Genome-wide Identification And Functional Exploration Of Ralstonia Solanacearum Genes Required For Survival In Tomato Plants

Bacterial wilt is a devastating soil-borne bacterial disease caused by Ralstonia solanacearum,causing extremely damaging to agricultural production.R.solanacearum is genetically diverse,infecting more than hundreds plant species over a broad geographical range.An effective method to control bacterial wilt is still lacking.R.solanacearum invades from plant roots and colonizes rapidly in xylem vessels,resulting in xylem vessels obstruction and dysfunction,and finally leads to plant wilting and death.Tomato xylem is a nutritionally limiting and dynamically changing habitat.The reason for the rapid colonization of R.solanacearum in xylem is not clear.In this study,genes of R.solanacearum required for survival in tomato plants were identified at the genome-wide level,which promoted the comprehensive understanding of the pathogenic mechanism of R.solanacearum.At the same time,the function of ATP dependent protease ClpP encoded by gene clp P of R.solanacearum required for survival in tomato plants was also deeply studied,so as to provide a new potential drug target for the control of bacterial wilt.This study includes the following aspects:1.In this study,a nearly saturated transposon insertion library of R.solanacearum GMI1000 was constructed,and the whole genome coverage of the insertion site was as high as 70.44%-80.96%,which can be used to identify the genes of R.solanacearum required for survival in different environments.2.The transposon insertion library was injected into tomato to screen the genes of R.solanacearum required for survival in tomato plants.The results showed that 130 genes were identified as genes required for the survival of R.solanacearum GMI1000 in tomato plants.Among these 130 genes,20 genes are related to "cell wall / membrane / envelope biogenesis";19 genes were annotated as "amino acid transport and metabolism";13genes involved "energy production and conversion" and 11 genes are associated with "posttranslational modification,protein turnover and chaperones".3.In this study,16 potential genes of R solanacearum required for survival in tomato plants were selected,knocked out one by one,and the corresponding gene deletion mutants were obtained.The growth curve and pathogenicity of 16 mutants were measured.It was found that the growth of 8 mutants exhibited impaired in rich BG medium;The pathogenicity of9 mutants was weaker than that of wild-type GMI1000.4.The colonization and adaptive competition index of 7 mutants in tomato xylem were measured.It was found that the gene RS＿RS01965,the gene RS＿RS04475,the gene RS＿RS09970,the gene RS＿RS04490,the gene RS＿RS10340 and the gene RS＿RS08645 is required for R.solanacearum to survival in tomato plants,which is consistent with the results of Tn-seq.5.The gene RS＿RS08645 encodes ATP dependent protease ClpP.In this study,the biological phenotype of ΔRS＿RS08645 was measured.It was found that ClpP protease positively regulated the growth and pathogenicity of R.solanacearum,negatively regulated motility and biofilm formation.The critical involvement of regulate at serine 120 of ClpP in R.solanacearum.The ATPase Clp X and Clp A,which are dependent on ClpP protease,positively regulate the growth,pathogenicity and motility of R.solanacearum,and negatively regulate the biofilm formation.