The in planta transcriptome of Ralstonia solanacearum: conserved physiological and virulence strategies during bacterial wilt of tomato

Abstract

Plant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogen Ralstonia solanacearum is well adapted to it, growing to 10(8) to 10(9) CFU/g tomato stem. To better understand how R. solanacearum succeeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinct R. solanacearum strains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 10(8) CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly altered in planta versus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in the scrABY cluster. A UW551 scrA mutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed host Solanum dulcamara. Functional scrA contributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose. scrA expression was induced by sucrose, but to a much greater degree by growth in planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulated in planta at high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle. IMPORTANCE Ralstonia solanacearum is a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions that R. solanacearum expresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen's dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further, R. solanacearum was highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops.

FIG 1

Microarray experimental design and flowchart. We compared transcriptional profiles of phylogenetically and ecologically distinct R. solanacearum strains GMI1000 (orange) and UW551 (green) during growth in tomato stems and in rich medium (CPG) at comparable cell densities. Bacteria grown in culture were treated with a transcriptional stop solution and pelleted. Bacteria were harvested from infected tomato xylem by centrifugation from cut stems into a transcriptional stop solution. Bacterial RNA was isolated from four biological replicates per strain per condition (16 replicates total).

FIG 2

Gene expression heat map of experimentally determined bacterial wilt virulence and fitness factors. (A) Absolute log₂ gene expression levels of known virulence genes in R. solanacearum UW551 (551) and GMI1000 (GMI) was artificially clustered with The Institute for Genomic Research (TIGR) multiple experiment viewer (MeV). Asterisks (*) designate genes previously identified in an IVET screen of R. solanacearum genes induced in tomato (28). The color indicates low (7.0 [blue]) to high (14.0 [yellow]) absolute log₂ expression levels. (B and C) Absolute expression per cell (AEU) of hrpB (B) and popA (C) was quantified using qPCR at low and high cell densities both in planta and in culture. For gene expression in culture, R. solanacearum strain GMI1000 was grown in rich medium (CPG) to cell densities of 2 × 10⁷ CFU/ml and 7 × 10⁸ CFU/ml. The strain was also grown in minimal medium to a final cell density of 8 × 10⁸ CFU/ml (BMM). For in planta gene expression, susceptible Bonny Best tomato plants were inoculated with strain GMI1000 via soil soak. RNA was isolated from plant stems showing early wilt symptoms and containing 1 × 10⁹ CFU/g stem (in planta high) and from asymptomatic plants containing 3 × 10⁷ CFU/g stem (in planta low). Purified RNA was reverse transcribed into cDNA and measured by quantitative real-time PCR. The transcript cycle threshold (C_T) values of hrpB and popA were plotted on their respective cDNA standard curves, and the amount of transcript per cell was used to determine the arbitrary absolute expression unit (AEU). Each point represents the mean absolute expression value of three biological replicates; the error bars indicate the standard error (P < 0.05 by ANOVA).

FIG 3

The scrRABY sucrose metabolic cluster is induced in planta and encodes a sucrose-specific PTS. (A) R. solanacearum converts sucrose to glucose-6-phosphate (glucose-6-P) and fructose-6-P with the components of a sucrose-specific PTS encoded by the scr cluster, which was highly expressed in planta. The absolute log₂ gene expression from bacteria growing in planta or in rich medium is shown in a heat map layered on the scr gene cluster. The location of the insertional mutation in scrA (scrA::Km^r or scrA) is indicated by a black triangle. (B) Relative fold change of scrA expression in R. solanacearum UW551 cells growing in culture media (CPS, broth containing Casamino Acids, peptone, and 55 mM sucrose; BMM, Boucher’s minimal medium; SUC, sucrose; GLUC, glucose) or in Bonny Best tomato stems. Gene expression was measured at a cell density of 1 × 10⁹ CFU/ml broth or 1 × 10⁹  CFU/g tomato stem and determined on a per-cell basis. The black bars represent the mean scrA expression fold change for each growth condition compared to the level in CPG medium, with three biological replicates for culture and four biological replicates for in planta expression. R. solanacearum UW551 scrA expression fold changes in planta were significantly different from expression levels in all culture media tested (P < 0.03 by Mann-Whitney test). (C) A UW551 scrA mutant grew as well as the wild type (WT) on minimal medium (BMM) plus glucose but failed to grow on sucrose as the sole carbon source. Growth on sucrose was restored by scrRABY in trans on plasmid pUFJ10.

