The Phosphorylation Mechanism Of Receptor-Like Cytoplasmic Kinase RIPK In Plant Immunity

Reactive oxygen species（ROS）signaling plays a crucial role in multiple layers of the plant immune system.In Arabidopsis,the production of ROS is mainly mediated by respiratory burst oxidase homolog D（RBOHD）localized in the plasma membrane.RIPK（RPM1-induced protein kinase）,a member of receptor-like cytoplasmic kinases（RLCKs）,regulates broad-spectrum ROS signaling.RIPK directly phosphorylates the N-terminal region of RBOHD and activates NADP-malic enzyme 2（NADP-ME2）to generate NADPH for RBOHD.In this study,we characterized the mechanisms of how RIPK is activated to regulate broad-spectrum RBOHD-mediated ROS signaling and the results are as follows:1.Identification of RIPK phosphorylation sites.Chitin receptor CERK1（chitin elicitor receptor kinase1）and 3-OH-C10:0 receptor LORE（lipopolysaccharide-specific reduced elicitation）could directly phosphorylate RIPK in vitro.LC–MS/MS identified the same phosphorylation sites of RIPK targeted by CERK1 and LORE,S251,T252 and T423.2.The S251A/T252A substitution completely abolished RIPK kinase activity.Complementation test of ripk-c33 mutants with phospho-dead and phospho-mimic variants of RIPK indicated that S251 and T252 of RIPK were vital for RIPK-mediated ROS production,while T423 was not.Moreover,different immune elicitors increased the phosphorylation levels of the RIPK S251 and T252 residues in vivo.In addition,we found that mutations of S251 and T252 of RIPK resulted in the loss of kinase activity.3.Loss of RIPK kinase activity had a dominant negative effect on ROS production.A point mutation of G237R in RIPK also abolished RIPK kinase activity.We introduced RIPK^G237R into Col-0 background and found that overexpression of RIPK^G237R reduced the ROS production,suggesting RIPK^G237R has a dominant negative effect on ROS signaling.Further analysis showed that RIPK^G237R did not affect the trans-phosphorylation of RBOHD by RIPK and other RLCKs,nor inhibited the in vivo phosphorylation of RBOHD and other RLCKs.However,compared with RIPK,RIPK^G237R showed stronger association with NADP-ME2,a NADPH generating enzyme.Collectively,the mechanism of dominant negative effect on ROS production by kinase-dead RIPK need to be further explored.