A plant natriuretic peptide-like molecule of the pathogen Xanthomonas axonopodis pv. citri causes rapid changes in the proteome of its citrus host

<p>Abstract</p> <p>Background</p> <p>Plant natriuretic peptides (PNPs) belong to a novel class of peptidic signaling molecules that share some structural similarity to the N-terminal domain of expansins and affect physiological processes such as water and ion homeostasis at nano-molar concentrations. The citrus pathogen Xanthomonas axonopodis pv. citri possesses a PNP-like peptide (XacPNP) uniquely present in this bacteria. Previously we observed that the expression of <it>XacPNP </it>is induced upon infection and that lesions produced in leaves infected with a XacPNP deletion mutant were more necrotic and lead to earlier bacterial cell death, suggesting that the plant-like bacterial PNP enables the plant pathogen to modify host responses in order to create conditions favorable to its own survival.</p> <p>Results</p> <p>Here we measured chlorophyll fluorescence parameters and water potential of citrus leaves infiltrated with recombinant purified XacPNP and demonstrate that the peptide improves the physiological conditions of the tissue. Importantly, the proteomic analysis revealed that these responses are mirrored by rapid changes in the host proteome that include the up-regulation of Rubisco activase, ATP synthase CF1 α subunit, maturase K, and α- and β-tubulin.</p> <p>Conclusions</p> <p>We demonstrate that XacPNP induces changes in host photosynthesis at the level of protein expression and in photosynthetic efficiency in particular. Our findings suggest that the biotrophic pathogen can use the plant-like hormone to modulate the host cellular environment and in particular host metabolism and that such modulations weaken host defence.</p

A LOV Protein Modulates the Physiological Attributes of Xanthomonas axonopodis pv. citri Relevant for Host Plant Colonization

Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease

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

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

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

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