FIG 4

Differential virulence of the R. solanacearum UW551 scrA mutant on susceptible and resistant hosts. (A to D) The wild-type R. solanacearum UW551 (WT) (blue triangles) and scrA mutant (red squares) were inoculated by soil soaking onto the unwounded roots of susceptible tomato cultivar Bonny Best (A), quantitatively resistant tomato cultivar Hawaii7996 (B), potato cultivar Russet Norkotah (C), and solanaceous nightshade (D). Plant symptoms were rated daily on a disease index scale from 0 to 4 (0 for healthy or no leaf area wilted, 1 for 1 to 25% of the leaf area wilted, 2 for 26 to 50% of the leaf area wilted, 3 for 51 to 75% of the leaf area wilted, and 4 for 76 to 100% of the leaf area wilted). Each point indicates the mean disease index for three independent inoculations (A, B, and D) or one experiment (C); the scrA mutant was significantly less virulent than the wild type on each host (P < 0.001 by repeated measures ANOVA).

FIG 5

Sucrose metabolism provides R. solanacearum with a competitive advantage during xylem colonization. (A to C) Twenty-two-day-old wilt-resistant H7996 tomato plants were inoculated through a cut petiole with 4 µl of wild-type R. solanacearum UW551 (WT) or UW551 scrA::Km^r (UW551 scrA) (A and B) or with a 1:1 mixture of wild-type R. solanacearum UW551 Rif^r and scrA mutant (~2,000 CFU total) (C). (A) Wilt disease progress of wild-type UW551 and scrA mutant strains on stem-inoculated tomato plants that were rated as described in the legend to Fig. 4. (B) Stem colonization ability of wild-type UW551 and scrA mutant cells stem inoculated separately on tomato plants. Ten plants displaying a disease index of 1 (corresponding to 5 days after inoculation) were sampled per treatment (UW551 WT or UW551 scrA mutant), and the bacterial population sizes in the stems were quantified by dilution plating. Each symbol represents one plant; short horizontal lines represent the mean bacterial colonization in tomato stems. (C) Competitive fitness of wild-type and scrA mutant strains. The tomato plants were sampled 5 days postinoculation, and in planta population sizes of wild-type and scrA mutant strains were quantified by dilution plating. Data represent two biological replicates containing 30 plants each. The WT strain significantly outcompeted the scrA mutant in planta (P < 0.006 by paired Student’s t test).

FIG 6

Select biochemical pathways of the R. solanacearum core genome represented in the R. solanacearum in planta transcriptome. Bacterial processes previously not known to be active during tomato xylem colonization are shown in green. Abbreviations: PTS, phosphoenolpyruvate-carbohydrate phosphotransferase system; Suc-6-P, sucrose-6-phosphate; Fruc, fructose; G-6-P, glucose-6-phosphate; F-6-P, fructose-6-phosphate; F-1,6-P2, fructose-1,6-bisphosphate; PEP, phosphoenolpyruvate; CoA, coenzyme A; TCA, tricarboxylic acid; αKG, alpha-ketoglutarate; Ribulose-5-P, ribulose-5-phosphate; PPP, pentose phosphate pathway; EPS, exopolysaccharide; ROS, reactive oxygen species; T2SS, type 2 secretion system; CWDEs, cell wall-degrading enzymes; T3, type 3; T3SS, type 3 secretion system.