| Autor: |
Georgoulis SJ; Department of Plant Pathology, University of California Davis, Davis, California, USA., Shalvarjian KE; Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, California, USA., Helmann TC; Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, California, USA.; Emerging Pests and Pathogens Research Unit, Robert W. Holley Center, Agricultural Research Service, U.S. Department of Agriculture, Ithaca, New York, USA., Hamilton CD; Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin, USA., Carlson HK; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Deutschbauer AM; Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, California, USA.; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA., Lowe-Power TM; Department of Plant Pathology, University of California Davis, Davis, California, USA.; Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, California, USA. |
| Abstrakt: |
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 three strains spanning the genetic, geographical, and physiological range of plant-pathogenic Ralstonia : Ralstonia solanacearum IBSBF1503, Ralstonia pseudosolanacearum GMI1000, and Ralstonia syzygii PSI07. Contrasting mutant fitness phenotypes in culture media versus in xylem sap suggest that Ralstonia strains are adapted to ex vivo xylem 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. Each strain required many genes associated with envelope remodeling and repair processes for full fitness in xylem sap. These genes were associated with peptidoglycan peptide formation ( murI ), secretion of periplasmic proteins ( tatC ), periplasmic protein folding ( dsbA ), synthesis of osmoregulated periplasmic glucans ( mdoGH ), and lipopolysaccharide (LPS) biosynthesis. Mutant strains with mutations in four genes had strong, sap-specific fitness defects in all strain backgrounds: murI , thiC , purU , and a lipoprotein (RSc2007). Many amino acid biosynthesis genes were required for fitness in both minimal medium and xylem sap. Multiple mutants with insertions in virulence regulators had gains of fitness in culture media and neutral fitness in sap. Our genome-scale genetic screen identified Ralstonia fitness factors that promote growth in xylem sap, an ecologically relevant condition. IMPORTANCE Traditional transposon mutagenesis genetic screens pioneered molecular plant pathology and identified core virulence traits like the type III secretion system. TnSeq approaches that leverage next-generation sequencing to rapidly quantify transposon mutant phenotypes are ushering in a new wave of biological discovery. Here, we have adapted a genome-scale approach, random barcoded transposon mutant sequencing (RB-TnSeq), to discover fitness factors that promote growth of three related bacterial strains in a common niche, tomato xylem sap. Fitness of the wild type and mutants show that Ralstonia spp. are adapted to grow well in xylem sap from their natural host plant, tomato. Our screen identified multiple sap-specific fitness factors with roles in maintaining the bacterial envelope. These factors include putative adaptations to resist plant defenses that may include antimicrobial proteins and specialized metabolites that damage bacterial membranes. |
