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>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

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

<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

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

Using the Amino Acid Network to Modulate the Hydrolytic Activity of β-Glycosidases

<div><p>The active site residues in GH1 β-glycosidases are compartmentalized into 3 functional regions, involved in catalysis or binding of glycone and aglycone motifs from substrate. However, it still remains unclear how residues outside the active site modulate the enzymatic activity. To tackle this question, we solved the crystal structure of the GH1 β-glycosidase from <i>Spodoptera frugiperda</i> (Sfβgly) to systematically map its residue contact network and correlate effects of mutations within and outside the active site. External mutations neighbouring the functional residues involved in catalysis and glycone-binding are deleterious, whereas mutations neighbouring the aglycone-binding site are less detrimental or even beneficial. The large dataset of new and previously characterized Sfβgly mutants supports that external perturbations are coherently transmitted to active site residues possibly through contacts and specifically disturb functional regions they interact to, reproducing the effects observed for direct mutations of functional residues. This allowed us to suggest that positions related to the aglycone-binding site are preferential targets for introduction of mutations aiming to further improve the hydrolytic activity of β–glycosidases.</p></div

Effects on the <i>k</i>_cat/<i>K</i>_m for the hydrolysis of NPβglc due to mutation of residues involved in the three functional regions of the Sfβgly active site.

<p>Effects on the <i>k</i>_cat/<i>K</i>_m for the hydrolysis of NPβglc due to mutation of residues involved in the three functional regions of the Sfβgly active site.</p

Spatial distribution of mutational effects outside the Sfβgly active site.

<p>Highlighted are the main chain (spheres) of amino acids whose mutations cause deleterious (decreases higher than 4-fold using NPβglc, dark orange spheres) and mild decreases (smaller than 4x, light purple spheres) or positive effects (cyan spheres) on Sfβgly activity. Residues outside the active site are labelled with their corresponding colour, as described above. Active site residues from GBS (red sticks), ABS (dark blue sticks) and CR (yellow sticks) are also labelled with their corresponding colour. A substrate molecule, <i>p</i>-nitrophenyl β-glycoside (NPβglc), is placed in the active site (green sticks). Note that positive mutations (cyan spheres), are located close to the ABS but not to the GBS.</p