Transcriptome analysis of quantitative resistance-specific response upon Ralstonia solanacearum infection in tomato

Abstract

Bacterial wilt, caused by the soil-borne bacterium Ralstonia solanacearum, is a lethal disease of tomato, but the molecular mechanisms of the host resistance responses to R. solanacearum remain unclear. In this study, we report the first work describing the transcriptome of cultivar resistance and susceptible tomato cultivar after inoculation with R. solanacearum. To elucidate the characteristics of resistance early in the interaction, we analyzed microarrays for resistant cultivar LS-89 and susceptible cultivar Ponderosa 1 day after stem inoculation. No change in gene expression was detected for Ponderosa, but expression levels of over 140 genes, including pathogenesis-related, hormone signaling and lignin biosynthesis genes, increased in LS-89. Expression of β-1,3-glucanase genes increased substantially. In an immunohistochemical study, glucanase in LS-89 accumulated in the xylem and pith tissues surrounding xylem vessels filled with R. solanacearum. The expression of these genes also increased in four other resistant cultivars, but changed little in four susceptible cultivars in response to R. solanacearum, suggesting that similar reactions occur in other cultivars. These gene expression profiles will serve as fundamental information to elucidate the molecular mechanisms in the resistance response to R. solanacearum in tomato.

Figure 1. Symptoms on susceptible tomato cv. Ponderosa and resistant tomato cv. LS-89 after inoculation with Ralstonia solanacearum or water (mock) and bacterial density over time.

(A) Symptoms at 1 and 7 dpi. (B) Bacterial density, determined on selective medium, in stems of four to eight randomly selected plants at 1, 2, 3, 4, 6 and 8 dpi.

Figure 2. Scatter plots of mean logarithmic values for gene expression in LS-89 and Ponderosa at 1 dpi after inoculation with Ralstonia solanacearum or water (mock).

In triplicate experiments using GeneChip, tomato probe sets with less than three ‘present’ calls were removed from the plot. Red squares: genes with significantly increased expression, blue diamonds: genes with significantly suppressed expression, closed circles: genes with unaltered expression. Gray lines: regression lines, red lines: 2-fold induction, blue lines: 2-fold suppression. Regression equation and correlation coefficient (R) are also shown.

Figure 3. Real-time quantitative RT-PCR analysis of time course of relative transcription levels of β-1,3-glucanase genes in LS-89 and Ponderosa after inoculation with Ralstonia solanacearum or water (mock).

Total RNA was extracted from stems to analyze expression of acidic class III (A), basic class III (B) and class II (C) β-1,3-glucanase genes at 0, 12, 24 and 48 hpi. Sample from Ponderosa at 0 hpi was used for calibration.

Figure 4. Molecular functional groups of genes that were differentially expressed in resistant tomato cultivar LS-89 after inoculation with Ralstonia solanacearum.

Up- (A) and down- (B) regulated genes were classified according to Molecular Function in Gene Ontology. Genes with ‘catalytic activity’ were further subdivided into enzymatic function. Numbers indicate the number of genes found for each group.

Figure 5. Comparison of autofluorescence of lignin in stem sections of susceptible tomato cv. Ponderosa (A) and resistant cv. LS-89 (B) after inoculation with water (mock) or Ralstonia solanacearum.

In stems illuminated with UV and observed with a fluorescence microscope, green autofluorescence of xylem was present in all samples because lignin is naturally extremely high in xylem; the green autofluorescence of xylem in the pith increased only in the bacteria-inoculated LS-89 stems. Chloroplasts autofluoresce red. X, xylem; V, vessel; P, pith. Bar = 100 µm.

Figure 6. Immunohistochemical detection of Ralstonia solanacearum or tobacco PR-N protein, a class II acidic β-1,3-glucanase, in stem sections from susceptible tomato cv. Ponderosa at 4 dpi (A) and resistant cv. LS-89 at 2 dpi (B) and 4 dpi (C) after inoculation with R. solanacearum.

Sections were exposed to antibodies against the bacteria or the protein, then stained with a VECTASTAIN ABC kit to localize the antibodies seen as blue signals (arrows). X, xylem; V, vessel; P, pith. Bar = 100 µm.

Figure 7. Real-time quantitative RT-PCR of relative transcription levels of β-1,3-glucanase genes in resistant and susceptible tomato cultivars at 1 dpi with water (mock, M) or Ralstonia solanacearum (R).

Acidic class III (A), basic class III (B) and class II (C) β-1,3-glucanase genes were analyzed in total RNA from stems of five resistant cultivars LS-89, Volante (Vola), Anchor T (Anch), Ganbarune (Gan) and Hawaii7996 (Haw) and five susceptible cultivars (S) Ponderosa (Pon), Micro-Tom (Micro), Momotaro (Mom), House-Momotaro (H-Mo) and Bonny Best (Bonny). Sample from mock-inoculated Ponderosa stems was used for calibration.