Knocking out SOBIR1 in Nicotiana benthamiana by CRISPR/Cas9, abolishes the functionality of the transgenic receptor-like protein Cf-4, recognizing the Avr4 effector of the fungus Cladosporium fulvum.

The first layer of plant immunity is formed by pattern recognition receptors (PRRs) that are present at the cell surface and perceive extracellular immunogenic patterns. Receptor-like proteins (RLPs), such as the tomato (Solanum lycopersicum) PRR Cf-4 that provides resistance to the fungus Cladosporium fulvum secreting the matching avirulence factor Avr4, have an extracellular receptor domain consisting of leucine-rich repeats, but lack a cytoplasmic kinase domain for downstream signaling. RLPs constitutively interact with the receptor-like kinase SUPPRESSOR OF BIR1-1 (SOBIR1), thereby providing the receptor with a kinase domain, and recruit the co-receptor BRI-ASSOCIATED KINASE 1 (BAK1) upon their activation by a matching ligand. Trans-phosphorylation events, which can take place between the kinase domains of SOBIR1 and BAK1 after their association with the RLP, are thought to initiate downstream defense signaling. Currently, our knowledge on RLP/SOBIR1/BAK1-mediated defence initiation is limited and to understand the role of SOBIR1 in RLP function, we knocked out SOBIR1 and its close homolog SOBIR1-like in the model plant Nicotiana benthamiana, as well as in transgenic N. benthamiana stably expressing Cf-4. We observed that Cf-4 function is completely abolished in the knock-out mutants, and we show that these plants can be used to perform transient complementation studies with SOBIR1 mutants. Thereby, these mutants are an important tool to study the fundamentals of plant immunity mediated by RLPs.

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.

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