Extended Data Fig. 6: PBL19C3A interacts with and phosphorylates WRKY8 and WRKY28.
a, BiFC assay reveals that PBL19C3A constitutively interacts with WRKY8 and WRKY28 in protoplasts. HY5-mCherry labels the nucleus. IAA28 was used to replace WRKYs as a negative control. Scale bar = 5 μm. b, In vitro kinase assay indicates that PBL19 can phosphorylate WRKY8 and WRKY28 but not WRKY48. The kinase-dead (Km) PBL19 is a negative control. c, PBL19-mediated WRKY8 phosphorylation sites identified by mass spectrometry. Identified phosphosites with peptide ion scores over 20 are considered reliable. Mass error in parts per million (ppm) = [1-(experimentally determined mass)/(predicted mass)]×106. d, Thr177 and Thr227 (red) are conserved residues in the DNA-binding domain of WRKY8 and WRKY28. Note that Thr209 and Thr210 (black) of WRKY8 can be phosphorylated by CPK5. e, Phosphomimetic T177D/T227D mutations can enhance the potency of WRKY8 in activating the PBL19 promoter. The basal pPBL19 activity was normalized as 1 in each replicate. Data are shown as mean ± s.d. of three biological replicates with P values indicated (one-way ANOVA with Tukey’s multiple comparisons test). The experiments in (a) and (b) were repeated three times with similar results.
