Salicylic acid, yersiniabactin, and pyoverdin production by the model phytopathogen Pseudomonas syringae pv. tomato DC3000: synthesis, regulation, and impact on tomato and Arabidopsis host plants

Abstract

A genetically tractable model plant pathosystem, Pseudomonas syringae pv. tomato DC3000 on tomato and Arabidopsis thaliana hosts, was used to investigate the role of salicylic acid (SA) and iron acquisition via siderophores in bacterial virulence. Pathogen-induced SA accumulation mediates defense in these plants, and DC3000 contains the genes required for the synthesis of SA, the SA-incorporated siderophore yersiniabactin (Ybt), and the fluorescent siderophore pyoverdin (Pvd). We found that DC3000 synthesizes SA, Ybt, and Pvd under iron-limiting conditions in culture. Synthesis of SA and Ybt by DC3000 requires pchA, an isochorismate synthase gene in the Ybt genomic cluster, and exogenous SA can restore Ybt production by the pchA mutant. Ybt was also produced by DC3000 in planta, suggesting that Ybt plays a role in DC3000 pathogenesis. However, the pchA mutant did not exhibit any growth defect or altered virulence in plants. This lack of phenotype was not attributable to plant-produced SA restoring Ybt production, as the pchA mutant grew similarly to DC3000 in an Arabidopsis SA biosynthetic mutant, and in planta Ybt was not detected in pchA-infected wild-type plants. In culture, no growth defect was observed for the pchA mutant versus DC3000 for any condition tested. Instead, enhanced growth of the pchA mutant was observed under stringent iron limitation and additional stresses. This suggests that SA and Ybt production by DC3000 is costly and that Pvd is sufficient for iron acquisition. Further exploration of the comparative synthesis and utility of Ybt versus Pvd production by DC3000 found siderophore-dependent amplification of ybt gene expression to be absent, suggesting that Ybt may play a yet unknown role in DC3000 pathogenesis.

FIG. 1.

P. syringae pv. tomato DC3000 produces SA and Ybt under iron-limiting conditions. (A) Overlaid HPLC chromatograms showing SA extracted from iron-limited (black) and high-iron (gray) DC3000 cultures. Fluorescence at excitation of 305 nm and emission of 407 nm, optimal for SA, is shown. The structure of SA is shown in the upper right corner. (B) Overlaid HPLC chromatograms showing Ybt-Fe³⁺ with Ybt extracted from iron-limited (black) and high-iron (gray) DC3000 cultures, measured at a Ybt-Fe³⁺ absorbance maximum (385 nm). Extracted Ybt and Ybt-Fe³⁺ was saturated with Fe³⁺ prior to HPLC analysis to facilitate comparison. The absorbance profile of Ybt (at peak maximum, 17.8 min) is shown in the inset. (C) LC-MS (positive-ion mode) analysis of the DC3000 Ybt HPLC fraction. Black arrows indicate the major ion intensities (m/z) associated with the iron-bound monomer (m/z, 535.1 [FeM+H]⁺), dimer (1,068.7), and trimer (1,603.5); gray arrows indicate dominant cleavage products. The structure of Ybt is shown in the upper right corner.

FIG. 2.

Organization of the putative Ybt cluster in the genome of P. syringae pv. tomato DC3000. Gene names are modeled after the Ybt cluster in Y. pestis, except for pchA and pchB, which are named after the P. aeruginosa orthologs. P. syringae pv. tomato DC3000 genes from PSPTO2595 (pchA) to PSPTO2606 (ybtA) are shown. ybtA is shaded light gray and is an AraC-type transcriptional regulator. Transport genes are shaded black and consist of the TonB-dependent receptor fyuA and two ABC transporters, ybtP and ybtQ. Biosynthetic genes are shaded dark gray and include the isochorismate synthase gene pchA and the salicyl-AMP ligase gene ybtE. ybtX is shaded white and has no identified function in Y. pestis.

FIG. 3.

Synthesis of SA and Ybt requires a functional pchA gene. Shown are SA (gray) and Ybt (black) measured in iron-limited culture supernatants at 24 hpi with wild-type (wt) P. syringae pv. tomato DC3000 or the pchA DC3000 mutant. The DC3000 pchA mutation is in the isochorismate synthase gene of the putative Ybt biosynthetic cluster. nd, not detected (below detection limit).

