A novel two-component response regulator links rpf with biofilm formation and virulence of Xanthomonas axonopodis pv. citri

Abstract

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a serious disease that impacts citrus production worldwide, and X. axonopodis pv. citri is listed as a quarantine pest in certain countries. Biofilm formation is important for the successful development of a pathogenic relationship between various bacteria and their host(s). To understand the mechanisms of biofilm formation by X. axonopodis pv. citri strain XW19, the strain was subjected to transposon mutagenesis. One mutant with a mutation in a two-component response regulator gene that was deficient in biofilm formation on a polystyrene microplate was selected for further study. The protein was designated as BfdR for biofilm formation defective regulator. BfdR from strain XW19 shares 100% amino acid sequence identity with XAC1284 of X. axonopodis pv. citri strain 306 and 30-100% identity with two-component response regulators in various pathogens and environmental microorganisms. The bfdR mutant strain exhibited significantly decreased biofilm formation on the leaf surfaces of Mexican lime compared with the wild type strain. The bfdR mutant was also compromised in its ability to cause canker lesions. The wild-type phenotype was restored by providing pbfdR in trans in the bfdR mutant. Our data indicated that BfdR did not regulate the production of virulence-related extracellular enzymes including amylase, lipase, protease, and lecithinase or the expression of hrpG, rfbC, and katE; however, BfdR controlled the expression of rpfF in XVM2 medium, which mimics cytoplasmic fluids in planta. In conclusion, biofilm formation on leaf surfaces of citrus is important for canker development in X. axonopodis pv. citri XW19. The process is controlled by the two-component response regulator BfdR via regulation of rpfF, which is required for the biosynthesis of a diffusible signal factor.

Figure 1. Schematic diagram of bfdS and bdfR and their homologues in X. axonopodis pv. citri strains XW19 and 306.

The open arrows show the locations and orientations of the genes. The position of EZ-Tn5 in the mutant is indicated by an inverted red triangle. The construction of the complementation plasmids pbfdSR and pbfdR is described in Materials and Methods. The primers used to construct the plasmids for complementation are shown on the top of the solid arrows.

Figure 2. Alignment of the Xanthomonas axonopodis pv. citri XW19 two-component response regulator with its homologues in various organisms

The putative signal receiver domain (REC) of the protein is depicted with a red line. Active sites (solid triangles) are present at amino acid (a.a.) positions 11, 12, 65, 85, 101,104, and 105 in X. axonopodis pv. citri; a phosphorylation site (open triangle) is present at a.a. position 56; and the dimerization interface (solid circle) is located at a.a. positions 104, 105, and 106. Identical (shading), highly conserved (:) and less conserved (.) a.a. residues are indicated. The GenBank accession number of the two-component response regulator homologue in Xanthomonas vesicatoria is ZP_08177161.1; that in Xanthomonas campestris pv. campestris is NP_636561.1; that in Rhodopseudomonas palustris is YP_567467.1; that in Stenotrophomonas maltophilia is YP_002029492.1; that in Agrobacterium tumefaciens is NP_356752.2; that in Pseudomonas fluorescens is ZP_07777428.1; and that in Pseudomonas putida is YP_001749312.1.

Figure 3. Xanthomonas axonopodis pv.citri biofilm formation in a 24-well polystyrene microplate.

Experiments were performed three times with six replicates for each strain. The data presented are the means and standard deviations (error bars) from one representative experiment. *, significantly different (p<0.05) from strain TPH2 based on one-way ANOVA and Tukey's HSD test.

Figure 4. Epifluorescence micrographs of Xanthomonas axonopodis pv.citri biofilms on grapefruit, Mexican lime and navel orange leaf discs.

X. axonopodis pv. citri TPH2, TPH3 and TPH5 were tagged with green fluorescent protein and expressed using the plasmid pGTKan. The culture suspensions (OD₆₂₀  = 0.05) were inoculated in a 24-well polystyrene plate containing grapefruit, Mexican lime and navel orange leaf discs and incubated at 27°C with shaking at 50 rpm for 2 days. Scale bars, 10 μm.

Figure 5. Confocal laser scanning micrographs of biofilms on Mexican lime leaf discs.

X. axonopodis pv. citri TPH2, TPH3 and TPH5 were tagged with green fluorescent protein and expressed using the plasmid pGTKan. The culture suspensions (OD₆₂₀  = 0.05) were inoculated in a 24-well polystyrene plate containing Mexican lime leaf discs and incubated at 27°C with shaking at 50 rpm for 2 days. A, Horizontal (xy-axis) biofilm section with lines indicating the positions of the xz-axis and yz-axis shown at the top and right margins of the images, respectively. B, A simulated projection shows a field of 141.56 μm ×141.56 μm ×9.55 μm (xyz) in TPH2/pGTKan, 141.56 μm ×141.56 μm ×9.18 μm (xyz) in TPH3/pGTKan and 141.56 μm ×141.56 μm ×10.65 μm (xyz) in TPH5/pGTKan. Scale bars, 10 μm.

Figure 6. Epiphytic growth of Xanthomonas axonopodis pv.citri on Mexican lime leaves.

Bacterial suspensions (OD₆₂₀  = 0.3) were sprayed on the leaves of Mexican lime plants in a greenhouse. Bacterial populations were determined by homogenizing the leaves in Milli-Q water followed by dilution and plating at 0, 1, 2, 4, and 6 days post-inoculation. All experiments were performed three times with similar results. The results shown are the means and standard deviations (error bars) of triplicates from one representative experiment.

Figure 7. The two-component response regulator BfdR plays a role in the virulence of Xanthomonas axonopodis pv.citri in Mexican lime plants.

A, Symptoms on the upper (top panel) and lower (bottom panel) leaf surfaces of Mexican lime leaves at two months post-inoculation with strains TPH2, TPH3 and TPH5. Bacterial suspensions (OD₆₂₀  = 0.3) were inoculated on leaf surfaces using the spray method. B, Number of cankers per cm² on each leaf. All experiments were performed three times with similar results. The results are given as the means and standard deviations (error bars) of six replicates from one representative experiment. *, significantly different (p<0.05) from strain TPH2 based on one-way ANOVA and Tukey's HSD test. Scale bars, 1 cm.

Figure 8. Virulence-related gene expression in Xanthomonas axonopodis pv.citri measured by RT-PCR analysis.

RNA was isolated from cultures of strains TPH2, TPH3 and TPH5 in TSB or XVM2 medium, the latter of which mimics cytoplasmic fluids in planta, at 27°C for 18 hr with shaking at 100 rpm. RT-PCR was performed with primers specific for rfbC (113 bp), hrpG (747 bp), rpfF (870 bp), katE (127 bp) and rpoD (263 bp). The mRNA level of rpoD was used as loading control. The experiments were performed three times with similar results, and representative results from one experiment are shown.