The LysR-Type Transcriptional Regulator CrgA Negatively Regulates the Flagellar Master Regulator flhDC in Ralstonia solanacearum GMI1000

Abstract

The invasion and colonization of host plants by the destructive pathogen Ralstonia solanacearum rely on its cell motility, which is controlled by multiple factors. Here, we report that the LysR-type transcriptional regulator CrgA (RS_RS16695) represses cell motility in R. solanacearum GMI1000. CrgA possesses common features of a LysR-type transcriptional regulator and contains an N-terminal helix-turn-helix motif as well as a C-terminal LysR substrate-binding domain. Deletion of crgA results in an enhanced swim ring and increased transcription of flhDC In addition, the ΔcrgA mutant possesses more polar flagella than wild-type GMI1000 and exhibits higher expression of the flagellin gene fliC Despite these alterations, the ΔcrgA mutant did not have a detectable growth defect in culture. Yeast one-hybrid and electrophoretic mobility shift assays revealed that CrgA interacts directly with the flhDC promoter. Expressing the β-glucuronidase (GUS) reporter under the control of the crgA promoter showed that crgA transcription is dependent on cell density. Soil-soaking inoculation with the crgA mutant caused wilt symptoms on tomato (Solanum lycopersicum L. cv. Hong yangli) plants earlier than inoculation with the wild-type GMI1000 but resulted in lower disease severity. We conclude that the R. solanacearum regulator CrgA represses flhDC expression and consequently affects the expression of fliC to modulate cell motility, thereby conditioning disease development in host plants.IMPORTANCERalstonia solanacearum is a widely distributed soilborne plant pathogen that causes bacterial wilt disease on diverse plant species. Motility is a critical virulence attribute of R. solanacearum because it allows this pathogen to efficiently invade and colonize host plants. In R. solanacearum, motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum motility is more complex than previously thought and enhances our understanding of flagellum regulation in R. solanacearum.

Keywords: CrgA; LysR; Ralstonia solanacearum; flhDC; swimming motility; virulence.

FIG 1

Swimming motility and growth of GMI1000 and crgA mutants. (A) Swimming motility of the crgA mutant on semisolid agar. The cultured cells were prepared to the same concentration (OD₆₀₀ = 0.7), and swimming motility was examined. The swim rings were recorded 3 days postinoculation (dpi). WT, wild type. (B) Measurement of colony diameter. Each histogram bar represents the mean (±standard error [SE]) for three replicates; different letters indicate a significant difference based on Tukey’s honestly significant difference test (at P = 0.05). (C) Growth curve for GMI1000 and crgA mutants grown in rich broth. (D) Growth curve for GMI1000 and crgA mutants grown in BMM-glucose broth. Diamonds, GMI1000; squares, ΔcrgA mutant; triangles, CΔcrgA mutant. Three independent experiments, each with three replicates, were performed for each strain. Results represent the mean value for all replicates at each time point.

FIG 2

Flagellar morphology and fliC expression in GMI1000 and crgA mutants. (A) Transmission electron micrographs of flagella in GMI1000, ΔcrgA, and CΔcrgA cells. Scale bar, 1 μm. (B) Transcript levels of fliC. Total RNA was extracted from cells cultured in NB and BMM-glucose broth. Values are means ± SE for at least three replicates. Different letters indicate a significant difference based on Tukey’s honestly significant difference test (at P = 0.05).

FIG 3

qRT-PCR analysis of transcript levels of flhD. Total RNA was extracted from cells cultured in NB and BMM-glucose broth. Values are the means ± SE for at least three replicates. Different letters indicate a significant difference based on Tukey’s honestly significant difference test (at P = 0.05).

FIG 4

CrgA binds directly to the flhDC promoter region. (A) Y1H experiment data showing the interaction between CrgA and the flhDC promoter. β-Galactosidase activity was quantified using the Miller method, after cotransformation of pYF503-crgA and pG221-PflhDC into yeast. Enzymatic activities are presented as the mean ± SE from three independent performances. Different letters indicate a significant difference based on Tukey’s honestly significant difference test (at P = 0.05). (B) Electrophoretic mobility shift assays. Two reaction mixtures containing Cy5-labeled probe alone or together with GST protein were used as negative controls (first two lanes). To confirm the binding specificity, 40-, 120-, and 360-fold excesses of unlabeled flhDC probe were supplied to the binding reaction mixtures containing 3 μg purified GST-CrgA protein and 40 fmol Cy5-labeled probe (last three lanes). All reaction mixtures were electrophoresed and visualized by fluorescence imaging.

FIG 5

Virulence and growth of crgA mutant in tomato plants. (A) Disease index using the soil-soaking inoculation method. Each point represents the mean from four independent assays with 11 plants per treatment. Diamonds, GMI1000; squares, ΔcrgA mutant; triangles, CΔcrgA mutant. Bars indicate the SE. Asterisks indicate days on which the wild-type and ΔcrgA mutant disease indices differed significantly based on ANOVA (P < 0.05). (B) Disease index using petiole inoculation. Each point represents the mean from four independent repeats with a total of 44 plants per treatment. Diamonds, GMI1000; squares, ΔcrgA mutant; triangles, CΔcrgA mutant. Bars indicate the SE. (C) Bacterial proliferation in tomato stems. The R solanacearum cells were counted at 2, 4, and 6 days postinoculation. Each assay consisted of four independent trials, and each trial contained six plants per strain. Values are means ± SE.

FIG 6

Expression pattern of crgA. (A) Promoter region of the crgA gene, where boxed regions include the putative promoter sequence. The putative LTTR boxes (T-N₁₁-A) are shown in red. The bent arrow denotes the start of the crgA open reading frame. (B) β-Glucuronidase activities driven by the crgA promoter at different cell densities. The crgA promoter was fused to the GUS gene, and β-glucuronidase activity was analyzed in GMI1000. Values are means ± SE for three replicates. (C) qRT-PCR analysis of crgA autoregulation in NB and BMM-glucose broth. Values are means ± SE for three replicates. Different letters indicate a significant difference based on Tukey’s honestly significant difference test (at P = 0.05).