Stomatal immunity against fungal invasion comprises not only chitin-induced stomatal closure but also chitosan-induced guard cell death

Abstract

Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca²⁺, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca²⁺ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.

Keywords: Ca2+ signaling; chitin oligosaccharide; chitosan oligosaccharide; fungal resistance; stomatal immunity.

Fig. 1.

(GlcNAc)₈ but not (GlcN)₈ induces stomatal closure mediated by CERK1. (A and B) Dose effect of (GlcNAc)₈ (A) and (GlcN)₈ (B) on Col-0 stomatal aperture. (C) (GlcNAc)₈-induced stomatal closure in cerk1 knockout and complemented plants. Averages from three independent experiments (90 total stomata per bar) are shown. Data are mean ± SEM (n = 3). Student’s t test: *P < 0.05; N.S., no significant difference.

Fig. 2.

(GlcNAc)₈ activates I_Ca channels mediated by CERK1 in guard cells. (A) Representative whole-cell I_Ca channel current recordings in GCPs. (B–D) Average current-voltage curves of whole-cell I_Ca channel current recordings in A. Data are mean ± SEM (n = 5). (E) Average currents at −180 mV. Data are mean ± SEM (n = 5). Different letters indicate statistical significance (P < 0.05, ANOVA with Tukey’s test).

Fig. 3.

(GlcNAc)₈ induces [Ca²⁺]_cyt elevations in guard cells in a CERK1-dependent manner. (A) Representative traces of fluorescence emission ratios (535/480 nm) showing transient [Ca²⁺]_cyt elevations in Col-0 and cerk1-2 guard cells treated with 60 μM (GlcNAc)₈. (B) Percentage of number of guard cells showing different number of transient [Ca²⁺]_cyt elevations. (C) Area of transients of ratio (F535/F480) elevation in each guard cell showing [Ca²⁺]_cyt elevations in B. Area of ratio transients per guard cell was calculated as the sum of areas of ratio transients above the baseline ratio in each guard cell and is considered proportional to the amount of Ca²⁺ mobilized. (D) Baseline ratio (F535/F480) in guard cells. (E) Traces of fluorescence emission in Col-0 guard cells treated with 60 μM (GlcNAc)₈ in A. Note that fluorescence at 485 nm (white circles) decreases while fluorescence at 530 nm (black circles) increases, confirming fluorescence resonance energy transfer of the YC3.6 reporter. (GlcNAc)₈ was added 5 min after the start of imaging. The average fluorescence ratio in the first 5 min of imaging was considered as the baseline ratio in D. [Ca²⁺]_cyt elevations were counted when changes in fluorescence ratios were more than or equal to 0.1 U from the baseline ratio. Data are mean ± SEM. Different letters indicate statistical significance (P < 0.05, ANOVA with Tukey’s test). N.S., not significant.

Fig. 4.

(GlcNAc)₈ activates S-type anion channels mediated by CERK1 and Ca²⁺ in guard cells. (A) Representative whole-cell S-type anion channel current recordings in GCPs. (B) Average steady-state current-voltage curves of whole-cell S-type anion channel current recordings in A. Data are mean ± SEM (n = 5). (C) Average steady-state S-type anion channel currents at −145 mV. Data are mean ± SEM (n = 5). Different letters indicate statistical significance (P < 0.05, ANOVA with Tukey’s test).

Fig. 5.

SLAC1 is required for (GlcNAc)₈-induced stomatal closure and activation of S-type anion channels in guard cells. (A) (GlcNAc)₈-induced stomatal closure in slac1-1 and complemented lines. Averages from three independent experiments (90 total stomata per bar) are shown. Data are mean ± SEM (n = 3). Different letters indicate statistical significance (P < 0.05, ANOVA with Tukey’s test). (B) Representative whole-cell S-type anion channel current recordings in GCPs. (C) Average steady-state current-voltage curves of whole-cell S-type anion channel current recordings in A. Data are mean ± SEM (n = 5). (D) Average steady-state S-type anion channel currents at −145 mV. Data are mean ± SEM (n = 5). Different letters indicate statistical significance (P < 0.05, ANOVA with Tukey’s test). (E) (GlcNAc)₈-induced stomatal closure in Col-0, cpk6-2, and ost1-3. Averages from three independent experiments (90 total stomata per bar) are shown. Data are mean ± SEM (n = 3). Student’s t test: *P < 0.05. N.S., no significant difference.

Fig. 6.

CERK1 interacts with OST1. (A) Confocal microscopy of N. benthamiana leaves transiently expressing the indicated split-YFP constructs. Representative images are shown. (Scale bar: 100 µm.) (B) Interaction analysis of CERK1 kinase domain (CERK1^KD) with OST1, CPK6, and SLAC1 N terminus (SLAC1-NT) and C terminus (SLAC1-CT) using yeast two-hybrid assays.

Fig. 7.

(GlcN)₈ induces guard cell death in a manner dependent on Ca²⁺ but not CERK1. (A–C) FDA staining rate of guard cells treated with oligosaccharides for 2 h. A and C are results from Col-0 guard cells. Averages from three independent experiments (100 to 200 guard cells per bar) are shown. Data are mean ± SEM (n = 3). Asterisks indicate statistical significance compared to mock treatment in the same genotype (P < 0.05, Student’s t test). (Scale bar: 20 µm.)

Fig. 8.

(GlcN)₈ activates I_Ca channels in guard cells. (A) Representative whole-cell I_Ca channel current recordings in GCPs. (B) Average current-voltage curves of whole-cell I_Ca channel current recordings in A. Data are mean ± SEM (n = 5 for Col-0; n = 3 for cerk1-2).

Fig. 9.

(GlcN)₈ induces [Ca²⁺]_cyt elevations in guard cells. (A and B) Representative traces of fluorescence emission ratios (535/480 nm) in Col-0 (n = 14) and cerk1-2 guard cells (n = 7) treated with 60 μM (GlcN)₈. (C and D) Traces of fluorescence emission (535 nm, black circles; 480 nm, white) in A and B. Note that all of the guard cells monitored showed similar patterns of change of fluorescence with the timing of ratio burst varying from cell to cell.