Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants

Abstract

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps(-) mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps(-) mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps(-) mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.

Figure 1. Virulence of Ralstonia solanacearum strains GMI1000 and UW551 on resistant and susceptible tomato plants.

Unwounded susceptible (cv. Bonny Best) and horizontally resistant (H7996) tomato plants were soil-soak inoculated to a concentration of ∼1×10⁸ CFU/g soil and incubated at 28°C. Plants were rated daily on a 0 to 4 disease index scale where 0 = healthy and 4 = 100% wilted. Each point represents the mean disease index (± SE) for four independent experiments, each containing 16 plants per treatment.

Figure 2. Expression of tomato defense genes following soil-soak inoculation with Ralstonia solanacearum strains GMI1000 or UW551 in susceptible cultivar Bonny Best or horizontally resistant line H7996.

Genes represent activation of the jasmonic acid (JA) pathway (A: Pin2, B: LoxA), the ethylene (ET) pathway (C: PR-1b, D: Osm), and the salicylic acid (SA) pathway (E: GluA, F: PR-1a). Gene expression was measured by qRT-PCR in response to three pathogen cell densities: 1×10⁷ CFU/g stem (symptomless plants, white bars), 3×10⁸ CFU/g (symptomless or first wilting signs, grey bars), and 1×10⁹ CFU/g (early disease corresponding to DI = 1, black bars). Asterisks above bars indicate significant differences (P>0.05) in gene expression between mock and R. solanacearum inoculated tomatoes. P-values reflecting differences between cell densities (CFU), tomato cultivars and strains are shown in Table S2. Bars show normalized mean fold induction relative to mock-inoculated control plants (± SE). N = 6 to 12 plants for each cell density and strain, >3 independent experiments.

Figure 3. Expression of tomato defense genes following petiole inoculation with Ralstonia solanacearum wild-type strains and extracellular polysaccharide-deficient ΔepsB mutants.

Gene expression was measured in A: BW-susceptible (S) cv. Bonny Best infected with UW551 or UW551ΔepsB; B: BW-susceptible cv. Bonny Best infected with GMI1000 or GMI1000ΔepsB; and C: horizontally resistant (R) line H7996 infected with UW551 or UW551ΔepsB; D: horizontally resistant line H7996 infected with GMI1000 or GMI1000ΔepsB. Plants were inoculated through the cut petiole of the first true leaf. Genes represent activation of ET pathway (PR-1b), SA pathway (GluA), and JA pathway (Pin2). Gene expression was measured in response to two pathogen cell densities in tomato stem tissue: 1 to 5×10⁸ CFU/g stem and 6×10⁸ to1×10⁹ CFU/g. Asterisks above bars indicate significant differences in gene expression between wild-type strain and ΔepsB mutant (*  =  P>0.05, **  =  P = 0.001). Bars show normalized mean fold induction relative to mock-inoculated control plants (± SE). UW551: N = 8 to 15 plants per treatment, with 4 independent experiments; GMI1000: N = 6 to 11 plants per treatment, with 3 independent experiments.

Figure 4. Expression of tomato defense genes in response to purified Ralstonia solanacearum extracellular polysaccharide.

Gene expression was measured in bacterial wilt-susceptible cv. Bonny Best (S) and horizontally resistant line H7996 (R) by qRT-PCR 24 h after injection of 20 µg purified EPS through the cut petiole of the first true leaf directly into the vascular system. Genes represent activation of the ET pathway (PR-1b), the SA pathway (GluA), and the JA pathway (Pin2). Asterisks above bars indicate significant differences in gene expression between BW susceptible Bonny Best and horizontally resistant H7996 (***  =  P>0.0001). Bars show normalized mean fold induction relative to mock-inoculated control plants (± SE). N = 40 plants per treatment, in four independent experiments.

Figure 5. Accumulation of reactive oxygen species (ROS) in tomato stem tissue.

ROS were determined in A: horizontally BW-resistant tomato H7996 and B: susceptible cv. Bonny Best 48 h after infection with Ralstonia solanacearum wild-type strain UW551 or UW551ΔepsB or water (mock-inoculated control). At 48 h post-inoculation, stem cross-sections containing 10⁵ CFU/g bacteria were stained with 50 µM dihydrorhodamine 123 (DHR 123) and fluorescence microscopy was used to visualize the green fluorescence of rhodamine 123 generated by oxidizing DHR 123 by ROS. Three independent experiments each contained eight plants per treatment; representative results are shown.

Figure 6. Reactive oxygen species (ROS) accumulation in tomato leaves.

ROS were determined in A: horizontally BW-resistant line H7996 and B: BW-susceptible cv. Bonny Best 48 h after infusion with 1×10⁹ CFU/ml R. solanacearum strain UW551 or EPS I mutant UW551ΔepsB. ROS appears as a brown precipitate after leaves were stained with the in situ endogenous peroxidase-dependent histochemical stain 3,3′-diaminobenzidine (DAB). The experiment was repeated five times; representative results are shown.