XerR, a negative regulator of XccR in Xanthomonas campestris pv. campestris, relieves its repressor function in planta

Abstract

We previously reported that XccR, a LuxR-type regulator of Xanthomonas campestris pv. campestris (Xcc), activates the downstream proline iminopeptidase virulence gene (pip) in response to certain host plant factor(s). In this report, we further show that the expression of the xccR gene was repressed in the culture medium by an NtrC-type response regulator, which we named XerR (XccR expression-related, repressor), and that this repression was relieved when the bacteria were grown in planta. Such a regulatory mechanism is reinforced by the observations that XerR directly bound to the xccR promoter in vitro, and that mutations at the phosphorylation-related residues of XerR resulted in the loss of its repressor function. Furthermore, the expression level of xccR increased even in XerR-overexpressing Xcc cells when they were vacuum infiltrated into cabbage plants. We also preliminarily characterized the host factor(s) involved in the above mentioned interactions between Xcc and the host plant, showing that a plant material(s) with molecular weight(s) less than 1 kDa abolished the binding of XerR to the xccR promoter, while the same material enhanced the binding of XccR to the luxXc box in the pip promoter. Taken together, our results implicate XerR in a new layer of the regulatory mechanism controlling the expression of the virulence-related xccR/pip locus and provide clues to the identification of plant signal molecules that interact with XerR and XccR to enhance the virulence of Xcc.

Figure 1

XerR is required for repression of xccR and pip transcription in medium. (A) The domain organization of XerR and the sequence of receiver domain. Three putative modular components of XerR are shown in the diagram. Multiple amino acid sequence alignments between XerR and NtrC in S. typhimurium, CheY in E. coli and LuxO in V. harveyi are shown at the bottom of the diagram. The residues altered by site-directed mutagenesis are shaded in black, and the putative phosphorylation site (Asp-60) is marked. (B) GUS expression levels in different Xcc strains were assayed by enzymatic activities. xerR in-frame deletion mutant Xcc 8099 increased the GUS activity compared to that of Xcc 8177. Xcc 8099 and Xcc 8177 carrying the xerR gene in pHM1 plasmid (Xcc 8099/pFR423 and Xcc 8177/pFR423) exhibited reduced GUS activities. All the strains were harvested at OD₆₀₀ of 2.0 in NYG medium. Relative GUS activity units were defined as nM 4-methylumbelliferyl/min/10⁹ cells. The means and standard deviations were calculated from the data derived from at least nine independent experiments. (C) Expression of xccR-P/gusA in Xcc 8099 was density dependent when grown in NYG medium. GUS activities of different strains were assayed at different time points. The mean and standard deviation were calculated from the data derived from three independent experiments. (D) Expression levels of pip in different Xcc strains in medium and in planta. Relative transcriptional levels of pip were quantified by real-time RT-PCR. In NYG medium, RNA were extracted from the cultured strains at a cell density of OD₆₀₀ = 1.5−2.0. In planta, RNA was isolated from vacuum-infiltrated cabbage leaves 30 h post infiltration. Measurements were normalized by the wild-type values and fold differences were plotted. Each sample was assayed in triplicate.

Figure 2

EMSA shows that XerR binds to the upstream region of the xccR gene directly. (A) Schematic of the upstream region of xccR gene according to a promoter prediction program NNPP version 2.2 (1999). The putative −35/−10 and SD (Shine-Dalgarno sequence) elements are boxed, and an asterisk denotes the xccR transcriptional start site. The locations of two different probes that have a 10-bp overlap are denoted by lines. (B) EMSA assay of R1 probe with purified MBP-XerR. Isotope-labeled probe (8 fmol) was incubated for 30 min with indicated concentrations of protein (in μM) at room temperature. The shifted bands could be competed by excess of the unlabeled probe. The folds of unlabeled probe were indicated above. The migrated DNA-protein complexes and free probe R1 are indicated by arrows, and the bands marked with an asterisk indicate a possible higher structure of R1 probe formed during annealing step.

Figure 3

Phosphorylation-related residues of XerR are essential for its repressor function. (A) The conserved phosphorylation-related residues and the regulatory domains of XerR were indispensable for regulation of xccR expression in vivo. xerR ΔRR, xerR ΔHTH and different site-directed mutants exhibited increased GUS activities when grown in NYG medium. Plasmid-containing (pFR423) strains of different mutants reduced the GUS activities compared with that of Xcc 8177. Bacteria cultured in NYG medium were assayed at an OD₆₀₀ of 2.0. The experiments were repeated eight times with similar results. (B and C) EMSA assays of MBP-XerR^D60A and MBP-XerR^D60E with biotin-labeled R1 probe and plant extract. The two proteins presented the same binding characteristics to R1 probe, in which the plant extract of molecular weights < 1 kDa released the protein and DNA interactions. In the diagram, the concentration of purified protein and the volumes of plant signal(s) are indicated. (D) EMSA binding of phosphorylated and unphosphorylated MBP-XerR to the R1 probe. MBP-XerR was phosphorylated in vitro with acetyl phosphate and the R1 probe was end-labeled with ³²P at its 5′ termini. The bands marked with an asterisk indicate a possible higher structure of R1 probe formed during annealing step.

Figure 4

XerR relieves its inhibition on xccR expression in planta. (A) In planta cultivation did not significantly increase the GUS activity from Xcc 8099, while it had the opposite influence on that of Xcc 8099/pFR423 and Xcc 8177/pFR423. The bacteria were recovered from vacuum-infiltrated cabbage leaves 30 h post infiltration, and GUS activities were assayed. Data and standard deviation represented the mean of three independent measurements. (B and C) Plant signal(s) alleviated the binding activity of XerR protein to the xccR upstream regulatory sequence. EMSA assays with biotin-labeled probe were performed by MBP-XerR with plant extracts (< 1 kDa) at two dilutions.

Figure 5

EMSA binding of MBP-XccR protein to the luxXc box of the pip promoter. The band of XccR and DNA complex was intensified by adding different volumes of plant extracts in the EMSA assay. The migrated DNA-protein complexes and isotope-labeled luxXc box probe are indicated by arrows.

Figure 6

A model for expression regulation of xccR/pip locus by XerR. When Xcc is grown in NYG medium, XerR represses the expression of xccR and pip. The activity of XerR is dependent on the phosphorylation by an unknown two-component signaling transduction system. After entering the host plant, XerR relieves its inhibition to xccR expression in response to specific plant small molecule(s). The released XccR binds to luxXc box in the presence of the same or different plant signal(s) to induce the transcription of the pip gene for bacterial virulence. S1 and S2 denote the possible signal(s) from the host. OM and IM refer to the outside and inside membrane, respectively.