BIK1-mediated phosphorylation and SCF^SKIP31-mediated ubiquitination coordinately control CNGC3 homeostasis and...

Cyclic nucleotide-gated ion channels (CNGCs) are key components in pattern-triggered immunity (PTI) signaling. Tight control of CNGC homeostasis is crucial for maintaining a balance between plant growth and immunity. Nevertheless, the mechanisms for fine-tuning CNGC homeostasis remain largely unknown. Here, we report that Arabidopsis thaliana CNGC3 is a functional calcium channel to mediate pattern-induced Ca²⁺ influx, PTI, and resistance to Sclerotinia sclerotiorum. We identified a CNGC interactor, Skp1-interacting protein 31 (SKIP31). In the absence of a pathogen, SKIP31 ubiquitinates CNGC3 at Lys8 and Lys33 of the K-X-V-R motif for degradation to repress plant immunity. When a pathogen attacks, activated receptor-like cytoplasmic kinase (RLCK) BOTRYTIS-INDUCED KINASE1 (BIK1) phosphorylates SKIP31 to inhibit its ubiquitin ligase activity and interaction with the CNGC3 N-terminal region, thereby suppressing CNGC3 protein degradation to promote immunity. Phosphorylation within the F box of SKIP31 at Ser88 and Ser93 and at the C-terminal Ser261 prevents its interaction with Skp1 and CNGC3, respectively. These phosphorylation sites are conserved in SKIP31 of different plant species, and SKIP31 interacts with all examined CNGCs, suggesting a pivotal role of SKIP31 phosphorylation in regulating CNGC stability and plant immunity. Moreover, biochemical assays revealed that BIK1 directly phosphorylates the CNGC3 cytoplasmic C-terminal region at four Ser residues to enhance its Ca²⁺ channel activity, demonstrating dual roles of BIK1 in both promoting CNGC channel activity and stabilizing the channel protein. Collectively, our work unveils an SCF ubiquitin ligase-RLCK control system that fine-tunes the homeostasis of CNGCs for orchestrating plant immunity.