The PAS domain-containing histidine kinase RpfS is a second sensor for the diffusible signal factor of Xanthomonas campestris

Abstract

A cell-cell signalling system mediated by the fatty acid signal DSF controls the virulence of Xanthomonas campestris pv. campestris (Xcc) to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon the sensor RpfC and regulator RpfG. Detailed analyses of the regulatory roles of different Rpf proteins have suggested the occurrence of further sensors for DSF. Here we have used a mutagenesis approach coupled with high-resolution transcriptional analysis to identify XC_2579 (RpfS) as a second sensor for DSF in Xcc. RpfS is a complex sensor kinase predicted to have multiple Per/Arnt/Sim (PAS) domains, a histidine kinase domain and a C-terminal receiver (REC) domain. Isothermal calorimetry showed that DSF bound to the isolated N-terminal PAS domain with a Kd of 1.4 μM. RpfS controlled expression of a sub-set of genes distinct from those controlled by RpfC to include genes involved in type IV secretion and chemotaxis. Mutation of XC_2579 was associated with a reduction in virulence of Xcc to Chinese Radish when assayed by leaf spraying but not by leaf inoculation, suggesting a role for RpfS-controlled factors in the epiphytic phase of the disease cycle.

Figure 1

Changes in gene expression of rpfF, rpfC and rpfFC mutants compared with the wild‐type Xcc 8004 as measured by RNA‐Seq. A and B. Venn diagrams showing the overlap of genes whose expression is (A) downregulated or (B) upregulated in different mutant backgrounds. Divergently regulated genes are not depicted in these Venn diagrams but can be found in Table S3. C. Comparison of relative fold changes between RNA‐Seq and qRT‐PCR results in (i) rpfF, (ii) rpfC and (iii) rpfFC mutant backgrounds. All qRT‐PCR results were normalized using the Cts obtained for the 16S rRNA amplifications run in the same plate. The relative levels of gene transcripts are determined from standard curves. Values given are the mean and standard deviation of triplicate measurements (three biological and three technical replicates).

Figure 2

DSF‐induced expression of XC_0107 and the role of XC_2579. A. Effects of mutation of XC_2579 in wild type and rpfFC mutant backgrounds on expression of XC_0107 gfp fusion induced by DSF. Bacteria carrying the promoter of XC_0107 fused to gfp were grown overnight in NYGB medium at 30°C and subcultured (1:100) in fresh NYGB medium (5 ml) containing 50 μM synthetic DSF. The cells were grown at 30°C with shaking. After 14 h of cultivation, the GFP expression level was determined. Error bars show the standard deviation of triplicate experiments. Values observed are significant as they attain a P‐value of less than 0.05. (B) Genomic organization of the region encoding XC_2579 in Xanthomonas campestris 8004 and (C) domain structure of XC_2579 as revealed by the SMART algorithm (http://www.smart.embl‐heidelberg.de).

Figure 3

Isothermal titration calorimetry analysis (ITC) of the interaction between DSF and XC_2579. A. Summary of the binding affinity of full‐length XC_2579 and truncations to DSF as derived by ITC. B. A representative ITC data plot for titration of 20 μM of the PAS domain from XC_2579 (Tk 4) with 20 or 200 μM DSF in PBS buffer at 25°C.

Figure 4

Changes in gene expression of rpfC, rpfFC and XC_2579 mutants compared with the wild‐type Xanthomonas campestris 8004 as measured by RNA‐Seq. Venn diagrams showing the overlap of genes whose expression is (A) downregulated or (B) upregulated in different mutant backgrounds. Divergently regulated genes are not depicted in these Venn diagrams but can be found in Table S3. Comparison of relative fold changes between RNA‐Seq and qRT‐PCR results in XC_2579 mutant (C) and rpfFC mutant (D) background. All qRT‐PCR results were normalized using the Cts obtained for the 16S rRNA amplifications run in the same plate. The relative levels of gene transcripts are determined from standard curves. Values given are the mean and standard deviation of triplicate measurements (three biological and three technical replicates).

Figure 5

Effect of mutation of XC_2579 on motility and virulence in Xcc. A. The motility of different Xcc strains was tested on Eiken agar. The XC_2579 strain had reduced motility (comparable to the rpfF mutant). Motility was restored in the complemented strain XC_2579 (c2579). B. Virulence of different Xcc strains assessed after spray inoculation. The percentage of the total number of inoculated leaves that showed the typical black rot disease symptoms at the leaf margin is given. Values are means and standard deviations of three replicates, each comprising 25 plants and approximately 100 leaves. Values observed are significant as they attain a P‐value of less than 0.05. C. Symptom production on leaves after 10 day spray inoculation with (from left to right) water (control), wild‐type, XC_2579 mutant and complemented strain XC_2579 (c2579).

Figure 6

Model for multiple pathways of DSF perception and signal transduction in Xanthomonas campestris. DSF perception and signal transduction involving the sensor RpfC and response regulator RpfG act to co‐ordinately regulate the expression of a sub‐set of genes that include engXCA, prtA and manA, which encode extracellular enzymes. DSF and RpfC also regulate expression of a number of genes including XC_1766 (encoding a transcriptional regulator) independently of RpfG, suggesting further signalling outputs from RpfC. The second DSF‐dependent sensor RpfS (XC_2579) regulates genes including XC_0107 (encoding a hypothetical protein) in a pathway that is independent of RpfC and RpfG. Not depicted in the current model is the sub‐set of genes co‐regulated by RpfC and RpfS (XC_2579). How these genes are directly regulated is currently under investigation.