A Filamentous Hemagglutinin-Like Protein of Xanthomonas axonopodis pv. citri, the Phytopathogen Responsible for Citrus Canker, Is Involved in Bacterial Virulence

Xanthomonas axonopodis pv. citri, the phytopathogen responsible for citrus canker has a number of protein secretion systems and among them, at least one type V protein secretion system belonging to the two-partner secretion pathway. This system is mainly associated to the translocation of large proteins such as adhesins to the outer membrane of several pathogens. Xanthomonas axonopodis pv. citri possess a filamentous hemagglutinin-like protein in close vicinity to its putative transporter protein, XacFhaB and XacFhaC, respectively. Expression analysis indicated that XacFhaB was induced in planta during plant-pathogen interaction. By mutation analysis of XacFhaB and XacFhaC genes we determined that XacFhaB is involved in virulence both in epiphytic and wound inoculations, displaying more dispersed and fewer canker lesions. Unexpectedly, the XacFhaC mutant in the transporter protein produced an intermediate virulence phenotype resembling wild type infection, suggesting that XacFhaB could be secreted by another partner different from XacFhaC. Moreover, XacFhaB mutants showed a general lack of adhesion and were affected in leaf surface attachment and biofilm formation. In agreement with the in planta phenotype, adhesin lacking cells moved faster in swarming plates. Since no hyperflagellation phenotype was observed in this bacteria, the faster movement may be attributed to the lack of cell-to-cell aggregation. Moreover, XacFhaB mutants secreted more exopolysaccharide that in turn may facilitate its motility. Our results suggest that this hemagglutinin-like protein is required for tissue colonization being mainly involved in surface attachment and biofilm formation, and that plant tissue attachment and cell-to-cell aggregation are dependent on the coordinated action of adhesin molecules and exopolysaccharides

Characterization of the residue Arg210 of HrpG in Xcc pathogenicity and HR induction.

<p>(A) Sequence alignment and secondary structure assignments of a region of the DNA-binding domain of OmpR from <i>E</i>. <i>coli</i> and HrpG from Xcc. Helices α1, α2 and α3 are depicted with grey boxes. Asterisks (*) indicate identical residues, colons (:) are conservative replacements and full stops (.) are semiconservative replacements. Arg209 in OmpR sequence and Arg210 in Xcc sequence are depicted in bold. This residue turns into Cys210 in HrpG-R210C sequence (B) Xcc wild type, deletion mutant ΔhrpG, complemented strains ΔhrpG-R210C and ΔhrpG-HrpG and Xcc carrying the wild type and R210C mutant copy, Xcc-HrpG and Xcc-R210C, respectively, were inoculated at 10⁷ CFU/ml in 10 mM MgCl₂ into the intercellular spaces of fully expanded citrus leaves. Representative leaves are shown 7 dpi (left panel) and 25 dpi (right panel). (C) RT-qPCR to determine <i>CsLOB1</i> expression levels in leaves after 24 hours of inoculation with Xcc strains. Bars indicate the expression levels relative to buffer infiltrations. Values are the means of four biological replicates with three technical replicates each. (D) Bacterial growth of the Xcc strains in citrus leaves. Values represent the mean of three samples from three different plants. Error bars indicate standard deviations. (E) Xcc variants were inoculated at 10⁷ CFU/ml in 10 mM MgCl₂ in tomato leaves and a representative photograph after 24 h is shown.</p

HrpG interacts with itself and with HrpG-R210C.

<p>(A) Pull-down assays showing <i>in vitro</i> interaction studies with HrpG fused to GST (GST-HrpG) and HrpG and HrpG-R210C fused to thioredoxin. Proteins eluted from the matrix were analyzed by immunoblotting using anti-His and anti-GST antibodies. (B) Western and Far-Western blots showing interactions between HrpG and HrpG and HrpG-R210C fused to thioredoxin. The Western blot was incubated with anti-GST (left panel) and in the Far-Western blot (right panel) the nitrocellulose membranes were overlayed with 50 μg of GST-HrpG and after washing, probed with anti-GST antibody.</p

A substitution of Arg210 by cysteine does not prevent binding to DNA promoter regions.

