Coronatine Facilitates Pseudomonas syringae Infection of Arabidopsis Leaves at Night

Abstract

In many land plants, the stomatal pore opens during the day and closes during the night. Thus, periods of darkness could be effective in decreasing pathogen penetration into leaves through stomata, the primary sites for infection by many pathogens. Pseudomonas syringae pv. tomato (Pst) DC3000 produces coronatine (COR) and opens stomata, raising an intriguing question as to whether this is a virulence strategy to facilitate bacterial infection at night. In fact, we found that (a) biological concentration of COR is effective in opening dark-closed stomata of Arabidopsis thaliana leaves, (b) the COR defective mutant Pst DC3118 is less effective in infecting Arabidopsis in the dark than under light and this difference in infection is reduced with the wild type bacterium Pst DC3000, and (c) cma, a COR biosynthesis gene, is induced only when the bacterium is in contact with the leaf surface independent of the light conditions. These findings suggest that Pst DC3000 activates virulence factors at the pre-invasive phase of its life cycle to infect plants even when environmental conditions (such as darkness) favor stomatal immunity. This functional attribute of COR may provide epidemiological advantages for COR-producing bacteria on the leaf surface.

Keywords: coronatine biosynthesis; pathogen penetration; phytotoxin; plant defense; stomatal immunity.

FIGURE 1

Similar to Pst DC3118, Pst DB29 cannot overcome stomatal immunity and has reduced virulence on surface-inoculated plants. (A) Stomatal aperture width in epidermal peels of Col-0 plants exposed to water, or Pseudomonas syringae pv. tomato (Pst) KP105 (DC3000 wild type parent) or Pst DB29 (COR defective mutant). (B) Bacterial enumeration in the apoplast of plants at 1 and 3 days after dip-inoculation (left graph) or vacuum-infiltration (right graph) with bacteria. Results are shown as the mean ± SE. Note that some error bars are too small to appear in the log scale graphs. Experiments were performed three times with similar results. (C) The graph shows stomatal aperture width in intact Arabidopsis leaves 2 h after dip-inoculation with bacterial suspension (Pst DC3118 and Pst DB29) with or without COR under light. Results in (A), (C) are shown as the mean (n = 60) ± SE. Statistical significance (all panels) were detected with ANOVA followed by Tukey–Kramer HSD at 95% confidence limit.

FIGURE 2

Biological concentrations of coronatine (COR) induce stomatal opening and prevent stomatal closure. (A) Stomatal aperture width of Arabidopsis epidermal peels (left) or leaves (right) incubated with purified COR under darkness. (B) The graph shows stomatal aperture width in intact Arabidopsis leaves 4 h after dip-inoculation with bacterial suspension (Pst DC3000, Pst DC3118, and Pst DB29) with or without COR in the dark. All results are shown as the mean (n = 60) ± SE. Statistical significance (all panels) were detected with ANOVA followed by Tukey–Kramer HSD at 95% confidence limit.

FIGURE 3

COR provides advantage for P. syringae infection in the dark. (A) Bacterial population in the plant apoplast of Col-0 plants dipped into a suspension (1 × 10⁸ CFU.ml^-1) of Pst DC3000, Pst DC3118, or Pst DC3118 supplemented with 1.5 μM COR at 8 and 24 h after inoculation under complete darkness. (B) The graph shows stomatal aperture width in intact Arabidopsis leaves 2 h after dip-inoculation with Pst DC3118 or mock control under light or darkness. Results are shown as the mean (n = 60) ± SE. (C) Bacterial population in the plant apoplast of Col-0 plants dipped into a suspension (1 × 10⁸ CFU.ml^-1) of Pst DC3000 or Pst DC3118 in the dark or under light at Day 1 and Day 3 after inoculation. Results in panels (A,C) are shown as mean of two biological replicates (n = 12) ± SE. Asterisks above the bars indicate statistical significance between the means within each time point (lower case letters = differences in the first time point; upper case letters = differences in the second time point). Statistical significance (A–C) were detected with ANOVA followed by Tukey–Kramer HSD at 95% confidence limit. (D) Symptoms were recorded 3 days after surface inoculation with the indicated bacteria and light conditions.

FIGURE 4

COR biosynthesis reporter strain (Pst DC3000-pHW01) is induced in contact with the Arabidopsis leaf surface. The pictures shown are representative of a time series fluorescence and bright field micrographs. (A) Micrographs of bacterial cell suspension showing that cells do not express GFP in the absence of leaf tissue. Top three pictures were taken with GFP filters and the bottom picture was taken under bright field. (B) Micrographs shows attached bacterial cells fluorescing after around 4 h of contact with the leaf surface. Black arrows on the middle micrograph show few fluorescing cells at 4 h. Insert on the middle micrograph shows a higher magnification that highlights well-defined glowing bacterial cells. (C) Micrographs show fluorescing bacterial cells in the inoculum exposed to the leaf surface.

FIGURE 5

Pst DC3000 produces comparable amounts of COR under light or darkness. Pst DC3000 was grown in COR inducing medium (HSC medium) for 24 h in constant light (70–80 μmol.m^-2.s^-1) or constant darkness and COR production was assessed by HPLC. (A–C) Chromatograms obtained by HPLC showing peaks of COR at the retention time of 9.4 min when the sample injected was 15 μg.ml^-1 COR (control) (A), Pst DC3000 grown in light (B) or Pst DC3000 grown in dark (C). mAU; milliAbsorbance Units at 208 nm. (D) COR concentration in Pst DC300 cells grown in light or dark calculated as μg COR per mg of total protein. Data points are shown as mean (n = 6) ± SE and no statistical significance was observed between the means.