<i>sX13</i> contributes to bacterial growth in culture and virulence.

<p>Growth of <i>Xcv</i> wild type 85-10 (wt), the <i>sX13</i> deletion mutant (Δ<i>sX13</i>) and Δ<i>sX13</i> containing chromosomally re-integrated <i>sX13</i> (Δ<i>sX13</i>+<i>sX13</i>_ch) in (A) complex medium NYG and (B) minimal medium MMA, respectively. Error bars represent standard deviations. Asterisks indicate statistically significant differences compared to wt (<i>t</i>-test; <i>P</i><0.05). (C) Growth of <i>Xcv</i> 85-10 (wt) and Δ<i>sX13</i> in leaves of susceptible ECW pepper plants. Data points represent the mean of three different samples from three different plants of one experiment. Standard deviations are indicated by error bars. (D) Plant infection assay. <i>Xcv</i> strains 85-10 (wt) and Δ<i>sX13</i> carrying the empty vector (pB) or the sX13 expression construct (p<i>sX13</i>) and strains additionally expressing HrpG* (p<i>hrpG*</i>) were inoculated at a density of 4×10⁸ (left panel) and 10⁸ cfu/ml (right panel), respectively, into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms in ECW were photographed 9 days post inoculation (dpi). The HR was visualized by ethanol bleaching of the leaves 3 dpi (left panel) and 18 hours post inoculation (right panel), respectively. Dashed lines indicate the inoculated areas. All experiments were performed at least three times with similar results.</p

sX13 accumulation is altered under stress conditions in <i>Xcv</i> 85-10.

<p>(A) Northern blot analysis of sX13. Exponential phase cultures of NYG-grown <i>Xcv</i> 85-10 were transferred to NYG medium or MMA containing the indicated additives or lacking a nitrogen or carbon source (ΔN; ΔC). Cultures were shaken for three hours at 30°C unless otherwise indicated. 5S rRNA was probed as loading control. (B) sX13 and selected sX13-regulated genes (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat-1003626-t001" target="_blank">Table 1</a>) were analyzed by qRT-PCR using RNA from <i>Xcv</i> 85-10 (wt) cultures shown in (A) and NYG-grown Δ<i>sX13</i>. Bars represent fold-changes (log₁₀) of mRNA amounts compared to <i>Xcv</i> 85-10 grown in NYG at 30°C. Experiments were performed twice with similar results.</p

Selected sX13-regulated genes validated by qRT-PCR analysis.

a<p>, bold letters indicate genes with known TSS <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat.1003626-Schmidtke1" target="_blank">[16]</a>.</p>b<p>, refers to Thieme <i>et al.</i> (2005) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat.1003626-Thieme1" target="_blank">[32]</a>.</p>c<p>, presence of a 4G-motif within the 5′-UTR or 100 bp upstream of translation start codon if TSS is unknown (a) and within 100 bp downstream of start codon (b) (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat.1003626.s004" target="_blank">Figure S4</a>).</p>d<p>, genes not detected as expressed are marked with —.</p>e<p>, values represent mean fold-change and standard deviation (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat-1003626-g004" target="_blank">Figure 4</a>);</p><p>n.t. - not tested.</p

Deletion of <i>sX13</i> derogates virulence gene expression.

<p>(A) <i>Xcv</i> strains 85-10 (wt) and the <i>sX13</i> deletion mutant (Δ<i>sX13</i>) carrying the empty vector (pB) or the sX13 expression construct (p<i>sX13</i>) and strains additionally expressing HrpG* (p<i>hrpG*</i>) were incubated for 3.5 hours in <i>hrp</i>-gene inducing medium XVM2. Total protein extracts were analyzed by immunoblotting using antibodies directed against HrpF, HrcN, HrcJ and GroEL. The experiment was repeated twice with similar results. (B) <i>Xcv</i> 85-10 (wt), Δ<i>sX13</i> and Δ<i>sX13</i>+<i>sX13</i>_ch and strains additionally expressing HrpG* were incubated for 3.5 hours in <i>hrp</i>-gene inducing medium XVM2. Total protein extracts were analyzed by immunoblotting using antibodies directed against HrcJ and GroEL. The experiment was repeated twice with similar results. (C) Indicated genes were analyzed by qRT-PCR using RNA from cultures described in (B). The amount of each RNA in <i>Xcv</i> 85-10 was set to 1. Data points and error bars represent mean values and standard deviations obtained with three independent biological samples. Asterisks indicate statistically significant differences compared to wt (<i>t</i>-test; <i>P</i><0.03).</p

sX13 loops differentially contribute to abundance of putative mRNA targets.

