Identification of a twin-arginine translocation system in Pseudomonas syringae pv. tomato DC3000 and its contribution to pathogenicity and fitness

Abstract

The bacterial plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) causes disease in Arabidopsis thaliana and tomato plants, and it elicits the hypersensitive response in nonhost plants such as Nicotiana tabacum and Nicotiana benthamiana. While these events chiefly depend upon the type III protein secretion system and the effector proteins that this system translocates into plant cells, additional factors have been shown to contribute to DC3000 virulence and still many others are likely to exist. Therefore, we explored the contribution of the twin-arginine translocation (Tat) system to the physiology of DC3000. We found that a tatC mutant strain of DC3000 displayed a number of phenotypes, including loss of motility on soft agar plates, deficiency in siderophore synthesis and iron acquisition, sensitivity to copper, loss of extracellular phospholipase activity, and attenuated virulence in host plant leaves. In the latter case, we provide evidence that decreased virulence of tatC mutants likely arises from a synergistic combination of (i) compromised fitness of bacteria in planta; (ii) decreased efficiency of type III translocation; and (iii) cytoplasmically retained virulence factors. Finally, we demonstrate a novel broad-host-range genetic reporter based on the green fluorescent protein for the identification of Tat-targeted secreted virulence factors that should be generally applicable to any gram-negative bacterium. Collectively, our evidence supports the notion that virulence of DC3000 is a multifactorial process and that the Tat system is an important virulence determinant of this phytopathogenic bacterium.

FIG. 1.

Complementation of E. coli with DC3000 tat genes. Microscopy (1,000× magnification) of (A) wild-type E. coli MC4100 carrying pTatC_DC3000; (B) B1LK0 (DtatC mutant derived from MC4100) carrying empty pTrc99A; and (C) B1LK0 carrying pTatC_DC3000.

FIG. 2.

Tat secretion signal sequence logo. This is the sequence logo representing the subsequences of known Tat substrates used in the HMM training set to find potential Tat substrates in DC3000. The vertical axis is information content in bits. The height of the amino acids reflects the representation of the residue at that position.

FIG. 3.

Multiple phenotypes of DC3000 tatC mutants. (A) Growth of DC3000 and PBTAT1 (tatC mutant derived from DC3000) (upper plate) and DC3000 pBS1-TatC_DC3000 and PBTAT1/pBS1-TatC_DC3000 (lower plate) on low-iron medium containing 50 μM of 2,2′-dipyridyl. Arabinose at 0.2% was added to the lower plate to induce tatC_DC3000 synthesis. DC3000 and PBTAT1 cells were also tested for (B) growth in the presence of 100 μM of the iron chelating agent 2,2′-dipyridyl; (C) formation of swarm halos on semisolid (0.4%, wt/vol) agar plates; and (D) cell division via microscopy (1,000× magnification). (E) Copper susceptibility assays of DC3000 (gray bars) and PBTAT1 (white bars) over a range (0 to 4 mM) of CuSO₄. All growth data was normalized to growth of DC3000 in the absence of CuSO₄. (F) Subcellular distribution of phospholipase (gray bars) and phosphatase (white bars) activity measured by pNPPC and pNPP assay, respectively. Supernatant and pellet fractions of DC3000 and PBTAT1 cells were prepared as described in Materials and Methods. Data were normalized to the phospholipase or phosphatase activity in supernatant fraction isolated from wild-type DC3000 cells. wt, wild type; sup, supernatant.

FIG. 4.

Infiltration of plants with tatC mutants of DC3000. (A) A. thaliana was infected by vacuum-infiltrating bacterial suspensions containing 10⁵ CFU ml⁻¹. Suspensions contained no bacteria (mock), wild-type DC3000, or PBTAT1. Images were captured 4 days postinoculation. (B) CFU of DC3000 and PBTAT1 per milligram of leaf cells determined by cutting leaf disks with a boring tool (inner diameter, 0.7 cm) and plating completely homogenized material, containing the bacteria, on KB plates with rifampin (50 μg ml⁻¹) and cycloheximide (2 μg ml⁻¹). (C) Elicitation of HR by DC3000 and PBTAT1 cells assayed by syringe infiltration of bacterial suspensions of various concentrations into N. tobacco cv. Xanthi. (D) Cya translocation assays performed by inoculating L. esculentum (tomato) plants via a blunt syringe with wild-type or PBTAT1 strains containing plasmids expressing HopPtoN-Cya. Plant tissue was sampled 8 h later using a boring tool (inner diameter, 0.7 cm), and cAMP levels were determined as described in Materials and Methods. All plant assay experiments were repeated at least three times with similar results. t, time; hpi, hours postinfection.

FIG. 5.

Broad-host genetic reporter of Tat transport based on GFP-SsrA. Flow cytometric analysis of (A) wild-type E. coli MC4100 and B1LK0 and (B) DC3000 and PBTAT1 cells all expressing pMMB-TGS. Histograms depict number of cells (events) versus their fluorescence intensity (FL1-H). Standard error of three replicate experiments was <10%. M, mean fluorescence.

FIG. 6.

Confirmation of DC3000 Tat substrates using GFP-SsrA reporter. (A) Predicted signal peptides for PlcA1 (PSPTO3648) and PlcA2 (PSPTOB0005). Gray shaded area denotes amino acid dissimilarities. Underlined, boldface text denotes a Tat consensus motif. Space between the last three amino acids (A/HE or S/HE) denotes peptidase cleavage site as predicted by SignalP, version 3.0 (http://www.cbs.dtu.dk/services/SignalP/). Mean fluorescence measured by flow cytometric analysis of MC4100 (gray bars) or B1LK0 (white bars) expressing (B) putative Tat signal peptides cloned in pMMB-GGS or (C) full-length Tat substrates cloned in pMMB-GGS. All mean fluorescence (M) data are the averages of three replicate experiments. Western blot analysis of cytoplasmic (cyt) and periplasmic (per) fractions generated from MC4100 cells expressing (D) ssPlcA1-GFP-SsrA or (E) full-length PlcA1 fused to GFP-SsrA. Blots were first probed with anti-GFP serum and then stripped and reprobed with anti-GroEL serum to ensure the quality of the fractionation procedure.

FIG. 7.

Extracellular secretion of Tat substrates by the Gsp system. (A) Subcellular distribution of phospholipase activity measured by the pNPPC assay. Expression of PlcA1 from plasmid pBS1-PlcA1 is denoted as PlcA+. Supernatant and pellet fractions were prepared as described in Materials and Methods. Data were normalized to the phospholipase activity in supernatant fraction isolated from DC3000. (B) A. thaliana was infected by vacuum infiltrating bacterial suspensions containing 10⁵ CFU ml⁻¹. Suspensions contained no bacteria (mock), wild-type DC3000, PBGspD, or PBGspE. Images were captured 4 days postinoculation. (C) Elicitation of HR by DC3000 and PBGspD cells assayed by syringe infiltration of bacterial suspensions of various concentrations into N. tobacco cv. Xanthi. All plant assay experiments were repeated at least three times with similar results.