Regulation of Cell Wall-Bound Invertase in Pepper Leaves by Xanthomonas campestris pv. vesicatoria Type Three Effectors

Xanthomonas campestris pv. vesicatoria (Xcv) possess a type 3 secretion system (T3SS) to deliver effector proteins into its Solanaceous host plants. These proteins are involved in suppression of plant defense and in reprogramming of plant metabolism to favour bacterial propagation. There is increasing evidence that hexoses contribute to defense responses. They act as substrates for metabolic processes and as metabolic semaphores to regulate gene expression. Especially an increase in the apoplastic hexose-to-sucrose ratio has been suggested to strengthen plant defense. This shift is brought about by the activity of cell wall-bound invertase (cw-Inv). We examined the possibility that Xcv may employ type 3 effector (T3E) proteins to suppress cw-Inv activity during infection. Indeed, pepper leaves infected with a T3SS-deficient Xcv strain showed a higher level of cw-Inv mRNA and enzyme activity relative to Xcv wild type infected leaves. Higher cw-Inv activity was paralleled by an increase in hexoses and mRNA abundance for the pathogenesis-related gene PRQ. These results suggest that Xcv suppresses cw-Inv activity in a T3SS-dependent manner, most likely to prevent sugar-mediated defense signals. To identify Xcv T3Es that regulate cw-Inv activity, a screen was performed with eighteen Xcv strains, each deficient in an individual T3E. Seven Xcv T3E deletion strains caused a significant change in cw-Inv activity compared to Xcv wild type. Among them, Xcv lacking the xopB gene (Xcv ΔxopB) caused the most prominent increase in cw-Inv activity. Deletion of xopB increased the mRNA abundance of PRQ in Xcv ΔxopB-infected pepper leaves, but not of Pti5 and Acre31, two PAMP-triggered immunity markers. Inducible expression of XopB in transgenic tobacco inhibited Xcv-mediated induction of cw-Inv activity observed in wild type plants and resulted in severe developmental phenotypes. Together, these data suggest that XopB interferes with cw-Inv activity in planta to suppress sugar-enhanced defense responses during Xcv infection

Regulation of Cell Wall-Bound Invertase in Pepper Leaves by <em>Xanthomonas campestris</em> pv. <em>vesicatoria</em> Type Three Effectors

<div><p><em>Xanthomonas campestris</em> pv. <em>vesicatoria (Xcv)</em> possess a type 3 secretion system (T3SS) to deliver effector proteins into its <em>Solanaceous</em> host plants. These proteins are involved in suppression of plant defense and in reprogramming of plant metabolism to favour bacterial propagation. There is increasing evidence that hexoses contribute to defense responses. They act as substrates for metabolic processes and as metabolic semaphores to regulate gene expression. Especially an increase in the apoplastic hexose-to-sucrose ratio has been suggested to strengthen plant defense. This shift is brought about by the activity of cell wall-bound invertase (cw-Inv). We examined the possibility that <em>Xcv</em> may employ type 3 effector (T3E) proteins to suppress cw-Inv activity during infection. Indeed, pepper leaves infected with a T3SS-deficient <em>Xcv</em> strain showed a higher level of cw-Inv mRNA and enzyme activity relative to <em>Xcv</em> wild type infected leaves. Higher cw-Inv activity was paralleled by an increase in hexoses and mRNA abundance for the <em>pathogenesis-related</em> gene <em>PRQ.</em> These results suggest that <em>Xcv</em> suppresses cw-Inv activity in a T3SS-dependent manner, most likely to prevent sugar-mediated defense signals. To identify <em>Xcv</em> T3Es that regulate cw-Inv activity, a screen was performed with eighteen <em>Xcv</em> strains, each deficient in an individual T3E. Seven <em>Xcv</em> T3E deletion strains caused a significant change in cw-Inv activity compared to <em>Xcv</em> wild type. Among them, <em>Xcv</em> lacking the <em>xopB</em> gene (<em>Xcv</em> Δ<em>xopB</em>) caused the most prominent increase in cw-Inv activity. Deletion of <em>xopB</em> increased the mRNA abundance of <em>PRQ</em> in <em>Xcv</em> Δ<em>xopB-</em>infected pepper leaves, but not of <em>Pti5</em> and <em>Acre31,</em> two PAMP-triggered immunity markers. Inducible expression of XopB in transgenic tobacco inhibited <em>Xcv</em>-mediated induction of cw-Inv activity observed in wild type plants and resulted in severe developmental phenotypes. Together, these data suggest that XopB interferes with cw-Inv activity <em>in planta</em> to suppress sugar-enhanced defense responses during <em>Xcv</em> infection.</p> </div

Content of soluble sugars in susceptible pepper leaves after infection with <i>Xcv</i> wild type or with the TTSS<i>-</i>deficient <i>Xcv</i> Δ<i>hrpB1</i> strain.