<p>Relative transcript levels of (A) <i>XCV2821</i>, (B) <i>XCV3927</i>, (C) <i>hfq</i>, (D) <i>pilH</i>, (E) <i>XCV3572</i> and (F) <i>XCV0612</i> were analyzed by qRT-PCR in total RNA of NYG-grown <i>Xcv</i> strains 85-10 (wt) and Δ<i>sX13</i> carrying pBRS (pB), p<i>sX13</i> or mutated sX13-derivatives (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat-1003626-g006" target="_blank">Figure 6</a>). The mRNA abundance in the wt was set to 1. Data points and error bars represent mean values and standard deviations obtained with at least three independent biological samples. Statistically significant differences are indicated (<i>t</i>-test; <i>P</i><0.015).</p

Small RNA sX13: A Multifaceted Regulator of Virulence in the Plant Pathogen <i>Xanthomonas</i>

<div><p>Small noncoding RNAs (sRNAs) are ubiquitous posttranscriptional regulators of gene expression. Using the model plant-pathogenic bacterium <i>Xanthomonas campestris</i> pv. <i>vesicatoria</i> (<i>Xcv</i>), we investigated the highly expressed and conserved sRNA sX13 in detail. Deletion of <i>sX13</i> impinged on <i>Xcv</i> virulence and the expression of genes encoding components and substrates of the Hrp type III secretion (T3S) system. qRT-PCR analyses revealed that sX13 promotes mRNA accumulation of HrpX, a key regulator of the T3S system, whereas the mRNA level of the master regulator HrpG was unaffected. Complementation studies suggest that sX13 acts upstream of HrpG. Microarray analyses identified 63 sX13-regulated genes, which are involved in signal transduction, motility, transcriptional and posttranscriptional regulation and virulence. Structure analyses of <i>in vitro</i> transcribed sX13 revealed a structure with three stable stems and three apical C-rich loops. A computational search for putative regulatory motifs revealed that sX13-repressed mRNAs predominantly harbor G-rich motifs in proximity of translation start sites. Mutation of sX13 loops differentially affected <i>Xcv</i> virulence and the mRNA abundance of putative targets. Using a GFP-based reporter system, we demonstrated that sX13-mediated repression of protein synthesis requires both the C-rich motifs in sX13 and G-rich motifs in potential target mRNAs. Although the RNA-binding protein Hfq was dispensable for sX13 activity, the <i>hfq</i> mRNA and Hfq::GFP abundance were negatively regulated by sX13. In addition, we found that G-rich motifs in sX13-repressed mRNAs can serve as translational enhancers and are located at the ribosome-binding site in 5% of all protein-coding <i>Xcv</i> genes. Our study revealed that sX13 represents a novel class of virulence regulators and provides insights into sRNA-mediated modulation of adaptive processes in the plant pathogen <i>Xanthomonas</i>.</p></div

sX13 activity does not require Hfq.

