Modifications of Xanthomonas axonopodis pv. citri lipopolysaccharide affect the basal response and the virulence process during citrus canker

Abstract

Xanthomonas axonopodis pv. citri (Xac) is the phytopathogen responsible for citrus canker, one of the most devastating citrus diseases in the world. A broad range of pathogens is recognized by plants through so-called pathogen-associated molecular patterns (PAMPs), which are highly conserved fragments of pathogenic molecules. In plant pathogenic bacteria, lipopolisaccharyde (LPS) is considered a virulence factor and it is being recognized as a PAMP. The study of the participation of Xac LPS in citrus canker establishment could help to understand the molecular bases of this disease. In the present work we investigated the role of Xac LPS in bacterial virulence and in basal defense during the interaction with host and non host plants. We analyzed physiological features of Xac mutants in LPS biosynthesis genes (wzt and rfb303) and the effect of these mutations on the interaction with orange and tobacco plants. Xac mutants showed an increased sensitivity to external stresses and differences in bacterial motilities, in vivo and in vitro adhesion and biofilm formation. Changes in the expression levels of the LPS biosynthesis genes were observed in a medium that mimics the plant environment. Xacwzt exhibited reduced virulence in host plants compared to Xac wild-type and Xacrfb303. However, both mutant strains produced a lower increase in the expression levels of host plant defense-related genes respect to the parental strain. In addition, Xac LPS mutants were not able to generate HR during the incompatible interaction with tobacco plants. Our findings indicate that the structural modifications of Xac LPS impinge on other physiological attributes and lead to a reduction in bacterial virulence. On the other hand, Xac LPS has a role in the activation of basal defense in host and non host plants.

Figure 1. Analysis of Xac LPSs by SDS-PAGE

LPSs were isolated from Xac wild-type (lane 1), Xacwzt (lane 2), XacCwzt (lane 3), Xacrfb303 (lane 4) and XacCrfb303 (lane 5) strains by the hot phenol method. The polyacrylamide gel was run with a tricine buffer system and subsequently silver-stained.

Figure 2. Sensitivity of Xac wild-type, Xacwzt and Xacrfb303 to oxidative stress and SDS

(A) Hydrogen peroxide resistance of bacterial cultures. Cells in early exponential phase of growth were exposed to the indicated concentrations of H₂O₂ for 15 min. The number of CFU was determined for each culture before and after the peroxide treatment by plating of appropriate dilutions. The percentage of survival is defined as the number of CFU after treatment divided by the number of CFU prior to treatment ×100. (B) Susceptibility to MV toxicity by the disk diffusion assay. The diameters of the inhibition zones were measured after 24 h of incubation. In (A) and (B) data are expressed as the mean ± standard deviation of three independent experiments. (C) Growth of Xac strains in SB-agar plates supplemented with different concentrations of SDS.

Figure 3. Bacterial motility assays.

The different strains were centrally inoculated on SB plates supplemented with 0.3% (w v⁻¹) agar for swimming (A) and 0.7% (w v⁻¹) agar for swarming assay (B) and incubated 4 days at 28°C to determine migration zones. (C) Analysis of flagellin expression of bacteria from the border and the center of swarming plates by Western Blot using Serratia marcescens anti-flagellin rabbit polyclonal antibodies (left panel). Expression profiles were obtained by densitometric quantification of band intensities (right panel). Experiments were performed in triplicate with similar results; bars indicate mean ± standard deviation. IOD, integrated optical density; A.U., arbitrary units; C: center and B: border zone of a swarming plate.

Figure 4. Bacterial adhesion to abiotic and biotic surfaces.

(A) Bacterial adhesion on plastic (PVC microtiter plate) surface of Xac wild-type, Xacwzt, Xacrfb303, XacCwzt and XacCrfb303 strains grown in SB or XVM2 medium. (B) Bacterial adhesion on abaxial orange leaves surfaces. In the left, representative images of CV staining of a PVC plate or a leaf are shown. Histograms in the right represents spectrophotometric quantifications of CV attached (Abs 540 nm). Data are expressed as the mean ± standard deviation of three independent experiments. Scale bar, 1 cm.

Figure 5. Biofilm formation.

GFP-labeled Xac strains were grown on chambered cover slides and visualized under CLS microscopy after 2 and 5 days of bacterial growth. For each time period the left panels show cell aggregation at the bottom of the chambered cover slides with a magnification of 40× and the right panels show a 2× zoom of the regions marked in the previous panels. Scale bars, 50 µm.

Figure 6. Expression of Xac LPS genes in the plant-mimicking XVM2 medium.

(A) Amplified products of the wzt and rfb303 genes by semiquantitative RT-PCR using RNA preparations from early exponential Xac cultures grown in SB and in XVM2. As a control for constitutive bacterial expression a fragment of 16S rRNA was simultaneously amplified. (B) Expression profiles obtained by densitometric quantification of band intensities. Data are expressed as the mean ± standard deviation of three independent experiments. IOD, integrated optical density; A.U., arbitrary units.

Figure 7. Effect of wzt and rfb303 disruption on pathogenicity in host plants

(A) Disease symptoms on orange leaves inoculated with Xac wild-type, the LPS mutants Xacwzt and Xacrfb303 and the complemented strains XacCwzt and XacCrfb303 at 10⁷ CFU ml⁻¹ in 10 mM MgCl₂. A representative leaf 13 days after inoculation is shown. Left panel, adaxial side; right panel, abaxial side. Scale bars, 1 cm. (B) Bacterial growth of Xac cells in orange leaves during 22 days. Values represent means ± standard deviations of three independent samples.

Figure 8. Expression analysis of C. sinensis defense-related genes

(A) Real Time PCR showing expression levels of PrxA, PR-1, MKK4 and GST genes from orange leaves infiltrated with Xac wild-type, Xacwzt and Xacrfb303 at 10⁷ CFU ml⁻¹ and 10 mM MgCl₂ as control. Leaves were harvested at 2, 6 and 24 hpi. Columns show the expression value relative to the control. To perform the analysis of relative expression we used the 2^−ΔΔCT method normalizing to actin expression levels. The results are expressed as mean ± standard deviation of three independent determinations.

Figure 9. Interaction of Xac strains with non host plants.

(A) Phenotype developed on tobacco leaves inoculated with Xac wild-type, Xacwzt and Xacrfb303 at 10⁷ CFU ml⁻¹ in 10 mM MgCl₂. Representative leaves are shown 24 hpi. (B) Bacterial growth of Xac wild-type, Xacwzt and Xacrfb303 in tobacco leaves during 9 days. Values represent the mean ± standard deviation of three independent experiments. (C) Effect of pre-inoculation of tobacco leaves with LPS from Xac wild-type. A tobacco leaf area was inoculated with LPS (100 µg ml⁻¹) and 20 h later, Xac strains at 10⁷ CFU ml^–1 were inoculated into the same area. A representative 24 hpi leaf and bacterial growth curves during 9 days are shown. Scale bars, 1 cm.