Modifications of <em>Xanthomonas axonopodis</em> pv. citri Lipopolysaccharide Affect the Basal Response and the Virulence Process during Citrus Canker

<div><p><em>Xanthomonas axonopodis</em> 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 (<em>wzt</em> and <em>rfb30</em>3) 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, <em>in vivo</em> and <em>in vitro</em> adhesion and biofilm formation. Changes in the expression levels of the LPS biosynthesis genes were observed in a medium that mimics the plant environment. Xac<em>wzt</em> exhibited reduced virulence in host plants compared to Xac wild-type and Xac<em>rfb303</em>. 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.</p> </div

Bacterial motility assays.

<p>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 <i>Serratia marcescens</i> 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.</p

Effect of <i>wzt</i> and <i>rfb303</i> disruption on pathogenicity in host plants

<p>(A) Disease symptoms on orange leaves inoculated with Xac wild-type, the LPS mutants Xac<i>wzt</i> and Xac<i>rfb303</i> and the complemented strains Xac<i>Cwzt</i> and Xac<i>Crfb303</i> 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.</p

Interaction of Xac strains with non host plants.

<p>(A) Phenotype developed on tobacco leaves inoculated with Xac wild-type, Xac<i>wzt</i> and Xac<i>rfb303</i> at 10⁷ CFU ml⁻¹ in 10 mM MgCl₂. Representative leaves are shown 24 hpi. (B) Bacterial growth of Xac wild-type, Xac<i>wzt</i> and Xac<i>rfb303</i> 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.</p

Bacterial adhesion to abiotic and biotic surfaces.

<p>(A) Bacterial adhesion on plastic (PVC microtiter plate) surface of Xac wild-type, Xac<i>wzt</i>, Xac<i>rfb303</i>, Xac<i>Cwzt</i> and Xac<i>Crfb303</i> 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.</p

Sensitivity of Xac wild-type, Xac<i>wzt</i> and Xac<i>rfb303</i> to oxidative stress and SDS

<p>(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.</p

Cell membrane injuries of orange and tobacco leaves produced by bacterial infection.

<p>The leaves were inoculated with Xac wild-type, Xac<i>wzt</i> and Xac<i>rfb303</i> at 10⁷ CFU ml^-1 and the assays were performed at 7 (orange) or 2 (tobacco) days post-infiltration.</p>a<p>Data represent mean ± standard deviation of three independent experiments.</p

Analysis of Xac LPSs by SDS-PAGE

<p>LPSs were isolated from Xac wild-type (lane 1), Xac<i>wzt</i> (lane 2), Xac<i>Cwzt</i> (lane 3), Xac<i>rfb303</i> (lane 4) and Xac<i>Crfb303</i> (lane 5) strains by the hot phenol method. The polyacrylamide gel was run with a tricine buffer system and subsequently silver-stained.</p

Biofilm formation.

<p>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.</p

Primers used in this work.

a<p>Capital letters correspond to nucleotides of the Xac genome sequence and small letters to nucleotides added to facilitate cloning.</p