Figure 1.
Infection of susceptible pepper leaves with Xcv wild type or with the T3SS-deficient Xcv ΔhrpB1 strain.
Impact on symptom development and transcript accumulation of cw-Inv, PRQ and RbcS. Fully mature leaves of young pepper plants were infected with the Xcv wild type (wt), the Xcv ΔhrpB1 using a concentration of 5×108 cfu ml−1, and as control with 10 mM MgCl2. A.) Formation of disease symptoms after Xcv 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 [32]P-labeled cDNA fragments of cw-Inv, PRQ, RbcS and cytosolic GAPDH as control. Results of a representative experiment are shown which have been repeated three times.
Figure 2.
Activities of cell wall-bound and vaculoar invertase in susceptible pepper leaves following infection with Xcv wild type or with the TTSS-deficient Xcv ΔhrpB1 strain.
Leaves of susceptible pepper plants were infected with the Xcv wild type (wt), the Xcv ΔhrpB1 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 Xcv wild type (wt) (black bars), Xcv ΔhrpB1 (grey bars) 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.
Figure 3.
Content of soluble sugars in susceptible pepper leaves after infection with Xcv wild type or with the TTSS-deficient Xcv ΔhrpB1 strain.
Contents of glucose, fructose and sucrose were determined following inoculation of pepper leaves with Xcv wild type (wt) or the Xcv ΔhrpB1 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).
Figure 4.
Changes in pepper leaf photosynthetic capacity after Xcv infection.
Rate of photosynthesis, represented as effective quantum yield of photosystem II, was measured in susceptible pepper leaves 2, 6, 24 and 48 hours post infection (hpi) with the Xcv wild type strain (wt, black bars) and Xcv ΔhrpB1 (grey bars) and compared to 10 mM MgCl2 (white bars) inoculated control leaves. Values represent the mean ± SE of four measurements performed with independent plants. The experiment was repeated twice with similar results.
Figure 5.
Cw-Inv activity of susceptible pepper leaves infected with heat-inactivated and protease-treated preparations of Xcv cells.
Xcv wild type (wt) and Xcv ΔhrpB1 were grown over night in NYG medium and prepared as described in material and methods. Subsequently, cells were heat-inactivated at 95°C for 20 min. An aliquot was of each cell culture was digested with proteases (proteinase K and trypsin) for 2 h at 60°C. After heat-inactivation for 10 min, cells were pelleted and re-suspended in 10 mM sterile MgCl2 and adjusted to OD600 = 1. Heat-inactivated Xcv cells with or without protease treatment were used for inoculation of pepper leaves. Samples were taken before (0 d), 1, 2 and 3 days post infections (dpi) and cw-Inv activity was measured. Values represent the mean of four independent samples ± SD. Similar results were obtained in an independent experiment.
Table 1.
Xanthomonas campestris pv. vesicatoria strains used in this study.
Figure 6.
Screening for Xcv T3Es involved in regulation of cw-Inv activity.
Leaves of pepper plants were infiltrated with wild type and mutant Xcv 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 Xcv wild type (wt) response which was set to 100% for each individual experiment. Mean cw-Inv activities after infection with Xcv 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 Xcv wild type response (ca. 42%) is illustrated as a dashed line. Values are the mean response (as percentage to Xcv wild type) ± SD from three to nine different experiments. Statistically significant differences from Xcv wild type response were determined using two-tailed t-test assuming normal distribution and are indicated by asterisks (**p<0.01); (***p<0.001).
Figure 7.
Expression of cw-Inv, PRQ and RbcS in susceptible pepper leaves in response to infection with Xcv ΔxopB.
Leaves of pepper plants were infected with the Xcv wild type (wt), Xcv ΔhrpB1, Xcv ΔxopB 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 [32]P dCTP-labelled cDNA fragments of cw-Inv, PRQ and RbcS. A representative experiment is shown. Similar results were obtained in two other experiments.
Figure 8.
Expression of PTI marker genes Pti5 and Acre31 in susceptible pepper leaves after infection with different Xcv strains.
Leaves of pepper plants were inoculated with Xcv wild type (wt), Xcv ΔhrpB1, Xcv ΔxopB 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 Pti5 (A.) and Acre31 (B.) mRNA was detected by qPCR. Data were analysed using MxPro software v4.1. The expression levels of Pti5 and Acre31 were normalized with Actin 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, Xcv wild type; black, Xcv ΔhrpB1, dark grey, Xcv ΔxopB.
Figure 9.
Expression of XopB in Xcv ΔxopB complements the effect on cw-Inv activity.
Susceptible pepper leaves were inoculated with Xcv wild type (wt), Xcv ΔxopB containing both the pBBR1MCS5 vector (EV), or Xcv ΔxopB derivatives in which a genomic fragment was introduced containing the putative xopB promoter and open reading frame in sense (+) or antisense (−) orientation relative to the lac 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.
Figure 10.
Inducible xopB expression in transgenic tobacco plants causes severe leaf abnormality.
A.) Analysis of xopB-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 xopB expression by Northern blotting. Total RNA was isolated 1 day after watering plants with 1% ethanol to induce xopB expression. Twenty µg of RNA were separated on a formaldehyde-containing agarose gel and analysed by hybridization with a xopB-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 xopB 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.
Figure 11.
Inducible xopB expression in transgenic tobacco leaves suppresses cw-Inv activity during Xcv infection.
Control plants and two selected transgenic tobacco lines with inducible xopB expression (#22, #71) were watered with 1% ethanol. After 24 h, plantlets were inoculated with a 109 cfu ml−1 suspension of Xcv wild type. Samples were taken directly before inoculation and 1 and 2 days post inoculation (1dpi +EtOH; 2dpi Xcv+ EtOH). Non-ethanol watered plants were also inoculated with Xcv and samples were taken accordingly (1dpi -EtOH; 2dpi -EtOH). For control purposes ethanol-treated and non-treated plants were inoculated with 10 mM MgCl2. Cw-Inv activity was determined from four independent samples and fold changes ± SD were calculated for each sample relative to values obtained before Xcv inoculation. The experiment was repeated with similar results.