Identification And Functional Studies Of Ralstonia Solanacearum Effectors Ripu

Ralstonia solanacearum is a Gram-negative pathogen of the genus Ralstonia,which is capable of infecting many important crops including tomato,potato,and tobacco.Similar to many plant pathogens,R.solanacearum is able to attack the host defense system through effector proteins(T3Es)secreted by its type III secretion system(T3SS),altering the host immune mechanism and helping it to complete its infestation and colonization.Although more and more researchers are now focusing on the function of effector proteins in R.solanacearum,the number of effector proteins with known functions in R.solanacearum is still small.RipU is an effector protein secreted by strain GMI1000 of R.solanacearum,but its physiological and biochemical functions are currently unknown.In this study,we firstly predicted the secondary structure domain,threedimensional structure and subcellular localization of RipU by using bioinformatics prediction and analysis;the gene encoding RipU was cloned into a heterologous expression vector and expressed in Saccharomyces cerevisiae and tobacco cells,and found that RipU did not produce significant growth inhibition in S.cerevisiae and did not cause hypersensitivity response in tobacco leaf tissue;The results of subcellular localization experiments revealed that RipU is localized in the cytoplasm and peroxisomes of tobacco and Arabidopsis;Through the omics network of the interaction between the effector protein of R.solanacearum and the protein of Arabidopsis and in vitro pull down experiment combined with proteome sequencing analysis,we obtained10 Arabidopsis proteins with potential interactions with RipU;through yeast twohybrid,bimolecular fluorescent complimentary and co-immunoprecipitation experiments,we confirmed the interaction between RipU and its potential interacting protein,TOPP9.The results of this study showed that RipU did not affect the growth of eukaryotic cells;RipU was localized in the cytoplasm and peroxisomes of plant cells;and RipU interacted with the Arabidopsis protein TOPP9.TOPP9 is a type one protein phosphatase that affects pollen tube and root hair growth in Arabidopsis thaliana,which can be used as an entry point for further studies on the effects of RipU on plant immune mechanisms.The results of this study laid the foundation for the subsequent in-depth investigation of the physiological and biochemical functions of RipU,and also enriched our understanding of the pathogenic mechanism of R.solanacearum.It is hoped that more in-depth studies will follow to elucidate the function and mechanism of RipU,making it an effective tool for studying plant immunity,providing new ideas for exploring the mechanism of plant infestation by Ralstonia solanacearum,and providing new entry points for exploring the functions of different components of plant immunity.