<p>(A) Northern blot analysis. Total RNA from NYG-grown <i>Xcv</i> strains 85-10 (wt), the <i>hfq</i> frameshift mutant (<i>hfq⁻</i>) and the <i>hfq</i> mutant ectopically expressing Hfq (p<i>hfq</i>) was analyzed using sX13- or sX14-specific probes. 5S rRNA was probed as loading control. The experiment was performed twice with two independent mutants and with similar results. (B) Plant infection assay. The <i>Xcv</i> wild-type 85-10 (wt) and <i>hfq</i> mutant strain (<i>hfq⁻</i>) were inoculated at 2×10⁸ cfu/ml into leaves of susceptible ECW and resistant ECW-10R plants. Disease symptoms were photographed 6 dpi. The HR was visualized 2 dpi by ethanol bleaching of the leaves. Dashed lines indicate the inoculated areas. The experiment was repeated three times with similar results. (C) GFP fluorescence of NYG-grown <i>Xcv</i> 85-10 (wt), the <i>hfq</i> mutant (<i>hfq⁻</i>), the <i>sX13</i> deletion mutant (Δ<i>sX13</i>) and the double mutant (Δ<i>sX13hfq⁻</i>) carrying pFX<i>3927</i> or pFX<i>hfq</i>. <i>Xcv</i> autofluorescence was determined by <i>Xcv</i> 85-10 carrying pFX0 (control). Data points and error bars represent mean values and standard deviations obtained with at least four independent experiments. GFP fluorescence of the wt was set to 1. Asterisks denote statistically significant differences (<i>t</i>-test; <i>P</i><0.01).</p

qRT-PCR analysis of sX13-regulated genes.

<p>Selected sX13-regulated genes (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat-1003626-t001" target="_blank">Table 1</a>) were analyzed by qRT-PCR using RNA from NYG- and MMA-grown <i>Xcv</i> strains 85-10 (wt) and Δ<i>sX13</i>. The amount of each mRNA in the wt was set to 1. Bars represent fold-changes of mRNA amounts in strain Δ<i>sX13</i> compared to 85-10 on a logarithmic scale (log₁₀). Data points and error bars represent mean values and standard deviations obtained with at least three independent biological samples. Asterisks denote statistically significant differences (<i>t</i>-test; <i>P</i><0.05). Dashed lines indicate a 1.5-fold change. Transcripts not detected in the microarray analyses are marked with ‘a’.</p

sX13 loops impact on <i>Xcv</i> virulence.

<p>(A) Secondary structure of sX13 based on prediction and probing (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat.1003626.s002" target="_blank">Figure S2</a>). sX13 consists of an unstructured 5′-, three double-stranded regions (S1; S2; S3) and three loops (loop 1–3). 4C-/5C-motifs are highlighted in red. Bold letters indicate unpaired bases and bars mark double-stranded regions deduced from structure probing. Mutations in loops are boxed, exchanged nucleotides are underlined. (B) Derivatives mutated in loops 2 and 3 fail to complement the plant phenotype of Δ<i>sX13</i>. Leaves of resistant ECW-10R plants were inoculated at 10⁸ cfu/ml with <i>Xcv</i> 85-10 (wt) and Δ<i>sX13</i> carrying pBRS (pB), p<i>sX13</i> or one of the following derivatives: sX13 lacking 14 terminal nucleotides (p<i>sX13</i>Δ5′), sX13 mutated in single loops (p<i>L1</i>, p<i>L2</i>, p<i>L3</i>) or in two loops (p<i>L1/2</i>, p<i>L1/3</i>, p<i>L2/3</i>). The HR was visualized by ethanol bleaching of the leaves 2 dpi. Dashed lines indicate the inoculated areas. The experiment was performed four times with similar results.</p

sX13-dependency of mRNA target::GFP synthesis requires a G-rich motif.

<p>GFP fluorescence of NYG-grown <i>Xcv</i> strains 85-10 (wt) and Δ<i>sX13</i> carrying pB, p<i>sX13</i> or mutated sX13-derivatives (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003626#ppat-1003626-g006" target="_blank">Figure 6</a>) and carrying GFP-reporter plasmids (A) pFX<i>3927</i>, (B) pFX<i>hfq</i>, (C) pFX<i>pilH</i> or (D) pFX<i>0612</i>. pFX<i>_MUT</i> derivatives contain a mutated 4G-motif. <i>Xcv</i> autofluorescence was determined using pFX0. GFP fluorescence of the wt was set to 1. Data points and error bars represent mean values and standard deviations obtained from at least four independent experiments. Statistically significant differences are indicated (<i>t</i>-test; <i>P</i><0.015).</p