Novel virulence gene of Pseudomonas syringae pv. tomato strain DC3000

Abstract

Previously, we conducted a mutant screen of Pseudomonas syringae pv. tomato strain DC3000 to identify genes that contribute to virulence on Arabidopsis thaliana plants. Here we describe the characterization of one mutant strain, DB4H2, which contains a single Tn5 insertion in PSPTO3576, an open reading frame that is predicted to encode a protein belonging to the TetR family of transcriptional regulators. We demonstrate that PSPTO3576 is necessary for virulence in DC3000 and designate the encoded protein TvrR (TetR-like virulence regulator). TvrR, like many other TetR-like transcriptional regulators, negatively regulates its own expression. Despite the presence of a putative HrpL binding site in the tvrR promoter region, tvrR is not regulated by HrpL, an alternative sigma factor that regulates the expression of many known DC3000 virulence genes. tvrR mutant strains grow comparably to wild-type DC3000 in culture and possess an intact type III secretion system. However, tvrR mutants do not cause disease symptoms on inoculated A. thaliana and tomato plants, and their growth within plant tissue is significantly impaired. We demonstrate that tvrR mutant strains are able to synthesize coronatine (COR), a phytotoxin required for virulence of DC3000 on A. thaliana. Given that tvrR mutant strains are not defective for type III secretion or COR production, tvrR appears to be a novel virulence factor required for a previously unexplored process that is necessary for pathogenesis.

FIG. 1.

Disease symptoms resulting from dip inoculation of 4-week-old Col-0 A. thaliana plants at 4 days postinoculation with DC3000 (A) or DB4H2 (B). (C) Growth of indicated dip-inoculated strains within 4-week-old Col-0 A. thaliana plants. Data points represent mean numbers of CFU per gram of leaf tissue ± standard deviations from three separate plant samples. Similar results were obtained for two additional experiments. Open squares, DC3000; closed squares, DC3000(pTvrR); diamonds, DB4H2; open circles, tvrR::Ω strain; closed circles, tvrR::Ω(pTvrR) strain. (D) Growth of indicated vacuum-infiltrated strains within 4-week-old Col-0 A. thaliana plants. Data points represent mean numbers of CFU per cm² of leaf tissue ± standard deviations from three separate plant samples. Similar results were obtained for two additional experiments. Open squares, DC3000; diamonds, DB4H2.

FIG. 2.

Growth of DC3000 and the tvrR::Ω strain in HDM supplemented with 10 mM sucrose at various pHs. Open squares, DC3000 at pH 5.7; closed squares, the tvrR::Ω strain at pH 5.7; open circles, DC3000 at pH 7.0; closed circles, the tvrR::Ω strain at pH 7.0. Similar results were observed for two additional experiments. OD₆₀₀, optical density at 600 nm.

FIG. 3.

(A) Schematic diagram of the tvrR region of the DC3000 genome, including adjacent ORFs (bold arrows). tvrR (PSPTO3576 [accession no. Q87Z55]) is predicted to encode a 207-amino-acid protein with significant similarity to TetR-like regulatory proteins. The sequence of a putative hrp box located 94 nucleotides upstream of the predicted tvrR translation start site is indicated. The portion of the genome used to construct the complementing clone (pTvrR) is indicated by the double-headed arrow. PSPTO3575 (Q87Z56) is predicted to encode a 173-amino-acid protein of unknown function. PSPTO3577 (Q87Z54) is predicted to encode a 542-amino-acid protein with similarity to methyl-accepting chemotaxis proteins. PSPTO3578 (Q87Z53) is predicted to encode a 173-amino-acid protein of unknown function. (B) ClustalW (50) alignment of TvrR with similar putative proteins. The horizontal line indicates the PROSITE PS01081 TetR-type helix-turn-helix domain signature. TetR-like transcriptional regulators are indicated as follows: Psyringae, P. syringae pv. syringae strain B728a (Psyr_3347); Vcholera, Vibrio cholerae El Tor biotype strain N16961 (Q9K552); Xcampestris, Xanthomonas campestris pv. campestris ATCC 33913 (Q8P7C7); AefR, P. syringae pv. syringae strain B728a (Q7WU46); and PSPTO3549, the presumed DC3000 aefR homolog (Q87Z81). (C) ClustalW (50) alignment of TvrR with several known TetR-like transcriptional regulators, i.e., TetR(C) from E. coli (P03039), AcrR from E. coli (P34000), UidR from E. coli (Q59431), and BetI from E. coli (P17446). The horizontal line indicates the PROSITE PS01081 TetR-type helix-turn-helix domain signature.