Study On The Biological Functions Of Effectors RipAX1 And RipAX2 Of Ralstonia Solanacearum

Bacterial wilt caused by Ralstonia solanacearum can harm many Solanaceae economic crops and cause serious economic losses in agricultural production due to the difficulty of control.Ralstonia solanacearum uses many different virulence factors to cause disease in host plants,among which the effector proteins secreted by the type III secretion system are key for Ralstonia solanacearum pathogenicity.Two effector proteins RipAX1 and RipAX2 with unknown functions were previously screened in our laboratory.In this paper,the biological functions of the two proteins were systematically studied by analyzing the structure of effector proteins,observing the subcellular localization,detecting plant immune response changes upon the transient expression of these effectors,constructing knockout strains to detect the pathogenicity,and screening transgenic Arabidopsis thaliana and Solanum lycopersicum to detect the resistance to pathogens.The main results are as follows:By analyzing the structure of RipAX1 and RipAX2 proteins,it was found that RipAX1 and RipAX2 proteins contain a HEXXH motif,which presents in typical zinc metallopeptidases;The subcellular localization in tobacco showed that RipAX1 was located in the nucleus and cell membrane,and RipAX2 was located in the nucleus and nucleolus;Transient expression of RipAX1 and RipAX2 in Arabidopsis protoplasts attenuated the burst of reactive oxygen species(ROS)induced by flg22;Transient expression of RipAX2 in tobacco enhanced the flg22-induced ROS burst.In order to explore their functions in the pathogenicity of Ralstonia solanacearum,ΔripAX1 and ΔripAX2 single and double mutants were constructed.It was found that the deletion of RipAX1 and RipAX2 did not affect the growth rate,motility and extracellular polysaccharide level of Ralstonia solanacearum,but might affect some metabolic pathways of Ralstonia solanacearum.The pathogenicity of single mutant strains ΔripAX1 andΔripAX2 to Arabidopsis thaliana was weaker than that of wild-type strain.The pathogenicity of the double mutant strains was decreased not only to Arabidopsis thaliana,but also to Solanum lycopersicum,which might be caused by the proliferation difference in tomato stem base.In order to further study the functions of the two effector proteins in plants,overexpression transgenic Arabidopsis thaliana and Solanum lycopersicum were generated.Ox RipAX1 in Arabidopsis had no significant difference in resistance to Ralstonia solanacearum and Pseudomonas syringae compared with the wild type plants,but showed attenuated flg22-induced ROS burst and MAPKs activation,and cpmpromised expression of early immune genes.Arabidopsis oxRipAX2 plants showed comparable flg22-indued immune responses and similar reisistance to Ralstonia solanacearum and Pseudomonas syringae with the wild type plants.However,the expression of early immune genes in oxRipAX2 transgenic Arabidopsis was significantly down-regulated.It was also found that oxRipAX2 transgenic Solanum lycopersicum exibited not only attenuated resistance to Ralstonia solanacearum,but also compromised flg22-induced MAPKs activation.In conclusion,two effector proteins RipAX1 and RipAX2 function as virulence factors to participate in the pathogenicity of Ralstonia solanacearum to Arabidopsis and Solanum lycopersicum,which interfere with host immune responses and affect especially the expression of plant immune genes.The results of this study provide clues for further revealing the pathogenesis of Ralstonia solanacearum.