Ectopic expression of <i>hrpG</i> under control of a constitutive promoter restores full pathogenicity and HR of the <i>rsmA</i> mutant of <i>Xanthomonas citri</i> subsp. citri.

<p><b>A</b>) XCC strains 306 (WT) and the <i>rsmA</i> mutant carrying the empty plasmid pBBR5 (ΔrsmA) or pBBR5 with <i>hrpG</i> wild-type (hrpG) or <i>hrpG</i> alleles with E44K and D60N mutations were inoculated into <b>A</b>) sweet orange (<i>Citrus sinensis</i>) leaves or <b>B</b>) tobacco leaves by infiltrating bacterial cells at a concentration of 10⁶ CFU/ml. Sweet orange leaves inoculated with WT and <i>rsmA</i> mutant strains harboring both <i>hrpG</i> and hrpG-E44K alleles showed canker symptoms 7 days after inoculation, while a strong HR was observed in tobacco leaves only 2 days after infiltration. However, constitutive expression of the hrpGD60N allele was not able to recover the pathogenicity and HR in the <i>rsmA</i> mutant. <b>C</b>) <i>In plant</i> growth curve experiments confirmed that the <i>rsmA</i> mutant transformed with both empty pBBR5 (ΔrsmA) and pBBR5Lac-<i>hrp</i>GD60N-6His (D60N) have impaired growth in host plants. Error bars represent standard deviations. <b>D</b>) Western-blotting analysis of HrpG-His protein levels in the wild-type and <i>rsmA</i> mutant strains carrying the <i>hrpG</i> (hrpG) or mutated <i>hrpG</i> alleles (E44K and D60N) under control of a constitutive promoter. Equal amounts of total cell extracts were analyzed by immunoblotting with anti-6HisTag antibodies (MBL, USA).</p

RsmA regulates protein levels of T3SS in <i>X. citri</i> subsp. citri.

<p>Immunoblotting analyses of the total protein extracts of the wild-type (Wt), the <i>rsmA</i> mutant (Δ<i>rsmA</i>) harboring the empty plasmid pUFR047 and the complemented strain (pUFRrsmA) are shown. Bacterial cells were grown in the XVM2 medium and collected at OD_{600 nm} = 0.5. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The blots were probed with <b>A</b>) HrpB1, <b>B</b>) HrpD6 or <b>C</b>) HrcU and HrpB2 polyclonal antibodies, respectively. Protein-A conjugated with horseradish peroxidase was used to detect the blots. Beneath the panels are presented the values of the relative levels of detected proteins in <i>rsmA</i> mutant and complemented strains which were estimated according to wild-type results. The estimated values were normalized with the values obtained to unspecific protein bands also recognized by the antibodies. <b>D</b>) and <b>E</b>) GUS assays using translational fusion constructs. The different constructs used in this assay are represented by diagrams bellow of the graphics. D) Wild-type and <i>rsmA</i> mutant cells harboring plasmid-borne promoterless <i>gusA</i> in-frame fused to the native promoters and the first codons of the <i>hrp</i> genes. E) Wild-type and <i>rsmA</i> mutant cells transformed with translational fusions driven by the constitutive P<i>lac</i> promoter. Values presented are means ± standard deviations of three independent experiments. * represents the significant difference between the wild-type and Δ<i>rsmA</i> values by using ANOVA. The GUS assay was repeated twice with similar results.</p

