Activation and signal transduction of SOBIR1/BAK1-containing immune complexes present at the plant cell surface

Plants have evolved a two-layered innate immune system to cope with invading microbes from the surrounding environment. Receptors present at the plasma membrane, which are either receptor-like kinases (RLKs) or receptor-like proteins (RLPs), form the first layer of the plant innate immune system and play an important role in mediating host resistance against various pathogens. Our research focuses on the interaction between the extracellular pathogenic fungus Cladosporium fulvum and tomato (Solanum lycopersicum, Sl). Previously, we have shown that the cell-surface receptor Cf-4, which is a leucine-rich repeat (LRR)-RLP, specifically recognizes the apoplastic effector Avr4 secreted by C. fulvum. This recognition leads to the resistance of tomato to C. fulvum. In contrast to RLKs, RLPs lack an intracellular kinase domain for downstream signaling. Interestingly, our subsequent research has found SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR, further referred to as SOBIR1), which is an LRR-RLK, to constitutively interact with Cf-4 and to thereby provide Cf-4 with a kinase domain. Consistent with LRR-RLK-mediated immune signaling, the Cf-4/SOBIR1 complex recruits the LRR-RLK BRI-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3, further referred to as BAK1) to initiate plant immunity, upon the perception of Avr4 by Cf-4. Notably, BAK1 recruitment was later found to be a general phenomenon upon ligand-mediated activation of the LRR-RLP/SOBIR1 complex. The research described in this thesis was aimed to elucidate the molecular mechanisms behind the activation of the Cf-4/SOBIR1/BAK1 complex and to identify essential receptor-like cytoplasmic kinases (RLCKs) that play a role in immune signaling downstream of the Cf-4/SOBIR1 complex.

SOBIR1 is a positive regulator of LRR-RLP-mediated immune signaling and appears to be present throughout the plant kingdom. Overexpression of Arabidopsis (Arabidopsis thaliana, At) SOBIR1 results in constitutive activation of cell death and associated defense responses in both Arabidopsis and the model Solanaceous plant Nicotiana benthamiana (Nb). Nevertheless, no symptoms of auto-immunity were observed when NbSOBIR1, SlSOBIR1, or SlSOBIR1-like was overexpressed in leaves of N. benthamiana plants. To generate the material that can be used to characterize the function of SOBIR1 in planta and to study the fundamentals of plant immunity triggered by Avr4/Cf-4, we employed the CRISPR/Cas9 system to knock out SOBIR1 in N. benthamiana. We show that we successfully knocked out SOBIR1 and its homolog SOBIR1-like both in wild-type N. benthamiana and in N. benthamiana stably expressing the Cf-4 transgene. Strikingly, N. benthamiana sobir1 (/sobir1-like) knock-out plants are non-responsive to the Avr4/Cf-4 combination, and consistently, N. benthamiana:Cf-4 sobir1 (/sobir1-like) knock-out plants are also non-responsive to Avr4.

The sobir1 (/sobir1-like) knock-out plants were subsequently implemented for complementation studies, combined with a site-directed mutagenesis screen of putative phosphorylation sites present in SOBIR1. One serine (Ser/S) and four threonine (Thr/T) residues that are present in the activation segment of the kinase domain of SOBIR1 were studied. NbSOBIR1T522, as well as its analogous residues in SlSOBIR1 and SlSOBIR1-like, was identified to be essential for Avr4/Cf-4-induced reactive oxygen species (ROS) accumulation, the activation of a mitogen-activated protein kinase (MAPK) cascade, and the hypersensitive response (HR). Further in vitro phosphorylation assays demonstrated that this particular Thr residue is required for the intrinsic kinase activity of SOBIR1. Additionally, we provide in vitro evidence to support the SOBIR1/BAK1 activation model that was proposed before.

Recently, Tyr phosphorylation has been recognized to be a common feature of RLK activation in plants. Therefore, all Tyr residues present in the kinase domain of NbSOBIR1, SlSOBIR1, and SlSOBIR1-like were individually changed into the non-phosphorylatable amino acid phenylalanine (Phe/F) and the obtained mutants were included in the complementation study. We show that NbSOBIR1Y469, as well as its analogous residues in SlSOBIR1 and SlSOBIR1-like, plays a crucial role in Avr4/Cf-4-triggered MAPK activation and the HR, whereas this residue is not essential for the Avr4/Cf-4-induced ROS production or the intrinsic kinase activity of SOBIR1. However, no phosphorylated Tyr residue was detected in the kinase domain of either AtSOBIR1 or NbSOBIR1 by mass spectrometry. Therefore, we propose that SOBIR1 employs this important Tyr residue in its kinase domain to trigger plant immunity by binding a specific substrate, instead of being important in a phosphorylated state.

Cell-surface receptors deploy a large number of downstream RLCKs to relay the immune signals from the extracellular space into the plant cells. We show that eight members from the RLCK class VII subfamily 6 (RLCK-VII-6) in N. benthamiana:Cf-4 plants play a pivotal role in regulating the production of ROS that is stimulated by multiple extracellular immunogenic patterns (ExIPs), including the Avr4 protein. Strikingly, these eight members are dispensable for Avr/Cf-4-triggered MAPK activation and the HR. More importantly, despite their different subcellular localization, these eight members appear to function redundantly as positive regulators of the Avr4/Cf-4-induced ROS accumulation. Furthermore, members from the RLCK-VII-7 and -8 subfamilies might also be essential for the Avr4/Cf-4-triggered ROS burst. However, the molecular mechanisms by which the various members from the RLCK-VII-6 and RLCK-VII-7/8 subfamilies regulate the production of ROS in N. benthamiana are not known.