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

Resistance to limited proteolysis of the two protein variants.

<p><b>(A)</b> HrpG and HrpG-R210C were subjected to trypsin proteolysis for 30 min at room temperature. Lanes 1 and 5 are the undigested samples (5 μg), lanes 2 and 6, proteins were digested with 1:250, lanes 3 and 7 with 1:125 and lanes 4 and 8 with 1:62.5 (w/w) trypsin. (B) A mixture 1:1 of HrpG and HrpG-R210C was subjected to trypsin proteolysis for 30 min at room temperature. Lanes 1 is the undigested sample (5 μg), in lanes 2–4 proteins were digested with 1:250, 1:125 and 1:62.5 (w/w) trypsin, respectively.</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