Final Report Summary - PHOSPHINNATE (Signaling initiation and specificity in BAK1-dependent receptor kinase-mediated innate immunity in Arabidopsis) Plant receptor kinases (RKs) represent one the largest families of plant proteins, and control all aspects of plant life, ranging from development and growth to biotic and abiotic stress responses. Many plant RKs are pattern recognition receptors (PRRs) that perceive pathogen-associated molecular patterns to induce innate immune responses, which fend off most microbes and restrict the growth of pathogens. The best studied plant PRRs are the Arabidopsis thaliana leucine-rich repeat RKs FLS2 and EFR that perceive bacterial flagellin (or its peptide immunogenic epitope flg22) and EF-Tu (or its peptide immunogenic epitope elf18), respectively. In addition to being important for anti-bacterial immunity, FLS2 and EFR represent excellent model receptors to study RK-mediated signaling in plants.We have revealed that plant RKs, such as the immune receptor FLS2 or the growth receptor BRI1, form specific plasma membrane nanodomains to form pre-formed signaling platforms, providing novel insights into how plant cells initiate signaling and potentially maintain signaling specificity. We have also contributed to the definition of the molecular basis of the ligand-induced complex formation between FLS2 and BAK1 through structural studies. In addition, we have contributed to the findings that the ligand-induced complex formation between the immune receptor FLS2 and its co-receptor BAK1 (a step essential for the initiation of immune signaling) is controlled by a suite of regulatory RKs. In particular, we have found that the malectin-like RK FERONIA (FER) also regulates positively ligand-induced FLS2-BAK1 complex formation, and that this regulation is controlled by the perception of antagonistic endogenous RALF peptides.We have also contributed to the demonstration that the ligand-induced complex formation between FLS2 and BAK1 is a critical step for the phosphorylation-dependent activation of the complex and subsequent immune signaling. We identified a critical in vivo Tyr phosphosite, Tyr836, that is essential for EFR function in immunity. Notably, we also revealed that the phytopathogenic bacterium Pseudomonas syringae secretes within plant cells the type-III secreted effector HopAO1 (a tyrosine phosphatase), which directly interact with the kinase domains of EFR and other immune receptors to suppress their activity and immune signaling, thus contributing to bacterial virulence. This work also contributed to the emerging realization that tyrosine phosphorylation of plant RKs plays a much more important role than previously thought, and thus that these proteins are actually dual-specificity kinases despite being annotated as serine/threonine kinases. In addition to our work on EFR, we have also defined a number of BAK1 in vivo phosphosites that play a critical role in the induction of immune signaling by FLS2 and EFR. Interestingly, we also identified protein phosphatases that negatively regulates BAK1 activity. In addition, we characterize several substrates of the central immune kinase BIK1 (that is part of the FLS2/EFR-BAK1 complex), including the NADPH oxidase RBOHD, the calcium-dependent protein CPK28 and the protein phosphatase type 2C PP2C38. We also performed a forward-genetic screen, the ‘modifier of bak1-5’ (mob) mutant screen to identify novel regulator on plant immune signaling. The underlying genes were unambiguously identified in 4 out of 10 mob mutants, and for 2 additional mob mutants, strong candidate genes are currently being confirmed. Manuscripts describing the allelic MOB1/MOB2 genes (that encode the calcium-dependent protein kinase CPK28), and MOB6 (which encodes the subtilisin-like serine protease S1P) have been published. Importantly, we showed that CPK28 phosphorylates the key immune kinase BIK1 and controls its turnover via the proteasome. The study on MOB6/S1P revealed a very interesting regulatory module that involves the subtilase S1P, the endogenous peptide RALF23 and the malectin-like RK FER, and which regulates the ligand-induced complex formation between FLS2/EFR and BAK1. We have also begun to study the role of MOB4, which encodes a perforin-like protein, in immune signaling. The top candidates for MOB3 and MOB7 are being currently confirmed genetically, while the genetic analysis of the remaining 4 mob mutants is still ongoing.Together, the results obtained during the PHOSPHinnATE project have thus enabled a step-change in our understanding of the ligand-induced activation of plant receptor kinases and of the initiation and specificity of innate immune signaling. In addition, models and hypotheses established during this project provide a potential paradigmatic template for the molecular basis of signaling mediated by receptor kinases in other important plant processes, such as growth, development, and responses to the environment.