P-014 - Calcium-dependent protein kinases regulate sustained ROS production downstream of helper NLR activation

Plant immune responses are composed of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is a transient response that is initiated by the recognition of pathogen-associated molecular patterns (PAMPs), while ETI is induced by the effector recognition by sensor nucleotide-binding leucine-rich repeat receptor (NLR). After effector recognition by sensor NLRs, helper NLRs form a multimeric resistosome acting as Ca²⁺ channels. During both PTI and ETI, NADPH oxidase respiratory burst oxidase homolog (RBOH) -dependent reactive oxygen species (ROS) production is induced by post-translational modifications and transcriptional reprogramming of RBOH. However, detailed mechanisms connecting pathogen recognition and ROS production are still unknown. Here, we show that calcium-dependent protein kinases (CDPKs), NbCDPK4/5 phosphorylate NbRBOHB Ser-123, and induce ROS production in Nicotiana benthamiana. The phosphorylation level of the Ser-123 increased during AVRblb2-induced ETI and INF1-induced ETI-like responses, but not flg22-induced PTI. We further found that an autoimmune helper NLR, NLR required for cell death 4 (NRC4), induced NbRBOHB-dependent ROS production and the Ser-123 phosphorylation, depending on Ca²⁺ and its conserved N-terminal motif. These findings reveal the critical role of NbCDPK4/5-NbRBOHB in sustained ROS production during ETI downstream of helper NLR activation.