RNA mobility shift assays with purified 6HisRsmA of <i>X. citri</i> subsp. citri

<p><b>A</b>) 6HisRsmAxcc (65 nM) binds to the high affinity RNA target R9-43. Biotin 3′-end-labeled R9-43 (6.25 nM) was incubated with 6HisRsmAxcc (65 nM) for 30 minutes at room temperature, followed by analysis on a 5% native polyacrylamide gel. A competitive assay in which unlabeled R9-43 RNA (6.25 nM) was added to the reaction reduced the signal resulting from the biotinylated nucleotide. <b>B</b>) 6HisRsmAxcc directly interacts with the 5′ UTRs of <i>hrpG</i> and <i>hrpD</i>. The leader sequences of <i>hrpD</i>, <i>hrpE</i> and <i>hrpG</i> cloned were transcribed <i>in vitro</i> and biotinylated with RNA Labeling kit (Roche). Biotinylated RNA probes were incubated with 6HisRsmAxcc and resolved in a 5% native polyacrylamide gel. The addition of unlabeled competitor R9-43 to the reactions reduced the intensity of the shifted band, which confirmed the specificity of the RsmAxcc-<i>hrpG</i> and RsmAxcc-<i>hrpD</i> interactions. <b>C</b>) 3′-end-biotin-labeled RNA probes encoding the leader sequences of <i>hrpB</i>, <i>hrpC</i>, <i>hrpF</i> and <i>hrpX</i> were tested for interactions with 6HisRsmAxcc (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945.s007" target="_blank">Table S4</a>). In addition, 3′-end-biotin–labeled RNA probes <i>hrpG1</i> and <i>hrpG2</i>, which bear the GGA motifs encoded by the 5′ leader sequence of <i>hrpG</i>, were used to map the interaction RsmAxcc-<i>hrpG</i>. Only the GGA motif between nucleotides 80 and 120 in the <i>hrpG</i> leader sequence (<i>hrpG</i>2 probe) interacted with 6HisRsmAxcc. <b>D</b>) To determinate the apparent equilibrium binding constant (K_d), 3′ end-labeled <i>hrpG</i>2 RNA (6.25 nM) was incubated with increasing concentrations of 6HisRsmAxcc as noted at the bottom of each lane. The binding curve for the 6HisRsmAxcc-<i>hrpG</i>2 interaction was determined as a function of 6HisRsmAxcc concentration and shifted band intensity. The average pixel value of each shifted band was calculated with ImageJ software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Girish1" target="_blank">[68]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Hartig1" target="_blank">[69]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Jensen1" target="_blank">[112]</a>. The apparent equilibrium binding constant (K_d) for this reaction was 0.18±0.2 µM 6HisRsmAxcc. Samples were loaded and resolved onto a 5% native polyacrylamide gel. All probes were transferred and cross-linked to a nylon membrane, incubated with streptavidin conjugated with horseradish peroxidase, and detected according to manufacturer's instructions (LightShiftChemiluminescent RNA EMSA Kit, Thermo Scientific). Signals + and − correspond to the presence and absence in the reaction, respectively. Positions of bound and free probes are shown.</p

Analysis of <i>hrpG</i> and <i>hrpD</i> mRNA stability in the wild-type and <i>rsmA</i> mutant strains by RT-PCR.

<p><b>A</b>) Cells of the XCC wild-type and Δ<i>rsmA</i> strains were grown in XVM2 medium to OD_{600 nm} = 0.6, treated with 10 µg/µL ciprofloxacin and harvested at several time points after treatment. Total RNA was isolated, and 2 µg of RNA was used for One Step RT-PCR (Qiagen) in 25 µL reactions. Reactions were subjected to PCR amplification for 26 cycles. Ten microliters of each reaction were resolved on a 1.5% agarose gel. The stability of the <i>hrpD</i> transcript was evaluated using primers annealing within the first orf <i>hrpQ</i>. The 16S RNA was analyzed as a control for normalizing the <i>hrpG</i> and <i>hrpD</i> amplification products. <b>B</b>) The relative values of <i>hrpG</i> and <i>hrpD</i> mRNA half-lives were estimated by determinating the average pixel value of each amplified product and subtracting the background using ImageJ software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Girish1" target="_blank">[68]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Jensen1" target="_blank">[112]</a>. The mean values were normalized to the corresponding 16S amplification product. Mean values derived from two independent experiments are shown. <b>C</b>) Model for the predicted secondary structure of the <i>hrpG</i> and <i>hrpD</i> leader sequences obtained with MFold software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Zuker1" target="_blank">[66]</a>. The positions of GGA motifs in the structures are indicated with arrows. AUG is shown in an open box.</p

Determination of <i>hrp/hrc</i> transcriptional start sites of <i>X. citri</i> subsp. citri.

