Identification Of Effectors Of Ralstonia Solanacearum Inducing Plant Cell Death And Preliminary Function Analysis Of Effectors Rip54 And Rip5

Ralstonia solanacearum has a broad host range,which can infect more than 400 plant species from 50 different families.R.salanacearum is considered as“species complex”due to significant variation at different levels,and effective disease-resistant varieties is lacking in agricultural production.R.solanacearum has a large number of type III effectors（T3SEs）,which play a key role in plant-R.solanacearum interactions.These effectors induce plant defense in host plants containing the corresponding resistance genes,whereas they suppress plant basal defense in susceptible hosts,and promote pathogen infection.Using effectors as probes to explore plant-R.solanacearum interactions is of great theoretical significance for establish novel disease prevention and control strategies.In this study,Agrobacterium-mediated transient expression approach was employed to analyze cell death induction activity of R.solanacearum T3SEs and their recognition in tobacco.We then focused on two of these cell-death-inducing effectors Rip54 and Rip5,and preliminarily characterized their functions and mechanisms of action,providing clues for revealing the pathogenicity and recognition mechanism of R.solanacearum.The specific results are as follows:Characterization of cell-death-inducing activity of 69 GMI1000 T3SEs in tobacco:69 GMI1000 T3SE genes werecloned into plant expression vectors,and cell death induced by 69 T3SEs was analyzed using the Agrobacterium-mediated transient expression approach.Fourteen or thirteen GMI1000 T3SEs induce plant cell death in N.benthamiana or N.tabacum.Interestingly,Rip37,Rip54 and Rip67 only induce cell death in N.benthamiana,while Rip5 and Rip56 only induce cell death in N.tabacum.The reactive oxygen species（ROS）accumulation induced by these effectors was detected by using L-012 probe,and the result indicates that Rip2,Rip11,Rip16,Rip33,Rip63 and Rip67 are likely recognized by N.benthamiana.The defense signaling pathways were analyzed by using virus-induced gene silencing（VIGS）method,and the result suggests that the recognition of Rip16 and Rip67 is dependent on SGT1.Together,these results provide insights into tthe recognition mechanism of these effectors.Preliminary clarification of the function and mechanism of GMI1000 effector Rip54:Rip54 knockout mutants were generated by homology-mediated gene deletion approach.Inoculation assay showed that Rip54 deletion did not significantly affect the GMI1000 virulence.Transient expression of Rip54 in N.benthamiana suppressed the ROS burst induced by flg22,and promoted pathogen infection.The Rip54-interacting proteins in N.benthamiana were screened by yeast two-hybrid assay,and the protein interaction between Rip54 and Nb ERF77 or Nb PP2C was confirmed by luciferase complementation assay.By transient expression and gene silencing analyses,we showed that Nb ERF77promotes flg22-induced ROS burst,thereby inhibiting pathogen infection;while Nb PP2C significantly inhibits the ROS burst of and promotes the infection of pathogens.These results indicate that Nb ERF77 and Nb PP2C play an important role in plant immunity with opposite functions.It remains unknown how Rip54 interferes with plant defense by interacting with these two types of plant proteins.In addition,transgenic expression of Rip54 in Arabidopsis suppresses resistance to GMI1000,further indicating that Rip54 plays an important role in pathogen virulence.Preliminary elucidation of a role of Rip5 in plant-R.solanacearum interactions:Rip5 exists in both R.solanacearum GMI1000 and CQPS-1,and there are only 3 amino acid differences in the protein sequence.Rip5_GMI1000induces cell death in N.tabacum,but Rip5_CQPS-1cannot.By site-directed mutagenesis analysis,we showed that these three amino acidss are required for Rip5_GMI1000to induce cell death in N.tabacum.Expression of Rip5_GMI1000in N.tabacum significantly induced the expression of hypersensitivity-related genes.The above results indicate that Rip5_GMI1000can be recognized by N.tabacum,and reveal the mechanism by which CQPS-1 escapes tobacco recognition by mutating three amino acids.The expression of either Rip5_GMI1000or Rip5_CQPS-1in N.benthamiana leaves suppressed flg22-induced ROS burst,indicating that both of them have the activity of suppressing plant basal defense.