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

Area of Science:

• Plant immunity
• Molecular plant-microbe interactions
• Cellular signaling

Background:

• Plant immune responses involve pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).
• Nucleotide-binding leucine-rich repeat receptors (NLRs) mediate ETI, leading to hypersensitive response (HR) cell death.
• Reactive oxygen species (ROS) bursts, involving NbRBOHB, are critical during HR, but their regulation is unclear.

Purpose of the Study:

• To elucidate the regulatory mechanisms of ROS bursts during plant immune cell death.
• To investigate the role of calcium-dependent protein kinases (CDPKs) in ROS production during HR.
• To identify specific phosphorylation sites on NbRBOHB involved in ROS generation.

Main Methods:

• Luminol-based ROS assays in Nicotiana benthamiana.
• Immunodetection of phosphorylated NbRBOHB.
• Transient expression of autoactive NRC4^D478V.

Main Results:

• NbCDPK4 and NbCDPK5 directly phosphorylate NbRBOHB at Ser-123, inducing ROS bursts.
• Ser-123 phosphorylation is elevated during AVRblb2-ETI and INF1-triggered PTI-ETI, but not flg22-triggered PTI.
• Phosphorylation of Ser-123 depends on the N-terminal Ca²⁺ channel motif of NRC4.

Conclusions:

• NbCDPKs play a key role in activating NbRBOHB and sustaining ROS bursts during plant immune cell death.
• Specific phosphorylation of NbRBOHB Ser-123 by NbCDPKs is a critical regulatory step in ETI-associated ROS production.
• This mechanism highlights the interplay between calcium signaling and ROS generation in plant defense.