Phosphorylation and ubiquitination of immune sensory complexes in innate immune signaling Project Summary Plants and animals rely on pattern-recognition receptors (PRRs) to detect infection by recognizing microbe-associated molecular patterns (MAMPs). FLS2, a structural and functional ortholog of mammalian Toll-like receptors (TLRs) in plants, recognizes bacterial flagellin and initiates immune signaling by dimerizing with a co-receptor BAK1. With the previous NIH support, we have identified a plasma membrane-tethered receptor-like cytoplasmic kinase (RLCK) BIK1 as a convergent immune regulator by association with multiple PRR complexes. MAMP perception induces rapid BIK1 phosphorylation by BAK1, and subsequent release from PRR complexes to transduce diverse immune signaling. It remains unknown how PRR-associated BIK1 is activated and then bifurcates intracellular PRR signaling. Our preliminary data indicate that MAMP perception triggers rapid mono-ubiquitination of BIK1 and related RLCKs. A RING-type E3 ubiquitin ligase BMU1, that is phosphorylated by BIK1, mediates MAMP-induced BIK1 ubiquitination. BIK1 interacts and phosphorylates a diacylglycerol kinase DGK5 that regulates MAMP-induced phosphatidic acid (PA) production. Three over-arching aims are proposed to test a central hypothesis that mono-ubiquitination of BIK1 by BMU1 triggers release of BIK1 from PRR complexes, and subsequent endosomal trafficking and relaying intracellular signaling events, including activation of DGK5 for PA production. Three specific aims are 1. Ubiquitination of BIK1 by BIK1-phosphorylated BMU1 in plant immunity; 2. Ligand-induced BIK1 mono-ubiquitination in endosomal trafficking and plant immunity; 3. Dual phosphorylation of DGK5 by PRR-activated BIK1 and MAP kinase 4 in plant immunity. Recent advance on the molecular architecture of nonself recognition has revealed remarkable conservation in the mechanisms of microbial perception and innate immune signaling in multicellular eukaryotes. Phosphorylation and ubiquitination are two key mechanisms in regulating diverse cellular and organismal processes. Our project on pathogen- induced sequential and intertwined phosphorylation and ubiquitination orchestrating PRR complex activation and signal transduction in plant immunity will generate conceptual advance in understanding the biological functions of these two universal regulatory mechanisms at the whole organismal level. The proposed research will also contribute to the general understanding of innate immune signaling and immune sensory complex function.