a, BIK1(4D) phosphorylates CNGC4-NT but not CNGC2-NT in vitro. CNGC2-NT and CNGC4-NT were fused to the trigger factor (TF) chaperone as a soluble fusion tag. b, FLS2-KD or BAK1-KD (KD, kinase domain) does not phosphorylate CNGC4-CT in vitro. The in vitro phosphorylation reaction was performed using purified proteins and [γ-32P]ATP, resolved by SDS–PAGE, and phosphorylated proteins were detected by autoradiography. The Coomassie Brilliant Blue staining of a and b was used to verify the quality of samples and the loading consistency. c, flg22 induces CNGC4 phosphorylation in Arabidopsis mesophyll protoplasts. Col-0 mesophyll protoplasts expressing CNGC4–Flag were treated with 500 nM flg22 for the indicated time or H 2 O (0) as control. CNGC4–Flag proteins were purified by Flag-M2 beads. The bead-bounded CNGC4 at 5 min after flg22 treatment were incubated with λ protein phosphatase (PPase) alone, or PPase plus phosphatase inhibitors to dephosphorylate CNGC4. Proteins were eluted from beads by SDS loading buffer with boiling. CNGC4 phosphorylation was detected on a Phos-tag SDS–PAGE gel, indicating that CNGC4 can be phosphorylated in response to flg22 and reversibly dephosphorylated by PPase. BIK1–HA was co-expressed with CNGC4 in protoplasts as a positive control, which presented phosphorylation in response to flg22. Total proteins containing BIK1–HA were processed similarly as described above. Ponceau S staining was used as a loading control for BIK1–HA. d, flg22 does not induce CaM phosphorylation in Arabidopsis protoplasts. Col-0 protoplasts expressing the indicated constructs were treated with either flg22 (+) or H 2 O control (−) for 10 min. CaM mobility shift was not observed on a Phos-tag SDS–PAGE gel. The values to the sides of the SDS–PAGE gels in a–d indicate the molecular mass (kDa) of proteins. p, phosphorylated; np, non-phosphorylated. e, f, The CaM-gated CNGC2–CNGC4 calcium channel is not activated by FLS2 or RIPK in oocytes. e, Representative current traces from oocytes expressing CNGC2 + CNGC4, CNGC2 + CNGC4 + CaM, BIK1 + CNGC2 + CNGC4 + CaM, FLS2 + CNGC2 + CNGC4 + CaM or RIPK + CNGC2 + CNGC4 + CaM, perfused with 5 mM Ca2+. f, Current amplitudes at −140 mV from multiple recordings as in e. Data are presented as mean ± s.d., n = 12 biologically independent oocytes. P values are from two-sided Student’s t-tests. For e and f, experiments were repeated three times using different batches of oocytes and similar results were obtained. For images presented in a–d, experiments were repeated three times using different biological materials and one representative image is shown. Source data