Xanthan induces plant susceptibility by suppressing callose deposition

Abstract

Xanthan is the major exopolysaccharide secreted by Xanthomonas spp. Despite its diverse roles in bacterial pathogenesis of plants, little is known about the real implication of this molecule in Xanthomonas pathogenesis. In this study we show that in contrast to Xanthomonas campestris pv campestris strain 8004 (wild type), the xanthan minus mutant (strain 8397) and the mutant strain 8396, which is producing truncated xanthan, fail to cause disease in both Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) plants. In contrast to wild type, 8397 and 8396 strains induce callose deposition in N. benthamiana and Arabidopsis plants. Interestingly, treatment with xanthan but not truncated xanthan, suppresses the accumulation of callose and enhances the susceptibility of both N. benthamiana and Arabidopsis plants to 8397 and 8396 mutant strains. Finally, in concordance, we also show that treatment with an inhibitor of callose deposition previous to infection induces susceptibility to 8397 and 8396 strains. Thus, xanthan suppression effect on callose deposition seems to be important for Xanthomonas infectivity.

Figure 1.

Structures of the exopolysaccharides produced by Xcc. A, Xanthan produced by 8004 strain. B, Truncated xanthan produced by 8396 strain.

Figure 2.

Infection of N. benthamiana and Arabidopsis (Arabidopsis thaliana) with Xcc strains. Symptoms in N. benthamiana leaves (A) and Arabidopsis plants (B) 4 days after inoculation with either wild-type Xcc strain 8004 and Xcc xanthan mutant strains 8397 or 8396 (10⁷ cfu/mL). Photos of disease symptoms were taken 8 dpi. Bacterial population of Xcc strains in N. benthamiana leaves (C) and Arabidopsis plants (D). The mean and standard deviation of three independent experiments of bacterial numbers are given.

Figure 3.

Xanthan induces susceptibility to 8397 and 8396 strains in N. benthamiana and Arabidopsis. Symptoms in N. benthamiana leaves (A) and Arabidopsis plants (B) treated with xanthan (150 μg/mL) and then inoculated with 8004, 8397, and 8396 strains (10⁷ cfu/mL as in Fig. 2) 24 h after treatment. Photos of disease symptoms were taken 8 dpi. Bacterial populations were accounted at 0 and 4 dpi of N. benthamiana (C) and Arabidopsis (D). The mean and sd of three separate measurements of bacterial numbers are given. Data sets marked with an asterisk are significantly different from control (water-pretreated leaves) as assessed by the Student's t test: *P < 0.001.

Figure 4.

The susceptibility to Xcc induced by xanthan is dose dependent. A, Leaves of 4-week-old plants were preinfiltrated with either different concentrations of Xcc xanthan or water and 24 h later pretreated leaves were inoculated with the Xcc mutant strain 8397 (10⁷ cfu/mL). B, Immediate establishment of susceptibility to Xcc 8397 strain by the xanthan suppressor. Leaves were preinfiltrated with Xcc xanthan (150 μg/mL) 0 and 24 h before inoculation (0 and 24), or water 24 h before inoculation (water) and then pretreated leaves were infected with the Xcc mutant strain 8397 as before. Numbers of bacteria were assessed immediately upon infection and 4 d later. The mean and sd of three separate measurements of bacterial numbers are given. Data sets marked with an asterisk are significantly different from control (water-pretreated leaves) as assessed by the Student's t test: *P < 0.001.

Figure 5.

The structure of xanthan is essential for its effect in N. benthamiana and Arabidopsis. Symptoms in N. benthamiana leaves (A) and Arabidopsis plants (B) treated with the truncated xanthan (150 μg/mL) produced by 8396 mutant strain, and inoculated with 8004, 8397, or 8396 strains 24 h after treatment as before. Photos of disease symptoms were taken 8 dpi. Bacterial populations were accounted at 0 and 4 dpi of N. benthamiana (C) or Arabidopsis (D). The mean and sd of three separate measurements of bacterial numbers are given.

Figure 6.

Callose deposition in N. benthamiana leaves is associated with resistance and is suppressed by the Xcc exopolysaccharide xanthan. N. benthamiana leaves were pretreated with water, xanthan, or truncated xanthan and inoculated with strains of Xcc as before. A, The leaves were then stained for callose deposits 24 h post inoculation (white dots) and observed by light (left, light sections) and fluorescence microscopy (right, dark sections). Scale bars = 200 μm. B, Average numbers of callose deposits per field of view (0.45 mm²) are displayed. Error bars represent the sds from three leaves of each plant and three independent experiments. Data sets marked with an asterisk are significantly different from the control (water-treated leaves followed by 8004 infection and water-pretreated leaves followed by 8397 or 8396 infections) as assessed by the Student's t test.

Figure 7.

Inhibition of callose synthetase by 2 DDG restores mutant strain symptoms and bacterial populations. A, Disease symptoms on N. benthamiana leaves preinfiltrated with water, 25 or 250 μm of 2 DDG, and subsequently infected with Xcc mutant strains 8397 or 8396 (10⁷ cfu/mL). Photos of disease symptoms were taken 8 dpi. B, Microscopy pictures of callose deposition from leaves treated with 2 DDG followed by infection with 8397 and 8396 strains as in A. C, Average numbers of callose deposits per field of view (0.45 mm²) are displayed and data with asterisk are significantly different from the control (water-pretreated leaves followed by 8397 or 8396 infections) as assessed by the Student's t test. D, For every treatment bacterial populations were calculated at days 0 and 4, after inoculation as before. Error bars represent the sds and data sets marked with an asterisk are significantly different from the control (water-pretreated leaves followed by 8397 or 8396 infections) as assessed by the Student's t test.