Calcium (Ca²⁺)-dependent protein kinases (CDPKs or CPKs) are a unique family of Ca²⁺ sensor/kinase-effector proteins with diverse functions in plants. In Arabidopsis thaliana, CPK28 contributes to immune homeostasis by promoting degradation of the key immune signaling receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) and additionally functions in vegetative-to-reproductive stage transition. How CPK28 controls these seemingly disparate pathways is unknown. Here, we identify a single phosphorylation site in the kinase domain of CPK28 (Ser318) that is differentially required for its function in immune homeostasis and stem elongation. We show that CPK28 undergoes intermolecular autophosphorylation on Ser318 and can additionally be transphosphorylated on this residue by BIK1. Analysis of several other phosphorylation sites demonstrates that Ser318 phosphorylation is uniquely required to prime CPK28 for Ca²⁺ activation at physiological concentrations of Ca²⁺, possibly through stabilization of the Ca²⁺-bound active state as indicated by intrinsic fluorescence experiments. Together, our data indicate that phosphorylation of Ser318 is required for the activation of CPK28 at low intracellular [Ca²⁺] to prevent initiation of an immune response in the absence of infection. By comparison, phosphorylation of Ser318 is not required for stem elongation, indicating pathway-specific requirements for phosphorylation-based Ca²⁺-sensitivity priming. We additionally provide evidence for a conserved function for Ser318 phosphorylation in related group IV CDPKs, which holds promise for biotechnological applications by generating CDPK alleles that enhance resistance to microbial pathogens without consequences to yield.

钙 (Ca ²⁺ ) 依赖性蛋白激酶（CDPK 或 CPK）是一个独特的 Ca ²⁺传感器/激酶效应蛋白家族，在植物中具有多种功能。在拟南芥中，CPK28 通过促进关键免疫信号受体样细胞质激酶 BOTRYTIS 诱导激酶 1 (BIK1) 的降解来促进免疫稳态，并在营养期到生殖期的过渡中发挥额外作用。CPK28 如何控制这些看似不同的途径尚不清楚。在这里，我们鉴定了 CPK28 (Ser318) 激酶结构域中的单个磷酸化位点，该位点对其在免疫稳态和茎伸长中的功能有不同的需求。我们发现 CPK28 在 Ser318 上经历分子间自磷酸化，并且还可以通过 BIK1 在该残基上进行转磷酸化。^{对其他几个磷酸化位点的分析表明，Ser318 磷酸化是在 Ca 2+}生理浓度下引发 CPK28 激活 Ca ²⁺所必需的，可能是通过内在荧光实验表明的 Ca ²⁺结合活性状态的稳定来实现的。^{总之，我们的数据表明，Ser318 的磷酸化是在低细胞内 [Ca 2+} ]下激活 CPK28 所必需的，以防止在没有感染的情况下启动免疫反应。相比之下，茎伸长不需要 Ser318 的磷酸化，这表明基于磷酸化的 Ca ²⁺敏感性引发的途径特异性要求。我们还提供了相关 IV 组 CDPK 中 Ser318 磷酸化保守功能的证据，这通过生成增强对微生物病原体的抵抗力而不影响产量的 CDPK 等位基因，为生物技术应用带来了希望。