Motif-based substrate mapping of the receptor-like cytoplasmic kinase BIK1 reveals novel components and regulatory nodes of plant immunity.

Plant cell surface pattern recognition receptors (PRRs) perceive non- or altered-self elicitors to induce immune responses. PRRs relay information across the plasma membrane and trigger downstream signalling via receptor-like cytoplasmic kinases such as BOTRYTIS-INDUCED KINASE 1 (BIK1). BIK1 associates with several PRRs and acts as a key executor of immune responses through the phosphorylation of substrate proteins. However, a comprehensive understanding of how BIK1 targets specific substrates and a full repertoire of these substrates are lacking. Here we defined the substrate specificity of BIK1 and used these data to predict candidate substrates in Arabidopsis. Using high-throughput biochemical and genetic screening of these candidates, we confirmed many as direct BIK1 substrates in vitro and novel regulators of plant immunity. Among the BIK1 substrates identified are MULTIPLE C2 DOMAIN AND TRANSMEMBRANE REGION PROTEIN 3, which we reveal regulates flagellin 22 (flg22)-induced plasmodesmata closure and immunity, and members of the largely uncharacterized CYCLIN-DEPENDENT KINASE-LIKE family, which we uncover as novel negative regulators of immunity. In parallel, we interrogated intracellular NUCLEOTIDE-BINDING LEUCINE-RICH REPEAT (NLR) immune receptors for potential BIK1 phosphorylation motifs and identified multiple NLRs as direct BIK1 substrates. We reveal that BIK1 phosphorylation regulates NLR oligomerization, thus controlling a key activation step for these immune receptors. Together, our unbiased biochemical screens shed light on the central role of BIK1 as a key kinase shaping multiple layers of plant immune signalling.