Molecular analysis of leucine rich-repeat receptor protein-mediated immune signaling

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Leucine-rich repeat (LRR) ectodomain-containing PRRs mediate recognition of proteinaceous immunogenic patterns. LRR-type PRRs exist in two forms as LRR receptor kinases (LRR-RK), which carry an intracellular protein kinase domain, and LRR receptor proteins (LRR-RP) that lack a functional intracellular domain. It has recently been hypothesized that LRR-RP/SOBIR1 heteromeric complexes (SOBIR1 is an LRR-RK itself), constitutively formed in the absence of ligands, are equivalent to bi-molecular or bipartite LRR-RK-type PRRs. The proposed identity of molecular mechanisms underlying immune activation mediated through either LRR-RK or LRR-RP-type PRRs has yet to be shown. In fact, our recent findings have challenged this hypothesis to an extent that justifies a more in-depth comparative analysis of signaling networks and physiological outputs mediated through activation of either receptor type. Substantial differences were observed between Arabidopsis LRR-RK FLS2 and Arabidopsis LRR-RP RLP23-mediated cellular responses. For example, the cytoplasmic protein kinase BIK1, known as a positive regulator of flg22-induced ROS burst, acts as a negative regulator of nlp20-induced ROS burst and ethylene production; both amplitude and kinetics of flg22- and nlp20-mediated ROS burst differ substantially, and nlp20-treatment of Arabidopsis Col-0 resulted in the production of camalexin, whereas flg22-treatment did not. To characterize and compare LRR-RK and LRR-RP-mediated immune signaling, we propose to conduct a systematic analysis of pattern-specific signal transduction and of immune responses mediated through two LRR-RK-type PRRs (FLS2, EFR) or through two LRR-RPs (RLP23, RLP42). This research will reveal to which extent signaling pathways activated through LRR-RK and LRR-RP-type differ as well as whether there are differences in signaling networks and signal outputs mediated through activation of the same receptor types. We will further test whether chimeric receptors in which kinase domains of FLS2 and SOBIR1 were replaced with one another would turn flg22 perception into an nlp20 output response and vice versa. If so, this would suggest a strictly modular composition of these receptor types. We further propose to assess the mechanistic basis for BIK1-mediated negative regulation of nlp20 signaling. Here, we will analyze whether this novel function requires BIK1 protein kinase activity, whether BIK1 physically associates with SOBIR1 in ligand (in)dependent fashion, whether nlp20-specific phosphorylation of BIK1 is observed, whether BIK1 phosphorylates substrates (such as RbohD) in an nlp20-specific manner or whether putative novel BIK1-interacting proteins (substrates) can be identified that might explain the differential involvement of BIK1 in flg22/FLS2 and nlp20/RLP23-mediated immune signaling.