Receptor-like cytoplasmic kinases belonging to different subfamilies differentially regulate SOBIR1/BAK1-mediated immune responses in Nicotiana benthamiana

Abstract Cell-surface receptors, which are either receptor-like proteins (RLPs) or receptor-like kinases (RLKs), stand at the basis of the first layer of the plant innate immune system. The leucine-rich repeat (LRR)-RLKs SOBIR1 and BAK1 are the common regulatory co-receptors of LRR-RLPs. The tomato LRR-RLP Cf-4, which specifically detects the apoplastic effector Avr4 that is secreted by the pathogenic intercellular fungus Fulvia fulva (syn. Cladosporium fulvum), requires SOBIR1 and BAK1 to mediate resistance against this fungus. Here, by performing in vitro phosphorylation assays, we show that SOBIR1 directly phosphorylates BAK1, whereas BAK1, in turn, directly phosphorylates SOBIR1, which results in the full activation of the SOBIR1/BAK1-containing immune complex. The amino acid residues Thr522 and Tyr469, present in the kinase domain of Nicotiana benthamiana (Nb) SOBIR1, were found to be required for the intrinsic kinase activity of NbSOBIR1 and for interacting with specific downstream signalling partners, respectively. Receptor-like cytoplasmic kinases (RLCKs) are well-recognized to act as the initial cytoplasmic transducers, bridging cell-surface receptor complexes with their downstream signalling partners. Here, by knocking out multiple genes belonging to different RLCK-VII subfamilies in N. benthamiana:Cf-4, we show that members of RLCK-VII-6, -7, and -8 differentially regulate the Avr4/Cf-4-triggered biphasic reactive oxygen species (ROS) burst in N. benthamiana. This observation suggests that there are different downstream ROS regulatory mechanisms, involving different RLCKs, in N. benthamiana. In addition, members of RLCK-VII-7 play an essential role in the Avr4/Cf-4-triggered hypersensitive response (HR) and host resistance against the oomycete pathogen Phytophthora palmivora. Furthermore, SOBIR1 and BAK1 directly trans-phosphorylate members of RLCK-VII-6, -7 and -8 in vitro. Our study provides molecular evidence for the activation and signal transduction of SOBIR1/BAK1-containing immune complexes through RLCKs in N. benthamiana.