<p>The transcriptional start sites for <i>the genes hrpG, hrpX and hrpF, and the operons hrpB, hrpC, hrpD and hrpE</i> of XCC were determined by 5′RACE. <b>A</b>) Specific PCR products were detected after the amplification of reverse-transcribed cDNA with the gene-specific primers for <i>hrp/hrc genes</i> together with an adapter primer (Roche), respectively. <b>B</b>) Sequencing of the PCR products identified the nucleotides indicated by arrow as the transcription start sites of <i>hrpG, hrpX, hrpB, hrpC, hrpD, hrpE and hrpF</i> of XCC. Analysis of the 5′ leader sequences of the <i>hrp/hrc</i> transcripts suggests potential RsmA binding sites (highlighted) in <i>hrpG</i>, <i>hrpC</i>, <i>hrpD</i> and <i>hrpE</i>. However, the 5′ leader regions of the <i>hrpB</i>, <i>hrpF</i> and <i>hrpX</i> do not contain the GGA motifs. The +1 nucleotide is indicated with an arrow, putative RsmA binding sites (GGA) in the leader sequences are highlighted in red color, and the PIP-box motif within each promoter are in bold. The −35 and −10 sequences are shown in bold and italics. ATG or GTG are indicated in bold and underlined. The specific primers used to amplify the fragments are underlined.</p

<i>In vivo</i> phosphorylation of HrpG Asp60 residue is critical to restore the virulence in the Δ<i>rsmA</i> mutant of XCC.

<p><b>A</b>) Western-blotting analysis of XCC lysates on a 25 µM Phos-tag acrylamide gel (Wako, USA). Bacteria cells were grown in nutrient broth or XVM2 medium (T3SS inducible medium) to OD_{600 nm} = 0.6, and equal amounts of total cell extracts were analyzed by immunoblotting with anti-6HisTag antibodies (MBL, USA). <b>B</b>) Western-blotting analysis to determine HrpG-His protein levels in XCC cell extracts resolved in a 12% acrylamide gel system without Manganese(II)-Phos-tag. Strains analyzed: WT harboring the wild-type <i>hrpG</i>-6His allele; Δ<i>rsmA</i> carrying the wild-type <i>hrp</i>G-6His allele and the mutated <i>hrpG</i>-6His alleles D41N, E44K and D60N. All <i>hrpG</i>-6His constructs were cloned into the pBBR5 plasmid and placed under the control of a constitutive promoter.</p

<i>rsmA</i> is required for the pathogenicity of <i>Xanthomonas citri</i> subsp. citri in the host plant sweet orange and contributes to the hypersensitive response (HR) in tobacco leaves (<i>Nicotiana benthamiana</i>).

<p><b>A</b>) Disease symptoms on host sweet orange (<i>Citrus sinensis</i>) leaves 7 days post inoculation (D.P.I.) of bacterial cells at a concentration of 10⁶ CFU/ml. <b>B</b>) Growth assay in planta. Bacterial cells were inoculated into sweet orange leaves at a concentration of 10⁶ CFU/ml and recovered at different time points. The values represent the means of three replicates. The experiment was repeated three times with similar results. Means ± standard deviations are plotted. <b>C</b>) Macroscopic symptoms induced 7 D.A.I of tobacco leaves by infiltrating bacterial cells at a concentration of 10⁶ CFU/ml. <b>D</b>) Growth curve in the minimal medium XVM2 and Western-blotting assay using protein extracts of <i>rsmA</i> mutant cells harboring the pUFR047-<i>rsmA</i>-Flag construct. Cells were collected in different growth stages: EL, early log; ML, medium log); LL, late log; and ST, stationary phase. Wt = <i>X. citri</i> subsp. <i>citri</i> strain 306, Δrsm<i>A</i> = mutant with a deletion of XAC1743 (<i>rsmA</i>) harboring the empty plasmid pUFR047, pUFRrsmA = complementation of ΔrsmA with <i>rsmA</i> cloned in pUFR047, ΔhrpG = <i>hprG</i> mutant <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003945#ppat.1003945-Guo1" target="_blank">[9]</a>, pUFRhrpG = complementation of ΔhrpG with <i>hrpG</i> cloned into pUFR047, and Mock, 10 mM MgCl2. RNPβ: antibody to the β-subunit of RNA polymerase.</p