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

Stratton Georgoulis, Katie E. Shalvarjian, Tyler C. Helmann, Corri D. Hamilton, Hans K. Carlson, Adam M. Deutschbauer, Tiffany M. Lowe-Power
doi: https://doi.org/10.1101/2020.08.31.276741
Stratton Georgoulis
aDept Plant Pathology, UC Davis, Davis, California, USA
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Katie E. Shalvarjian
bDept Plant and Microbial Biology, UC Berkeley, Berkeley, California, USA
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Tyler C. Helmann
bDept Plant and Microbial Biology, UC Berkeley, Berkeley, California, USA
cEmerging Pests and Pathogens Research Unit, Robert W. Holley Center, Agricultural Research Service, U.S. Department of Agriculture, Ithaca, NY, USA
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Corri D. Hamilton
dDept Plant Pathology, UW-Madison, Madison, Wisconsin, USA
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Hans K. Carlson
eEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
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Adam M. Deutschbauer
bDept Plant and Microbial Biology, UC Berkeley, Berkeley, California, USA
eEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
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Tiffany M. Lowe-Power
aDept Plant Pathology, UC Davis, Davis, California, USA
bDept Plant and Microbial Biology, UC Berkeley, Berkeley, California, USA
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  • For correspondence: tlowepower@ucdavis.edu
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Abstract

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.

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 two related bacterial strains in a common niche, tomato xylem sap. Fitness of 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 are putative adaptations to resist plant defenses, including antimicrobial proteins and specialized metabolites that damage bacterial membranes.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted September 01, 2020.
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Genome-wide identification of tomato xylem sap fitness factors for Ralstonia pseudosolanacearum and Ralstonia syzygii
Stratton Georgoulis, Katie E. Shalvarjian, Tyler C. Helmann, Corri D. Hamilton, Hans K. Carlson, Adam M. Deutschbauer, Tiffany M. Lowe-Power
bioRxiv 2020.08.31.276741; doi: https://doi.org/10.1101/2020.08.31.276741
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Genome-wide identification of tomato xylem sap fitness factors for Ralstonia pseudosolanacearum and Ralstonia syzygii
Stratton Georgoulis, Katie E. Shalvarjian, Tyler C. Helmann, Corri D. Hamilton, Hans K. Carlson, Adam M. Deutschbauer, Tiffany M. Lowe-Power
bioRxiv 2020.08.31.276741; doi: https://doi.org/10.1101/2020.08.31.276741

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