Genome-wide identification of tomato xylem sap fitness factors for three plant-pathogenicRalstoniaspecies

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.ImportanceTraditional 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.

Trophic preferences of the pathogen Ralstonia solanacearum and consequences on its growth in xylem sap

“…The plants had the same age, and the xylem extraction technique and analysis of the xylem content were similar. The difference observed might be due to the cultivar (Georgoulisa et al, 2020 ), but also to the fertilizer (Bialczyk et al, 2004 ), since in the M82 experiment, tomato plants were grown in soil (SB2, Proveen, The Netherlands) supplemented with Osmocote® (Sierra Chemical Company), whereas in this study tomato plants were grown in the same soil without any supplementation by fertilizer.…”

“…The large variability in the C/N ratio depends on how much sugar and acids from the Krebs cycle are present in the xylem, and whether or not they were measured (due to different analytical techniques used). Age of the plants and culture growth conditions can also explain some of these differences and variations in xylem sap composition between two tomato cultivars have already been highlighted (Georgoulisa et al, 2020 ). The methods of extraction of xylem fluids can differ, some of which may lead to phloem fluid contamination.…”

“…The plants had the same age, and the xylem extraction technique and analysis of the xylem content were similar. The difference observed might be due to the cultivar (Georgoulisa et al, 2020), but also to the fertilizer (Bialczyk et al, 2004) The composition of xylem sap is quite conserved among plants (Andersen & Brodbeck, 1989;Gerlin et al, 2021a;Hocking, 1980;Anguita Maeso et al, 2021), with glutamine being the most abundant amino acid in most cases. As common xylem sap molecules can be efficiently metabolized by GMI1000, thus confirming it is overall well-adapted to the xylem sap environment, in which it can thrive easily (Gerlin et al, 2021a;Lowe-Power et al, 2018a).…”

Bacterial surface‐exposed lipoproteins and sortase‐mediated anchored cell surface proteins in plant infection

“…Surface-associated proteins participate in plant colonization and infection by bacteria, and some are important in phytopathogenic bacterial fitness. For example, gene RSc2007, encoding an unknown lipoprotein (UniProt Id: Q8XXV6), appears to be a xylem sap fitness factor in R. solanacearum (Georgoulis et al, 2021). Importantly, lipoproteins could be the target of potential biocontrol targets, such as the coumarins that inhibit the expression of the outer membrane lipoprotein OmlA in X. campestris (Fuangthong et al, 2008).…”

Genome-Wide Identification of Genes Important for Growth of Dickeya dadantii and Dickeya dianthicola in Potato (Solanum tuberosum) Tubers

“…and Ralstonia spp. ( Cole et al, 2017 ; Helmann et al, 2019 ; Georgoulis et al, 2021 ). A motivating factor for this study was to demonstrate the value of RB-TnSeq to characterize necrotrophic plant pathogenic bacteria in planta ; using both an established model strain as well as recently isolated and uncharacterized strains ( Liu and Deutschbauer, 2018 ).…”