<p>Contents of glucose, fructose and sucrose were determined following inoculation of pepper leaves with <i>Xcv</i> wild type (wt) or the <i>Xcv</i> Δ<i>hrpB1</i> using a concentration of 5×10⁸ cfu ml⁻¹ and compared to 10 mM MgCl₂ infiltrated control leaves. Samples were taken before (0 h), 24 and 48 hours post infection (hpi). Each value represents the mean ± SE of four different experiments each with four to six individual samples. Statistically significant differences to Mock-inoculated control plants were determined using two-tailed t-test assuming normal distribution and are indicated by asterisks (*p<0.05).</p

Screening for <i>Xcv</i> T3Es involved in regulation of cw-Inv activity.

<p>Leaves of pepper plants were infiltrated with wild type and mutant <i>Xcv</i> strains at 10⁹ cfu ml⁻¹ and cw-Inv activity was measured 2 and 3 days post infection (dpi) in independent experiments. Graphs represent values calculated relative to the <i>Xcv</i> wild type (wt) response which was set to 100% for each individual experiment. Mean cw-Inv activities after infection with <i>Xcv</i> wild type were 20.96 µmol min⁻¹ m⁻²±8.43 (100% ±40.2) and 57.57 µmol min⁻¹ m⁻²±23.92 (100% ±41.6) at 2 and 3 dpi, respectively. The variance of <i>Xcv</i> wild type response (ca. 42%) is illustrated as a dashed line. Values are the mean response (as percentage to <i>Xcv</i> wild type) ± SD from three to nine different experiments. Statistically significant differences from <i>Xcv</i> wild type response were determined using two-tailed t-test assuming normal distribution and are indicated by asterisks (**p<0.01); (***p<0.001).</p

Inducible <i>xopB</i> expression in transgenic tobacco plants causes severe leaf abnormality.

<p>A.) Analysis of <i>xopB</i>-specific transcript accumulation in transgenic tobacco lines. Seven different lines (No. 22, 26, 37, 44, 64, 71, 72) and two control plants (wt) were analysed for <i>xopB</i> expression by Northern blotting. Total RNA was isolated 1 day after watering plants with 1% ethanol to induce <i>xopB</i> expression. Twenty µg of RNA were separated on a formaldehyde-containing agarose gel and analysed by hybridization with a <i>xopB</i>-specific radioactively labelled probe. Ethidium bromide stained rRNA is shown as loading control. B.) Analysis of XopB protein accumulation upon watering with 1% ethanol in selected transgenic lines (#22, #71). XopB migrates at ∼70 kDa, while in tobacco a cross-reactive band appeared at ∼55kDa. Expression of RubisCO as stained by Coomassie Blue is shown as control for protein loading. C.) Phenotypic changes in transgenic tobacco plants caused by <i>xopB</i> expression. Upper panel: symptoms 2 days after ethanol-treatment; lower panel: phenotypic alterations 10 days after induction. Arrows indicate morphological changes of the leaf lamina and cell death of meristematic tissue, respectively. From left to right: control line, lines #22 and #71.</p

Expression of XopB in <i>Xcv</i> Δ<i>xopB</i> complements the effect on cw-Inv activity.

<p>Susceptible pepper leaves were inoculated with <i>Xcv</i> wild type (wt), <i>Xcv</i> Δ<i>xopB</i> containing both the pBBR1MCS5 vector (EV), or <i>Xcv</i> Δ<i>xopB</i> derivatives in which a genomic fragment was introduced containing the putative <i>xopB</i> promoter and open reading frame in sense (+) or antisense (−) orientation relative to the <i>lac</i> promoter. Samples were taken before (0), 1, 2 and 3 days post infection (dpi). A.) Cw-Inv activity was measured from the infected leaf tissue. Values represent the mean of four independent samples ± SD. B.) Expression of XopB was verified by Western blotting by probing with the anti-XopB antibody. XopB migrates at ∼70 kDa.</p

Activities of cell wall-bound and vaculoar invertase in susceptible pepper leaves following infection with <i>Xcv</i> wild type or with the TTSS-deficient <i>Xcv</i> Δ<i>hrpB1</i> strain.

