Calcium-dependent protein kinases participate in RBOH-mediated sustained ROS burst during plant immune cell death

Plant immune responses comprise two phases, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Sensing of the pathogen effector by a nucleotide-binding leucine-rich repeat receptor (NLR) induces robust and sustained immune responses, resulting in hypersensitive response (HR) cell death. During HR, the reactive oxygen species (ROS) burst is achieved through Nicotiana benthamiana NbRBOHB. However, the detailed regulatory mechanisms of the ROS burst during HR are unclear. Here, we show that calcium-dependent protein kinases (CDPKs) contribute to the ROS burst associated with ongoing cell death using luminol-based ROS assay together with immunodetection of the phosphorylated NbRBOHB in N. benthamiana. We found that NbCDPK4 and NbCDPK5 directly phosphorylate NbRBOHB Ser-123 and provoke ROS bursts. The phosphorylation of the Ser-123 was strongly increased during AVRblb2-ETI and INF1-triggered PTI-ETI responses. On the contrary, the Ser-123 was not phosphorylated during flg22-triggered PTI. Moreover, the transient expression of NRC4^D478V, an autoactive helper NLR, indicated that phosphorylation of Ser-123 was induced in a manner dependent on the N-terminal conserved motif required for Ca²⁺ channel activity of the NRC4. These findings demonstrate a pivotal role for NbCDPKs in activating RBOH and sustained robust ROS bursts during plant immune cell death.