Chemoperception of Specific Amino Acids Controls Phytopathogenicity in Pseudomonas syringae pv. tomato

Abstract

Chemotaxis has been associated with the pathogenicity of bacteria in plants and was found to facilitate bacterial entry through stomata and wounds. However, knowledge regarding the plant signals involved in this process is scarce. We have addressed this issue using Pseudomonas syringae pv. tomato, which is a foliar pathogen that causes bacterial speck in tomato. We show that the chemoreceptor P. syringae pv. tomato PscA (PsPto-PscA) recognizes specifically and with high affinity l-Asp, l-Glu, and d-Asp. The mutation of the chemoreceptor gene largely reduced chemotaxis to these ligands but also altered cyclic di-GMP (c-di-GMP) levels, biofilm formation, and motility, pointing to cross talk between different chemosensory pathways. Furthermore, the PsPto-PscA mutant strain showed reduced virulence in tomato. Asp and Glu are the most abundant amino acids in plants and in particular in tomato apoplasts, and we hypothesize that this receptor may have evolved to specifically recognize these compounds to facilitate bacterial entry into the plant. Infection assays with the wild-type strain showed that the presence of saturating concentrations of d-Asp also reduced bacterial virulence.IMPORTANCE There is substantive evidence that chemotaxis is a key requisite for efficient pathogenesis in plant pathogens. However, information regarding particular bacterial chemoreceptors and the specific plant signal that they sense is scarce. Our work shows that the phytopathogenic bacterium Pseudomonas syringae pv. tomato mediates not only chemotaxis but also the control of pathogenicity through the perception of the plant abundant amino acids Asp and Glu. We describe the specificity of the perception of l- and d-Asp and l-Glu by the PsPto-PscA chemoreceptor and the involvement of this perception in the regulation of pathogenicity-related traits. Moreover, a saturating concentration of d-Asp reduces bacterial virulence, and we therefore propose that ligand-mediated interference of key chemoreceptors may be an alternative strategy to control virulence.

Keywords: Pseudomonas syringae; chemoreceptors; virulence.

FIG 1

Chemoreceptor repertoire of P. syringae pv. tomato. LBDs were annotated according to the Pfam database (https://pfam.xfam.org/). The receptor studied is highlighted in boldface type.

FIG 2

Differential scanning fluorimetry-based ligand screening of PsPto-PscA-LBD. Shown are the melting temperature (T_m) changes for each of the 95 compounds present in the Biolog PM3B compound array of nitrogen sources with respect to the T_m of the ligand-free protein. The dashed line indicates the threshold of 2°C for significant hits. Data are the means and standard deviations from two assays.

FIG 3

Microcalorimetric studies showing the binding of different d- and l-amino acids to PsPto-PscA-LBD. (Top) Titration raw data for the injection of 8 μl of 0.5 to 1 mM ligand solutions into 15 μM PsPto-PscA-LBD. (Bottom) Integrated, dilution heat-corrected, and concentration-normalized peak areas fitted with the one-binding-site model of ORIGIN.

FIG 4

Quantitative assays of capillary chemotaxis of P. syringae pv. tomato (WT) and the PsPto-pscA mutant toward l-Asp (A), d-Asp (B), and l-Glu (C). The data have been corrected with the number of cells that swam into buffer-containing capillaries. Shown are means and standard errors from three independent experiments conducted in triplicate. Generalized linear models (GzLMs) were performed, followed by Fisher’s least significant difference (LSD) test (*, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001), with the exception of l-Asp at 0.5 mM and l-Asp at 5 mM, where ANOVA was performed, followed by Fisher’s LSD test (*, P < 0.05).

FIG 5

Biofilm formation in MGA medium. Total biofilm formation was quantified using the absorbance of crystal violet at 570 nm. Medium was supplemented with 0.5 mM d-Asp, 1 mM l-Glu, or 1 mM l-Asp. Shown are means and standard errors from at least three independent experiments conducted in triplicate. GzLM analysis was performed, followed by Fisher’s LSD test (****, P < 0.001).

FIG 6

Impact of pscA mutation on swarming motility. Five replicates of each strain were placed onto a single plate and examined after 16 h. (A) Photographs of representative swarm colonies. (B) Quantification of the lateral colony surface area in digital images of the colonies. Shown are means and standard errors from three independent experiments. GzLM analysis was performed, followed by Student’s t test (****, P < 0.001).

FIG 7

Effect of PsPto-pscA ligands on c-di-GMP levels. Fluorescence intensities of strains harboring the c-di-GMP biosensor plasmid pCdrA::gfp^S grown in M9 medium supplemented with d-Asp (A), l-Asp (B), and l-Glu (C) were determined. Shown are means and standard errors from three independent experiments. ANOVA was performed, followed by Fisher’s LSD test (*, P < 0.05). A.U., arbitrary units.

FIG 8

Effect of PsPto-PscA ligands on chemotaxis and c-di-GMP levels in a cheA2 mutant. (A) Quantitative capillary chemotaxis assay of P. syringae pv. tomato (WT) and the cheA2 mutant (PsPto-cheA2). (B) Fluorescence intensity of strains harboring the c-di-GMP biosensor plasmid pCdrA::gfp^S. Shown are means and standard errors from three independent experiments. GzLM analysis was performed, followed by Student’s t test (****, P < 0.001).

FIG 9

PsPto-PscA is required for the full virulence of P. syringae pv. tomato. (A) Virulence of P. syringae pv. tomato WT, mutant (PsPto-pscA), and complemented (PsPto-pscA-Comp) strains. (B) Plant colonization based on bacterial population sizes in tomato leaves at 6 days postinoculation, after spray inoculation of bacterial suspensions (10⁸ CFU/ml). Shown are means and standard errors from three independent experiments. ANOVA was performed, followed by Fisher’s LSD test (*, P < 0.05). (C) Virulence of P. syringae pv. tomato (WT) and a PsPto-pscA mutant when the indicated amino acids were added to the bacterial suspension before infection. (D) Plant colonization based on bacterial population sizes in tomato leaves at 6 days postinoculation, after spray inoculation of bacterial suspensions (10⁸ CFU/ml), in the presence of the indicated amino acids. Shown are means and standard errors from at least three independent experiments. GzLM analysis was performed, followed by Fisher’s LSD test (****, P < 0.001).