the N-terminal signal dominates the localization of the full-length protein on the cell membrane;XopXccR1 did not cleave under the NYG culture condition.