Figure 3.

The activity regulation of NADPH oxidases (NOXs/RBOHs) in plant immunity of rice. Two receptor-like cytoplasmic kinases (RLCKs), OsRLCK185 and OsRLCK176, can be directly phosphorylated by a plasma membrane-localized receptor-like kinase, OsCERK1 [201,210,211], which is associated with receptor-like kinases OsLYP4 or OsLYP6 when induced by PGN, but associated with OsLYP4, OsLYP6 or OsCEBiP under chitin treatment [211]. In addition, OsCEBiP can form a homodimer upon chitin binding that is followed by heterodimerization with OsCERK1, creating a signaling-active sandwich-type receptor system [213,214]. Then, the phosphorylated OsRLCK185 triggers a MAPK cascade: OsMAPKKK18/24-OsMKK4-OsMPK3/6, to activate the downstream proteins directly, or perhaps act as the upstream of NOXs/RBOHs to enhance ROS production [215,216]. Similar to OsRLCK185, OsRLCK176 also activates MAPK cascades [211], but the mechanism remains to be addressed as well. A ROP/RAC GTPase, OsRAC1, is also involved in the OsCERK1/OsCEBiP-mediated immune signaling, it activates OsRbohB for ROS production by directly interacting with the N-terminus of the NADPH oxidase [201,213]. Two other regulator factors, OsGEF1 and OsRACK1, also participate in this signaling pathway. OsGEF1 mediates the change between the RAC-GDP inactive form and the RAC-GTP active form as a molecular switch for the signaling [179,204], coupling with OsRACK1 [219], after being phosphorylated by OsCERK1 [28]. In addition, OsRACK1 also functions as a scaffolding protein linking heterotrimeric G proteins and the MAPK cascade [220]. Phosphorylation functions in important roles in activating NOXs/RBOHs and contributes to oxidative burst. However, the phosphorylation of NOXs/RBOHs is not sufficient for full activation of the proteins [221]. The binding of influx Ca²⁺ to EF-hand region and the phosphorylation in the N-terminal region of OsRbohB by CDPK5/13 perhaps induce the conformational change in EF-hand region of the NOX/RBOH and therefore, expose the site for interaction with RAC GTPase in the N-terminal region, and subsequently activate OsRbohB for ROS production. In turn, the ROS produced by the NOX/RBOH and other components induce more Ca²⁺ influx and the elevated Ca²⁺ in cytosolic play a positive role to induce more Ca²⁺ efflux from intracellular Ca²⁺ stores. Meanwhile, Ca²⁺ accumulation reaches a threshold, which may serve as a negative feedback mechanism to terminate the OsRAC1–OsRbohB interaction and ROS production [222]. These results imply that CDPK and RAC synergistically regulate the activity of NOXs/RBOHs in Ca²⁺-dependent manner. Furthermore, the Ca²⁺-related negative feedback mechanism exerts an important function in regulating the RAC–NOX/RBOH interaction for maintaining the ROS homeostasis in rice plants. CDPK, calcium-dependent protein kinase; CEBiP, chitin elicitor-binding protein; CERK, chitin-elicitor receptor kinase; G_pro, GTP-binding protein; GEF, guanine nucleotide exchange factors; LYP, homologs of LysM proteins in rice; MAPK, mitogen-activated protein kinase; PGN, peptidoglycan; RACK, receptor for activated C-kinase; RLCK, receptor-like cytoplasmic kinases; ROP/RAC GTPase, a subfamily of Rho-type GTPases, which belongs to the Rat sarcoma (Ras) superfamily of small GTP-binding proteins; SIK1/MAP4K, salt inducible kinase 1.