Response regulator VemR regulates the transcription of flagellar rod gene flgG by interacting with σ54 factor RpoN2 in Xanthomonas citri ssp. citri

Abstract

Xanthomonas citri ssp. citri, a polar flagellated bacterium, causes citrus canker disease worldwide. In this study, we found that the X. citri ssp. citri response regulator VemR plays a regulatory role in flagellum-derived cell motility. Deletion of the vemR gene resulted in a reduction in cell motility, as well as reductions in virulence and exopolysaccharide production. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that vemR is transcribed in an operon together with rpoN2 and fleQ. In the vemR mutant, the flagellar distal rod gene flgG was significantly down-regulated. Because flgG is also rpoN2 dependent, we speculated that VemR and RpoN2 physically interact, which was confirmed by yeast two-hybrid and maltose-binding protein (MBP) pull-down assays. This suggested that the transcription of flgG is synergistically controlled by VemR and RpoN2. To confirm this, we constructed a vemR and rpoN2 double mutant. In this mutant, the reductions in cell motility and flgG transcription were unable to be restored by the expression of either vemR or rpoN2 alone. In contrast, the expression of both vemR and rpoN2 together in the double mutant restored the wild-type phenotype. Together, our data demonstrate that the response regulator VemR functions as an RpoN2 cognate activator to positively regulate the transcription of the rod gene flgG in X. citri ssp. citri.

Keywords: RpoN2; VemR; Xanthomonas citri ssp. citri; cell motility; regulation.

Figure 1

Phenotypes of vemR mutants. (A) Citrus canker symptoms on Citrus paradisi Macf. cv. Duncan. Disease symptoms were scored every day and photographed at 7 days post‐inoculation (dpi). (B) Hypersensitive response (HR) on Solanum lycopersicum at 2 dpi. The Xanthomonas citri ssp. citri cells [10⁷ colony‐forming units (CFU)/mL] were inoculated into citrus and tomato plants for pathogenicity and HR assays, respectively. The inoculation areas of the vemR mutants are indicated by the broken lines. (C) Bacterial growth in nutrient‐rich nutrient broth (NB) liquid medium. Each strain was prepared at an initial concentration of OD₆₀₀ (optical density at 600 nm) = 0.01, and bacterial multiplication was measured from the OD₆₀₀ value every 6 h. The values shown are the means of three technical repeats with standard deviations. (D) Bacterial growth in citrus plants. Bacteria were recovered from leaves inoculated with X. citri ssp. citri cells at a concentration of 10⁸ CFU/mL every 2 days after inoculation. Values shown are the means of three technical repeats with standard deviations. WT, wild‐type.

Figure 2

Schematic representation of the fleQ‐vemR‐rpoN2 operon and the reverse transcription‐polymerase chain reaction (RT‐PCR) strategy. (A) Arrows with different colours depict the open reading frames of the operon and their lengths in base pairs. The threads represent the sizes and approximate locations in the PCR analysis with primer sets. (B) PCR products using cDNA and gDNA as templates. 1, 2, 3 and 4 represent the products of the corresponding junction fragments in (A). The DNA marker was DL2000.

Figure 3

Swimming motility of the vemR mutant and relative expression levels of eight flagellar biosynthesis genes. (A) Swimming motility of the vemR mutant on a semisolid plate. Cell suspensions (2 µL) were spotted onto 0.3% agar MMX plates, and bacterial swimming was determined from the diameter of each colony at 3 days post‐inoculation. (B) Measurement of colony diameters. The data were derived from triple repeats. The asterisks in the horizontal data column indicate significant differences in colony diameter at P = 0.01 according to Student’s t‐tests. (C) Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis of the expression of eight flagellar biosynthesis genes. Total RNA was extracted from Xanthomonas citri ssp. citri cells cultured in MMX liquid medium. For each gene, the expression level in the wild‐type (WT) was calculated as ‘1’ using gyrA as an internal control. Statistical analysis was conducted using Student’s t‐tests. **P ˂ 0.01 vs. WT.

Figure 4

VemR physically interacts with RpoN2. (A) Yeast two‐hybrid assays showing the interaction between VemR and RpoN2. The positive transformants were prepared to a cell density of OD₆₀₀ (optical density at 600 nm) = 1.0 and diluted to a 10‐fold series. For each concentration series, 2‐μL suspensions were spotted and incubated on synthetic defined SD/‐Ade/‐Leu/‐Trp/‐His plates supplied with 20 μg/mL X‐α‐galactosidase (X‐α‐gal) for 4 days. The interaction between Hpa2 and HrpF was used as a positive control. (B) VemR interacts with RpoN1 in vitro according to maltose‐binding protein (MBP) pull‐down assays. MBP‐RpoN2 (3 µg) and GST‐VemR were incubated overnight at 4 °C with 300 μL of amylose resin. After the eluted protein samples had been boiled, samples were separated on a 12% sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel and immunoblotted with anti‐MBP. The negative control included the incubation of 3 µg maltose binding protein (MBP) protein with GST‐VemR.

Figure 5

VemR and RpoN2 are both required for flgG transcription. (A) Phenotype of vemR and rpoN2 double mutant in citrus plants. Bacterial suspensions of 10⁷ colony‐forming units (CFU)/mL were inoculated onto citrus using an infiltration method. Disease symptoms were photographed at 7 days post‐inoculation. The weak canker symptoms caused by the mutants are highlighted by the broken lines. (B) Swimming motility of vemR and rpoN2 double mutant on low‐agar plates. Cell suspensions (2 µL) were spotted onto 0.3% agar MMX plates, and photographs were taken at 3 days post‐inoculation. (C) Transcription of flgG and promoter‐monitored GusA gene in the double mutant. Total RNA was extracted from cells cultured in MMX liquid medium. The expression of flgG or GusA in the wild‐type (WT) was set as ‘1’. Statistical analysis was conducted using Student’s t‐tests. *P ˂ 0.05, **P ˂ 0.01 vs. WT.

Figure 6

Proposed model for transcription of flgG synergistically controlled by VemR and RpoN2 in Xanthomonas citri ssp. citri. VemR contains a CheY‐like receiver domain that responds to environmental stimuli. As VemR does not have an output domain, a typical response regulator (RR) or transcriptional activator is probably recruited to interact with VemR. RpoN2 is responsible for recognizing the –24/–12 consensus sequence (TGGCACGGCACGTGCAT) in the flgG promoter. Using this protein–protein interaction module, the transcription of flgG is initiated by RNA polymerase, and the resulting protein plays a role in flagellar biosynthesis.