Genome-wide identification of tomato xylem sap fitness factors for Ralstonia pseudosolanacearum and Ralstonia syzygii

Plant pathogenic Ralstonia spp. colonize plant xylem and cause wilt diseases on a broad range of host plants. To identify genes that promote growth of diverse Ralstonia strains in xylem sap from tomato plants, we performed genome-scale genetic screens (random barcoded transposon mutant sequencing screens; RB-TnSeq) in Ralstonia pseudosolanacearum GMI1000 and R. syzygii PSI07. Contrasting mutant fitness phenotypes in culture media versus in xylem sap suggest that Ralstonia strains are adapted to sap and that culture media impose foreign selective pressures. Although wild-type Ralstonia grew in sap and in rich medium with similar doubling times and to a similar carrying capacity, more genes were essential for growth in sap than in rich medium. Multiple mutants lacking amino acid biosynthesis and central metabolism functions had fitness defects in xylem sap and minimal medium. Our screen identified > 26 genes in each strain that contributed to growth in xylem sap but were dispensable for growth in culture media. Many sap-specific fitness factors are associated with bacterial stress responses: envelope remodeling and repair processes such as peptidoglycan peptide formation (murI and RSc1177), LPS O-antigen biosynthesis (RSc0684), periplasmic protein folding (dsbA), drug efflux (tolA and tolR), and stress responses (cspD3). Our genome-scale genetic screen identified Ralstonia fitness factors that promote growth in xylem sap, an ecologically relevant condition.