Fig. 4. CPK3 activity is required for stomatal immunity and guard cell actin dynamics.

Deletion of CPK3 compromises stomatal immunity. Guard cells from the cpk3-2 mutant do not close in response to a flg22 or b Pst DC3000 treatment over a 6 h time course. Fourteen-day-old seedlings were stimulated with water or 10 μM purified flg22 peptide for 1 h, and images were collected by laser scanning confocal microscopy. c CPK3 kinase function is essential for guard cell response to flg22 treatment. Four-week-old WT Col-0, the cpk3-2 mutant, the complementation lines with full kinase function (i.e., cpk3-2/35S::CPK3^#1), and the kinase null line (cpk3-2/35S::CPK3^K107M#1) were stimulated with 10 μM purified flg22 peptide for 1 h. Images were collected by laser scanning confocal microscopy, and stomatal apertures (width/length) were calculated using ImageJ. Error bars represent means ± SD from three technical replicates of three independent biological repeats (n ≥ 24). Statistical analysis was performed using a two tailed Mann–Whitney U-test; *p < 0.05; ***p < 0.001; ****p < 0.0001; ns, not significant). Guard cell actin parameters of WT Col-0/35S::GFP-fABD2 and the cpk3-2/35S::GFP-fABD2 mutant from 14-day-old seedlings inoculated with d mock, e 3 × 10⁷ CFU mL⁻¹ Pst DC3000, and f 10 μM flg22. Left panels are representative images for guard cells. Stomatal images were collected over a 6 h time interval following inoculation. Images were collected from ≥24 cell pairs, and experiments were performed three times from multiple, independent, biological replicates. The box and whiskers plots represent minimum and maximum values. The line in the box plot represents the median value and the boundaries demonstrate the 25th percentile (upper) and the 75th percentile (lower). Statistical analysis was conducted using a two tailed Mann–Whitney U-test; *p-value < 0.05; **p-value < 0.01; ns, ***p < 0.001; ****p < 0.0001; ns, not significant.