Receptor-like cytoplasmic kinases belonging to different subfamilies mediate immune responses downstream of the Cf-4 resistance protein in Nicotiana benthamiana

Cell-surface receptors, which are either receptor-like proteins (RLPs) or receptor-like kinases (RLKs), form the first layer of the plant innate immune system. The LRR-RLKs SUPPRESSOR OF BIR1-1 (SOBIR1) and BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (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 Cladosporium fulvum, requires SOBIR1 and BAK1 to mediate resistance against this fungus. It is proposed that phosphorylation events take place between the cytoplasmic kinase domains of SOBIR1 and BAK1, which lead to the initiation of various downstream signaling outputs such as the rapid phosphorylation of receptor-like cytoplasmic kinases (RLCKs), the accumulation of reactive oxygen species (ROS), the activation of mitogen-activated protein kinase (MAPK) cascades, and in some cases, the hypersensitive response (HR). Here, by performing in vitro phosphorylation assays, we show that SOBIR1 directly phosphorylates BAK1, whereas BAK1, in its turn, directly phosphorylates SOBIR1, which results in the full activation of the Cf-4-containing immune complex. We found that threonine 522, present in the activation segment of the SOBIR1 kinase domain of Nicotiana benthamiana (Nb), is required for its intrinsic kinase activity, and is essential for the Avr4/Cf-4/SOBIR1/BAK1-triggered ROS burst, MAPK activation and the HR. Tyrosine residue Y469 was found to be crucial for the Avr4/Cf-4- triggered activation of MAPKs and the HR, but not for ROS production and NbSOBIR1 intrinsic kinase activity. RLCKs are well-recognized to act as the initial cytoplasmic transducers, bridging cell-surface receptor complexes with their downstream signaling partners. By knocking out multiple genes belonging to different RLCK-VII subfamilies in N. benthamiana plants stably expressing Cf-4, we show that members of the RLCK-VII subfamilies 6, 7 and 8 are required for the Avr4/Cf-4-triggered ROS production. Typically, the Avr4 protein triggers a biphasic ROS burst in leaf discs obtained from N. benthamiana:Cf-4 plants, in which the first transitory response is followed by a second, sustained ROS burst. Interestingly, the intensities of the first and second phase of the ROS burst are affected in different ways in the various rlck-vii knock-out plants, indicating that there are different downstream ROS regulatory mechanisms, involving different RLCKs in N. benthamiana. Further studies show that members from RLCK-VII-6, 7 and 8 also play an essential role in regulating the ROS production triggered by flg22, chitin, and the nlp20/RLP23 and the pg13/RLP42 combinations.