FIG. 4.

In planta bacterial growth of P. syringae pv. tomato DC3000 and the pchA mutant. Bacterial growth in planta is shown for infection of susceptible plant hosts: tomato (A), Arabidopsis thaliana (B), and the Arabidopsis ics1 mutant, which has a defect in the synthesis of pathogen-induced SA (C). Mature leaves of these plants were infiltrated with P. syringae pv. tomato DC3000 (black) or the pchA mutant (gray) at a dose of 5 × 10⁵ CFU/ml. In planta bacterial growth was assessed at 0, 1, and 3 dpi.

FIG. 5.

Detection of Ybt in planta. LC-MS analysis of Ybt from Arabidopsis leaf extracts harvested 5 dpi with P. syringae pv. tomato DC3000 (A), the pchA mutant (B), or control treatment (10 mM MgS0₄) (C). ESI (positive ion) of 200 to 1,200 for the elution time of Ybt-Fe³⁺ is shown. Major ion intensities (m/z) for the iron-bound Ybt monomer (m/z, 535.1; dimer, 1,068.7) are indicated with an arrow where present.

FIG. 6.

Production of Pvd by P. syringae pv. tomato DC3000. (A) HPLC chromatogram showing Pvd peaks, measured by absorbance at 400 nm for culture extracts isolated from iron-limited cultures (black) or high-iron cultures (gray). The absorbance profile of the dominant Pvd peak at its maximum (10.7 min) is shown in the inset. (B) LC-MS (positive-ion mode) analysis of the DC3000 dominant Pvd HPLC fraction. The relative abundance of the major ion intensities (m/z) from 1,120 to 1,240 is shown.

FIG. 7.

Bacterial growth and siderophore production in culture as iron is progressively more limited through the addition of 2,2-dipyridyl. Siderophores were measured in culture supernatants at 24 hpi. (A) P. syringae pv. tomato DC3000 growth (OD₆₀₀) and Ybt production (nmol). (B) P. syringae pv. tomato DC3000 growth (OD₆₀₀) and Pvd production (nmol). (C) Growth (OD₆₀₀) of P. syringae pv. tomato DC3000 compared with that of the pchA mutant. (D) Pvd production (nmol) by P. syringae pv. tomato DC3000 compared with that of the pchA mutant.

FIG. 8.

Conditional experiments varying temperature, redox conditions, and pH show enhanced growth of the pchA mutant compared with Pst DC3000 under stringent iron-limiting conditions. Bacterial growth (OD₆₀₀) was assessed for cultures at 24 hpi under iron-limiting conditions (with 150 μM dipyridyl) with varying levels of temperature, oxidant (H₂O₂), reductant (DTT), or pH to assess differences in bacterial growth under potentially environmentally relevant conditions. Data are normalized to growth of DC3000 under standard conditions (20°C, pH 7.2).

FIG. 9.

Time course of growth and siderophore production by P. syringae pv. tomato DC3000 and the pchA mutant under low-iron conditions. (A) Pvd and Ybt levels in culture supernatants of P. syringae pv. tomato DC3000. (B) Growth curve for P. syringae pv. tomato DC3000 and the pchA mutant. (C) Pvd levels in culture supernatants of DC3000 or the pchA mutant. Low-iron conditions are equivalent to no added dipyridyl or added FeCl₃.

FIG. 10.

Expression of SA, Ybt, and Pvd biosynthetic genes by DC3000 and the pchA mutant in high-iron or low-iron cultures. Shown is normalized expression of pchA (A), ybtE (B), and pvdA (C) in wild-type P. syringae pv. tomato DC3000 (black) and the pchA mutant (gray) at 24 hpi in high-iron (with 50 μM FeCl₃) or low-iron medium. pchA (PSPTO2595) is required for SA and Ybt biosynthesis. ybtE (PSPTO2597) is required for Ybt biosynthesis from SA. pvdA (PSPTO2135) is required for Pvd biosynthesis. RNA isolation and qPCR were performed as described in Materials and Methods. Gene expression was normalized to 16S rRNA for each sample and multiplied by 1,000 for ease of presentation. nd, not detected (below detection limit).