RpfC regulates the expression of the key regulator hrpX of the hrp/T3SS system in Xanthomonas campestris pv. campestris

Abstract

Background: The Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris recruits the hrp/T3SS system to inject pathogenicity effector proteins into host cells and uses the rpf/DSF cell-cell signaling system to regulate the expression of virulence factors such as extracellular enzymes and polysaccharide. Whether these two systems have any connection is unknown.

Methods: Positive regulator candidates affecting hrpX expression were identified by sacB strategy. The transcriptional expression was determined by qRT-PCR and GUS activity analysis. Transcriptome analysis was performed by RNA deep-sequencing. The hypersensitive response (HR) was determined in the nonhost plant pepper ECW-10R and electrolyte leakage assay.

Results: Mutation of the gene encoding the sensor RpfC of the rpf/DSF system significantly reduced the expression of hrpX, the key regulator of the hrp/T3SS system, all of the genes in the hrp cluster and most reported type III effector genes. Mutation of rpfG did not affect the expression of hrpX. The rpfC mutant showed a delayed and weakened HR induction.

Conclusions: RpfC positively regulates the expression of hrpX independent of RpfG, showing a complex regulatory network linking the rpf/DSF and hrp/T3SS systems.

Keywords: RpfC; Xanthomonas; hrpX.

Fig. 1

Identification of positive regulator candidates affecting hrpX expression by sacB strategy. Xcc wild type train 8004 and the deletion mutant strain ΔhrpG were used as controls. The principle in this strategy is that strain 8004/pL6hrpXsacB cannot grow on the minimal medium containing 5% sucrose, because the expression of the hrpX-promoter-driven sacB gene is lethal to the cells under these conditions, and only the strains with a mutation (i.e., deletion mutant of hrpG, ΔhrpG) impeding the expression of hrpX (i.e. strain ΔhrpG/pL6hrpXsacB, or disrupting the sacB gene, or the wild-type strain 8004 and the deletion mutant strain ΔhrpG can grow. a, wild-type strain 8004; b, 8004/pL6hrpXsacB; c, ΔhrpG/pL6hrpXsacB; d, the deletion mutant strain ΔhrpG

Fig. 2

RpfC positively affects the expression of hrpX in XCM1 minimal medium. β-Glucuronidase (GUS) activities of hrpG and hrpX promoter-gusA reporters in the rpfC mutant and the wild-type backgrounds. Strains were cultured in XCM1 medium for 24 h, and GUS activities were then determined by measurement of optical density at 415 nm (OD₄₁₅) using ρ-nitrophenyl-β-D-glucuronide as substrate. Data are mean ± standard deviations (SD) of triplicate measurements. The experiment was repeated twice and similar results were obtained. **, t-test, p < 0.01

Fig. 3

RpfC positively affects the expression of hrpG and hrpX in host plant. Xcc strains 8004/pGUShrpG, 8004/pGUShrpX, ΔrpfC/pGUShrpG, and ΔrpfC/pGUShrpX were cultured in NYG medium overnight and resuspended in water to an optical density at 600 nm of 0.01, and then inoculated into the Chinese radish cv. Manshenhong leaves by leaf clipping. At 5 days post-inoculation, the inoculated leaves were assayed. a, Leaves were taken and analyzed for bacterial numbers and GUS activity was measured with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide. GUS activity values per 10⁸ bacterial cells are the mean ± standard deviations of three independent measurements. b, GUS activity was measured using an in situ staining method, and bacterial cell numbers inside the infected leaves were measured in a parallel experiment. Average bacterial numbers inside the tested leaves are indicated. The experiments were repeated twice. Data presented are from a representative experiment, and similar results were obtained in the other independent experiment

Fig. 4

RpfC is involved in hypersensitive response. a, Hypersensitive response symptoms induced in pepper leaves (Capsicum annuum cv. ECW-10R) by the Xcc strains. Approximately 5 μl bacterial culture (1 × 10⁷ CFU/ml) suspended in 10 mM sodium phosphate buffer were infiltrated into the leaf mesophyll tissue with a blunt-end plastic syringe. Pictures of the pepper leaf were taken at 8, 16, and 24 h after infiltration. Three replications were done in each experiment, and each experiment was repeated three times. Results presented are from a representative experiment, and similar results were obtained in all other independent experiments. b, Electrolyte leakage from pepper leaves inoculated with Xcc strains. Results presented are from a representative experiment, and similar results were obtained in other independent experiments

Fig. 5

Comparison of RpfC and RpfG regulons. Venn diagrams showing the overlap of genes (a, Total regulated genes. b, hrp genes. c, Type III effector genes) whose expression is upregulated or downregulated in rpfC or rpfG deletion mutant backgrounds

Fig. 6

qRT-PCR verification of differently expressed genes in ΔrpfC (a) and ΔrpfG (b). The genes were chosen randomly from the transcriptome results. Two independent experiments were performed, and similar results were obtained. Results presented are from a representative experiment