CRK2-mediated control of ROS production by phosphorylation of the RBOHD C-terminus in Arabidopsis

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms. Extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase (RLK)-dependent signaling networks. This ROS production is tightly controlled by multiple mechanisms including phosphorylation by different kinases. Here we show that the cysteine-rich RLK CRK2 exists in a preformed complex with the NADPH oxidase RBOHD at the plasma membrane. Functional CRK2 is required for the full pathogen-induced ROS burst and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae. We identified phosphorylation sites in the C-terminal region of RBOHD and mutations of the phosphorylation sites modulate ROS production in response to biotic stimuli. Our work demonstrates that CRK2 occupies a central role in controlling ROS production and highlights that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region is an ancient mechanism which is conserved between animals and plants.

活性氧（Reactive Oxygen Species, ROS）是真核生物中重要的信号信使。植物中NADPH氧化酶介导的胞外活性氧产生，由类受体蛋白激酶（Receptor-like Protein Kinase, RLK）依赖的信号网络所触发。该活性氧产生过程受到多种机制的严格调控，其中涵盖不同激酶介导的磷酸化修饰。本研究发现，富半胱氨酸类受体蛋白激酶CRK2可与NADPH氧化酶RBOHD在质膜处形成预组装复合物。具备正常功能的CRK2对于病原菌诱导的完整活性氧爆发不可或缺，因此crk2突变体在抵御丁香假单胞菌（Pseudomonas syringae）这一细菌病原菌的防御过程中存在功能缺陷。我们鉴定出了RBOHD C端区域的磷酸化位点，且这些磷酸化位点的突变会调控植物响应生物刺激时的活性氧产生水平。本研究证实CRK2在活性氧产生的调控中发挥核心作用，并阐明通过磷酸化修饰RBOHD的C端区域以调控NADPH氧化酶活性，是一种在动植物间保守存在的古老调控机制。