Degradation of the Plant Defense Signal Salicylic Acid Protects Ralstonia solanacearum from Toxicity and Enhances Virulence on Tobacco

Abstract

Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule.

Importance: Plant pathogens such as the bacterial wilt agent Ralstonia solanacearum threaten food and economic security by causing significant losses for small- and large-scale growers of tomato, tobacco, banana, potato, and ornamentals. Like most plants, these crop hosts use salicylic acid (SA) both indirectly as a signal to activate defenses and directly as an antimicrobial chemical. We found that SA inhibits growth of R. solanacearum and induces a general stress response that includes repression of multiple bacterial wilt virulence factors. The ability to degrade SA reduces the pathogen's sensitivity to SA toxicity and increases its virulence on tobacco.

FIG 1

SA degradation is generally conserved in the R. solanacearum species complex. (A) The nag SA degradation pathway in R. solanacearum. (B) Conservation of SA degradation in the R. solanacearum species complex. (Left) A whole-genome comparison phylogenetic tree of R. solanacearum strains constructed using the maximal unique matches (MUM) index (77). Phylotypes (I to IV) are indicated at the dividing branch points. (Center) Genetic conservation (>80% amino acid identity by BLASTp and synteny with the GMI1000 locus) of nagAaGHAbIKL encoding the SA degradation pathway, RSc1092 (1092) encoding a LysR-type transcriptional regulator, and two genes encoding porins, pcaK and RSc1084 (1084). Dark gray indicates the gene is present at a single nag locus. Light gray indicates that the gene is present but located at a different genomic locus. White indicates that the gene is absent, and diagonal lines indicate the gene is present as a putative pseudogene. (Right) Ability of strains to grow on SA. +, growth was observed on minimal medium plates supplemented with 2 mM SA; −, growth was not observed; n.d., growth was not determined because the authors of the genome announcements would not share the strains.

FIG 2

The nag genes are required for growth on SA. (A) Structure of the SA degradation gene cluster in R. solanacearum strain GMI1000. Regions deleted in the mutants are indicated by dotted lines above the map. The complementation constructs and the region used to introduce SA degradation ability to K60 (KRNP) are indicated by solid lines below the map. (B and C) Growth of GMI1000 nag mutants on SA and gentisic acid. Strains were grown at 28°C in liquid minimal medium with the indicated carbon source and concentration. Cell density was measured after 48 h. The averages of three biological replicates are shown, with bars indicating standard errors. Asterisks indicate P was <0.05 (Student’s t test).

FIG 3

SA modulates R. solanacearum gene expression. (A) SA inhibits R. solanacearum growth. Strain GMI1000 was grown in minimal medium plus succinate with increasing SA concentration. Cell density was measured based on the A₆₀₀ after 48 h. The averages of three biological replicates with standard errors are shown. (B) A proportional Venn diagram of expression patterns created using BioVenn (78). Gene expression was measured on a custom-designed R. solanacearum strain GMI1000 microarray chip as previously described (28). ORFs with relative expression levels in SA medium greater than 2-fold different and adjusted P values of <0.05 were classified as differentially expressed. (C and D) Heat maps show absolute expression of genes induced (C) and repressed (D) by SA. The gene class is listed to the left of the gene name/gene locus. Heat maps indicating low absolute expression (blue; 4.0) to high absolute expression (yellow; 14.0) are shown to the right of gene names. The fold change (0 µM SA versus 500 µM SA) is shown to the right of each heat map. Heat maps were generated in MeV (version 4.9; Dana-Farber Cancer Institute; http://www.tm4.org/mev.html).

FIG 4

The Nag pathway protects R. solanacearum from toxicity of SA, but not from that of gentisic acid. Strains were grown in minimal medium plus succinate with increasing SA (A and C) or increasing gentisic acid supplemented with constant 10 µM SA (B) to induce nag gene expression. Cell density was measured based on the A₆₀₀ after 48 h. The average results of three biological replicates with standard errors are shown. At time points marked with an asterisk, the SA-degrading strains (GMI1000 or K60+KRNP) grew better than strains that cannot degrade SA (the ΔnagGH and ΔnagAaGHAbIKL mutants and wild-type K60) (P < 0.005; Student’s t test).

FIG 5

The Nag pathway does not contribute to virulence of R. solanacearum on tomato. (A) Seventeen-day-old tomato plants with unwounded roots were soil soak inoculated by pouring bacterial suspensions into the pots (1 × 10⁸ CFU/g soil). Symptoms were rated on a 0 to 4 disease index scale corresponding to the percentage of wilted leaves. (B) Twenty-one-day-old tomato plants were petiole inoculated by placing a suspension of 500 cells on a freshly cut branch. (C) Bacterial density in stem tissue was quantified by dilution plating stem tissue from soil soak inoculated plants. Each symbol represents the population size in a single plant, and horizontal lines represent the geometric means. (D) Seventeen-day-old tomato plants with unwounded roots were soil soak inoculated with SA-degrading strain K60+KRNP or the isogenic strain that does not degrade SA (K60). Virulence of strains (A, B, and D) were not significantly different (P > 0.05; repeated measures ANOVA). Bacterial populations in tomato stem (B) were not significantly different (P > 0.05 at 4 and 7 days post-soil soak inoculation; Mann-Whitney test).

FIG 6

SA degradation increases virulence of R. solanacearum strain K60 on tobacco. (A) In vitro growth of K60 wild-type (K60), K60 with nagAaGHAbIKL_GMI1000 genes (K60+N), K60 with pcaK_GMI1000, RSc1092_GMI1000, nagAaGHAbIKL_GMI1000, and RSc1084_GMI1000 (K60+KRNP), and GMI1000 wild type on SA as sole carbon source. Asterisks indicate P was <0.05 (Student’s t test). (B) Virulence of strains K60 and K60+KRNP following soil soak inoculation of 3- to 4-week-old tobacco with unwounded roots. Data are the average results for 6 experiments with 45 total plants per strain (P < 0.05; repeated measures ANOVA). (C) R. solanacearum density in tobacco stem following soil soak inoculation. Each symbol represents the bacterial population size in a single plant (P > 0.05 at 4, 7, and 10 days post-soil soak inoculation; Mann-Whitney test).