Fig. 3: P2K1 phosphorylates PAT5 and PAT9 to regulate immune responses.

a P2K1 directly phosphorylates PAT5 and PAT9. Purified P2K1 kinase domain recombinant proteins were incubated with PAT5/PAT9-CRD and -C domains in an in vitro kinase assay. Autophosphorylation and transphosphorylation were measured by incorporation of γ-[³²P]-ATP. MBP and GST-CD2b were used as positive and negative controls, respectively. The protein loading was measured by CBB staining. Red stars represent the trans-phosphorylated proteins. Experiments were repeated two times with similar results. b, c Phosphorylation of PAT9 regulates ATP-induced calcium influx and ROS production. The indicated genomic PAT9 variants were transformed into the pat9 mutant, and then examined for ATP-induced calcium influx and ROS production for 30 min. RLU relative luminescence units. Error bars indicate ±SEM; n = 8 (biological replicates); means with different letters are significantly different (p < 0.05); P-values indicate significance relative to Col-0 and were determined by one-sided ANOVA with multiple comparisons and adjusted using Benjamini–Hochberg post-test. d PAT9 Phosphorylation mediates ATP-induced restriction of bacterial growth. The indicated plant leaves were syringe-infiltrated with 10⁶ cfu/mL⁻¹ of Pst. DC3000 after 24 h water (mock) or 400 μM ATP treatment, which is different from surface inoculation (Fig. 1e). Bacterial numbers were determined 3 days post inoculation. Error bars indicate ±SEM; n = 12 (biological replicates); means with different letters are significantly different (p < 0.05); P-values indicate significance relative to Col-0 with mock treatment and were determined by one-sided ANOVA with multiple comparisons and adjusted using Benjamini–Hochberg post-test.