Fig. 5
DGK5α is defective in plant PTI and ETI [48]. DGK5β, but not DGK5α, restores flg22-induced ROS burst in the dgk5-1 mutant. Leaf disks from 4-week-old soil-grown WT, dgk5-1, and two independent transgenic lines carrying p35S::DGK5β-HA or p35S::DGK5α-HA in the dgk5-1 background were treated with or without 100 nM flg22, and the ROS production was measured as relative light units (RLU) by a luminometer for the indicated time (A). Total ROS levels between 0 and 30 min (ΣROS) are shown as mean ± S.E.M. (n = 24, biologically independent samples) analyzed by one-way ANOVA followed by Tukey’s test (B). Different letters (a, b, and c) indicate significant differences (p value < 0.05). C DGK5β, but not DGK5α, complements the dgk5-1 mutant defect of plant disease resistance against virulent bacterial pathogen Pst DC3000. Leaves from 4-week-old soil-grown WT, dgk5-1, p35S::DGK5β-HA/dgk5-1, or p35S::DGK5α-HA/dgk5-1 transgenic plants were hand-inoculated with Pst DC3000 bacterial suspension at 5 × 105 cfu/ml. Bacterial growth was measured at 0 and 3 days post-inoculation (dpi). Data are shown as mean ± S.D. (n = 6, biologically independent samples) analyzed by one-way ANOVA followed by Tukey’s test for multiple comparisons. Different letters (a and b) indicate significant differences (p value < 0.05). D DGK5β, but not DGK5α, complements the dgk5-1 mutant defect of plant disease resistance against avirulent bacterial pathogens Pst DC3000 carrying avrRpt2 or avrRpm1. The experiments were performed similarly as in C using the bacterial suspension of Pst DC3000 avrRpt2 or Pst DC3000 avrRpm1 at 5 × 105 cfu/ml. Data are shown as mean ± S.D. (n = 6, biologically independent samples) analyzed by one-way ANOVA followed by Tukey’s test for multiple comparisons. Different letters (a, b, and c) indicate significant differences (p value < 0.05). E A model of CPL3-mediated DGK5 AS in regulating PA homeostasis and plant immunity. MAMP perception by the PRR complex triggers BIK1 phosphorylation and activation of two MAPK cascades. BIK1 phosphorylates DGK5 at Ser506 and enhances its activity for PA production. In contrast, PRR-activated MPK4 phosphorylates DGK5 at Thr446, leading to reduced DGK5 activity and PA production. In addition, MAMP perception induces RNAPII CTD phosphorylation through MPK3/6-activated CDKCs, counteracted by the CTD phosphatase CPL3. The CDKC-CPL3-mediated CTD phosphorylation regulates DGK5 AS, producing two isoforms, DGK5β and DGK5α. DGK5β, which can be phosphorylated by BIK1, positively regulates DGK5 activity and plant immunity. In contrast, DGK5α, unable to be phosphorylated by BIK1, is nonfunctional and likely negatively regulates PA production through MPK4-mediated Thr446 phosphorylation to maintain PA homeostasis. Further, PA binds and stabilizes RBOHD in mediating ROS production in plant PRR-mediated PTI and NLR-mediated ETI. The figure was created with BioRender