Involvement of NpdA, a Putative 2-Nitropropane Dioxygenase, in the T3SS Expression and Full Virulence in Ralstonia solanacearum OE1-1

Abstract

Previously, we isolated several genes that potentially affected the expression of type III secretion system (T3SS) in Ralstonia solanacearum OE1-1. Here, we focused on the rsp0316, which encodes a putative 2-nitropropane dioxygenase (hereafter designated NpdA). The deletion of npdA substantially reduced the T3SS expression and virulence in OE1-1, and the complementation with functional NpdA could completely restore its reduced T3SS expression and virulence to that of wild type. The NpdA was highly conserved among diverse R. solanacearum species and the NpdA-dependent expression of T3SS was not specific to OE1-1 strain, but not the virulence. The NpdA was important for the T3SS expression in planta, while it was not required for the bacterial growth in planta. Moreover, the NpdA was not required for the elicitation of hypersensitive response (HR) of R. solanacearum strains in tobacco leaves. The T3SS in R. solanacearum is directly controlled by the AraC-type transcriptional regulator HrpB and regulated by a complex regulation network. The NpdA affected the T3SS expression mediated with HrpB but through some novel pathway. All these results from genetic studies demonstrate that NpdA is a novel factor for the T3SS expression in diverse R. solanacearum species in medium, but specifically for the T3SS expression in strain OE1-1 in planta. And the NpdA-dependent expression of T3SS in planta plays an important role in pathogenicity of R. solanacearum OE1-1 in host plants.

Keywords: Hrp regulon; NPD; Pathogenesis; Ralstonia solanacearum; type III secretion system.

FIGURE 1

The popA expression in Ralstonia solanacearum strains. Left, OE1-1 derivatives; right, RS1002 derivatives. Black column, wild type; white column, the npdA deletion mutants; gray column, complementation with OE1-1 NpdA. Cells were grown in hrp-inducing medium to an OD₆₀₀ of approximately 0.1 and subjected for β-galactosidase assay. The mean values of atleast 3 independent trials are presented in Miller units with SD (error bars). Significance level, ^∗∗P < 0.01 (t-test).

FIGURE 2

Relative expression of some T3Es genes in R. solanacearum OE1-1. Relative expression level of eight representative T3Es genes in hrp-inducing medium were determined by qRT-PCR and serC gene was used as reference gene. Normalized value was divided with that of wild type and relative values were presented. Each test was repeated in at least three independent trials and each trials included four replications. Mean values were averaged and presented with SD (error bars). Significance level, ^∗P < 0.01, ^∗∗P < 0.01 (t-test).

FIGURE 3

Pathogenicity test. R. solanacearum strains, (A–D) OE1-1 derivatives; (E,F) RS1002 derivatives. Inoculation methods: (A,C,E) soil soak inoculation, a bacterial suspension was poured into the soil of plants at a final concentration of 10⁷ cfu g^-1 of soil; (B,F) petiole inoculation, about 3 μl of bacterial suspension at 10⁸ cfu ml^-1 was dropped onto freshly cut surface of petioles; (D) Leaf infiltration in tobacco leaves, about 50 μl of bacterial suspension at 10⁸ cfu ml^-1 was infiltrated into tobacco leaves with a blunt-end syringe. Plants, (A,B,E,F) tomato plants; (C,D) tobacco plants. Closed circle, wild type; closed triangle, npdA mutant; closed square, complementation with OE1-1 NpdA. Plants were inspected daily for wilt symptoms, and scored on a disease index scale from 0 to 4 (0, no wilting; 1, 1–25% wilting; 2, 26–50% wilting; 3, 51–75% wilting; and 4, 76–100% wilted or dead). Each assay was repeated in four independent trials (each trial contains at least 10 plants) and mean values were averaged and presented with SD (error bars). Significance level, ^∗P < 0.01, ^∗∗P < 0.01 (t-test).

FIGURE 4

HR test. Approximate 50 μl of bacterial suspension at 10⁸ cfu ml^-1 was infiltrated into the leaf mesophyll tissue with a blunt-end syringe. (A) RS1002; (B) npdA mutant (RK10013); (C) RK10015 (complementation with OE1-1 NpdA); (D) distilled water. Development of necrotic lesions was observed periodically and pictures were taken. Each experiment were repeated in triple and each treatment contains four plants. The results presented are from a representative experiment, and similar results were obtained in all other independent experiments.

FIGURE 5

Bacterial growth test in planta. Tomato plants were inoculated with bacteria using petiole inoculation and stem species were removed at 4 and 7 dpi. Cells number was quantified by dilution plating. Black column, wild type (RK5050); gray column, npdA mutant (RK5628). Each experiment was repeated in triple and each treatment contains four plants. Mean values were averaged and presented with SD (error bars).

FIGURE 6

The popA expression in planta. (A) OE1-1 derivatives; (B) RS1002 derivatives. Black column, wild type strains; gray column, npdA mutants. Tomato plants were inoculated with bacteria using petiole inoculation and stem pecies were removed at 3–6 dpi for β-galactosidase assay in planta with Galacto-Light Plus kit and cells numbers were quantified by dilution plating. Each experiment was repeated at least in triple and each treatment contains four plants. Mean values were averaged and presented with SD (error bars). Significance level, ^∗P < 0.01, ^∗∗P < 0.01 (t-test).

FIGURE 7

Expression of some known regulators in R. solanacearum. Black column, wild type (RK5050); gray column, npdA mutant (RK5628). Cells were grown in hrp-inducing medium to an OD₆₀₀ of approximately 0.1 and subjected for β-galactosidase assay in vitro. Mean values were averaged and presented with SD (error bars). Significance level, ^∗∗P < 0.01 (t-test).