<p>(A) Electrophoretic mobility shift assay of ³²P-labeled <i>hrpX</i> promoter and purified HrpG and HrpG-R210C. Numbers in the top of the lanes indicate pmoles of protein added to the assay. (B) Complex competition assays with excess unlabeled DNA: Lanes labeled (-) show the complex between HrpG (left panel) and HrpG-R210C (right panel) with 8 pmol of protein. Competition was performed using the ratio indicated in each lane. (C) EMSA of ³²P-labeled <i>hrpX</i> promoter and a mixture 1:1 of HrpG and HrpG-R210C with the total pmol indicated on top of each lane. (D) Control EMSA of ³²P-labeled <i>hrpX</i> promoter and purified Trx in the same conditions as HrpG to discard unspecific interactions. Lane 1 shows the binding of 16 pmoles of HrpG to P_hrpX as control. Lanes 2 and 3: P_hrpX was incubated with 15 and 30 pmoles of Trx, respectively in the same conditions as for HrpG. Lanes 4 and 5: unspecific competition assay in which the HrpG-P_hrpX complex was challenged with 200 ng of poly-dIdC (lane 4) or salmon sperm DNA (lane 5). (E) Lane 1: free probe, lane 2: binding of 16 pmoles of HrpG to P_hrpX as control, lane 3: P_hrpX was incubated with 25 μg of a protein extract obtained from <i>E</i>. <i>coli</i> bearing the pET32 empty vector.</p

Three-dimensional structure models of HrpG and HrpG-R210C C-terminal domains.

<p>Modeling was done with the SwissModel-SPDViewer program based on the structure of OmpR from <i>E</i>. <i>coli</i>. Both structures are shown in a spatial orientation similar to that adopted by OmpR in its interaction with DNA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125516#pone.0125516.ref010" target="_blank">10</a>]. α-helices are shown in pink while β-sheets in blue. In black the side chains of the amino acids arginine (R210) and cysteine (C210) are shown. W1 and W2: indicate wings 1 and 2, respectively; and α-helices 2 and 3 and the α-loop are also indicated.</p

Expression of T3SS genes up-regulated by HrpG depends on the wild type copy of <i>hrpG</i>.

<p>(A) qRT-PCR of <i>hrpX</i>, <i>hrcC</i> and <i>hrpB2</i> of total RNA obtained from Xcc, deletion mutant ΔhrpG, ΔhrpG-R210C, ΔhrpG-HrpG, Xcc-R210C and Xcc-HrpG strains grown in XVM2 were assayed. (B) As in (A) but RNA was obtained from bacteria recovered from infiltrated tissue at 3 and 6 dpi before RNA extraction. As a reference gene, a fragment of <i>rpoB</i> gene was amplified. Values represent the means of four independent experiments. Error bars indicate standard deviations. Data were statistically analyzed using one-way ANOVA (p<0.05).</p

Analysis of the factors involved in the swarming motility of <i>X. axonopodis</i> pv. <i>citri</i> wild type and Δ<i>XacFhaB</i> and Δ<i>XacFhaC</i> strains.

<p>(A) For flagellar proteins immunodetection, whole-cell extracts from the XacWT, Δ<i>XacFhaB</i> and Δ<i>XacFhaC</i> strains isolated from the center (C) or the border (B) of the swarming colony grown as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004358#pone-0004358-g005" target="_blank">Figure 5A</a> were analyzed by Western blotting developed with anti-flagellin polyclonal antibodies. Samples were standardized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004358#s4" target="_blank">Materials and Methods</a>. (B) Cells of XacWT, Δ<i>XacFhaB</i> and Δ<i>XacFhaC</i> strains isolated from the center or the border of the advancing swarm were stained for flagellar structures (indicated by arrows) and observed under light microscopy at 100× magnification. The photographs are representative of three experiments in which several fields of view were observed. (C) Xanthan production in XOL medium of XacWT, Δ<i>XacFhaB</i> and Δ<i>XacFhaC</i> mutants strains. Each data point is the mean of three experiments, error bars indicate the standard error. (D) Total RNA was extracted from XacWT and Δ<i>XacFhaB</i> mutant grown in SB for 48 h at 28°C and <i>gumD</i> expression was analyzed by RT-PCR using specific primers. 16S rRNA was used as a constitutive control.</p