Activation of the CNGC2-CNGC4 channel complex by P2K1-mediated phosphorylation links extracellular ATP perception to calcium signaling in plant immunity.

Extracellular adenosine triphosphate (eATP) functions as a damage-associated molecular pattern in plant immunity. P2K1, a purinergic receptor with a cytoplasmic serine/threonine kinase domain, initiates ATP-responsive signaling cascades characterized by a rapid spike in cytosolic Ca²⁺, which acts as a critical second messenger. In this study, we identified the cyclic nucleotide-gated channel complex CNGC2-CNGC4 as essential for eATP-induced calcium signaling and bacterial resistance in plants. A biochemical link between eATP perception and CNGC2-CNGC4 function was established by demonstrating the physical association between the channel complex and the eATP receptor P2K1 at the plasma membrane. Furthermore, we discovered that P2K1 phosphorylates the CNGC2 subunit of the CNGC2-CNGC4 channel in response to eATP, establishing a phosphorylation-dependent mechanism that connects eATP perception to calcium influx. Through AlphaFold-Multimer prediction, electrophysiological assay, and genetic analysis, we identified serine residues S705 and S718 in CNGC2 as the key phosphorylation sites mediating P2K1-dependent channel activation and eATP-triggered immunity. Notably, P2K1 selectively phosphorylates CNGC2, in contrast to BIK1 that phosphorylates CNGC4 during pathogen-associated molecular pattern-triggered immunity. Together, these findings indicate that the CNGC2-CNGC4 channel complex serves as a core component of calcium-dependent plant immunity, with distinct kinases phosphorylating different subunits in response to specific immune elicitors.