<p>Leaves of susceptible pepper plants were infected with the <i>Xcv</i> wild type (wt), the <i>Xcv</i> Δ<i>hrpB1</i> using a concentration of 5×10⁸ cfu ml⁻¹, and as control with 10 mM MgCl₂. Activities of cell wall-bound acid invertase (cw-Inv) (A.) and acid soluble, vacuolar invertase (vac-Inv) (B.) were measured from source leaves before (0 h), 24 and 48 hours post infection (hpi) with <i>Xcv</i> wild type (wt) (black bars), <i>Xcv</i> Δ<i>hrpB1</i> (grey bars<i>)</i> or 10 mM MgCl₂ (white bars). Each value represents the mean ± SE of six samples taken from three different plants. The experiment was repeated three times with similar results.</p

Infection of susceptible pepper leaves with <i>Xcv</i> wild type or with the T3SS-deficient <i>Xcv</i> Δ<i>hrpB1</i> strain.

<p>Impact on symptom development and transcript accumulation of <i>cw-Inv, PRQ</i> and <i>RbcS</i>. Fully mature leaves of young pepper plants were infected with the <i>Xcv</i> wild type (wt), the <i>Xcv</i> Δ<i>hrpB1</i> using a concentration of 5×10⁸ cfu ml⁻¹, and as control with 10 mM MgCl₂. A.) Formation of disease symptoms after <i>Xcv</i> wild type inoculation of susceptible pepper leaves. Only the lower halves of leaves were infiltrated. Pictures were taken 3 days post infection. B.) For Northern Blot analysis total RNA was isolated from leaf material taken before (0 h) and 3, 6, 12, 18, 24 and 48 hours post infection (hpi). Thirty micrograms of total RNA were loaded per each lane. The Northern blot was probed with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051763#pone.0051763-Dean1" target="_blank">[³²]</a>P-labeled cDNA fragments of <i>cw-Inv</i>, <i>PRQ</i>, <i>RbcS</i> and cytosolic <i>GAPDH</i> as control. Results of a representative experiment are shown which have been repeated three times.</p

Expression of <i>cw-Inv</i>, <i>PRQ</i> and <i>RbcS</i> in susceptible pepper leaves in response to infection with <i>Xcv</i> Δ<i>xopB</i>.

<p>Leaves of pepper plants were infected with the <i>Xcv</i> wild type (wt), <i>Xcv</i> Δ<i>hrpB1</i>, <i>Xcv</i> Δ<i>xopB</i> using a concentration of 10⁹ cfu ml⁻¹, and as control with 10 mM MgCl₂.Total RNA was isolated from pepper leaves before (0), and 1, 2, 3 days post infection (dpi). Twenty five µg of total RNA was separated per each lane. Northern blots were hybridized with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051763#pone.0051763-Dean1" target="_blank">[³²]</a>P dCTP-labelled cDNA fragments of <i>cw-Inv</i>, <i>PRQ</i> and <i>RbcS.</i> A representative experiment is shown. Similar results were obtained in two other experiments.</p

Expression of PTI marker genes <i>Pti5</i> and <i>Acre31</i> in susceptible pepper leaves after infection with different <i>Xcv</i> strains.

<p>Leaves of pepper plants were inoculated with <i>Xcv</i> wild type (wt), <i>Xcv</i> Δ<i>hrpB1</i>, <i>Xcv</i> Δ<i>xopB</i> using a concentration of 10⁹ cfu ml⁻¹, and with 10 mM MgCl₂. Total RNA was isolated from samples taken before (0 h), 6 h and 24 h after infiltration and reverse transcribed into cDNA. Abundance of <i>Pti5</i> (A.) and <i>Acre31</i> (B.) mRNA was detected by qPCR. Data were analysed using MxPro software v4.1. The expression levels of <i>Pti5</i> and <i>Acre31</i> were normalized with <i>Actin</i> and displayed relative to the expression level at time point 0 h which was set to a value of 1. The average ± SE of three replicates is shown. Similar results were obtained in an independent experiment. White bars, MgCl₂; light grey, <i>Xcv</i> wild type; black, <i>Xcv</i> Δ<i>hrpB1</i>, dark grey, <i>Xcv</i> Δ<i>xopB</i>.</p