PROJECT SUMMARY Immune systems across kingdoms of life recognize pathogen-associated molecules through germline-encoded innate immune receptors. Receptor repertoires in plants have evolved to detect an especially diverse set of ligands due to massive expansion of the receptor kinase gene family with specialized ligand recognition functions. Pairing receptor sequence diversity with specific recognition functions across 100 million RK genes (350,000 plant species * 500 receptors per genome) is a grand challenge in plant molecular biology. It also presents the opportunity to develop a new class of protein-based sensors for biotechnology. The Steinbrenner lab aims to characterize and deploy this vast plant immunodiversity for ligand-induced modulation of engineered signaling pathways. First, we will define the full ligand space that is monitored by plant receptors by focusing on the large subfamily of leucine-rich repeat receptor kinases (termed receptors here) which bind small peptide epitopes to initiate immune signaling. We will combine evolution- and structure-guided approaches to decode the basis of receptor:ligand specificity, including an extensive phylogenomic analysis, peptide variant libraries, and ancestral sequence reconstruction. We hypothesize that transitions in ligand specificity are marked by amino acid substitutions in predicted ligand binding sites among ancestral receptor genes. For “orphan” receptors lacking defined functions, we will conduct a genomic screen using synthetic DNA libraries encoding candidate pathogen epitopes using both plant and yeast models as reporters for receptor activation. We hypothesize that most receptors involved in plant innate immunity will be activated by specific pathogen-derived peptide sequences. Combined, these approaches will provide basic insights into receptor:ligand specificity as well as a toolkit of extracellular sensor domains responsive to specific peptide agonists. Second, we will leverage the unique network architecture of plant immune networks to engineer synthetic signaling pathways that do not interfere with endogenous animal signaling pathways. The plant receptors studied here signal through heterodimerization with a common co-receptor called BAK1. Co-receptor activation culminates in phosphorylation of substrates based on defined phosphocode motifs. We are currently engineering the human inflammation signaling pathway to accept orthogonal input from plant receptors by incorporating plant kinase substrates into specific, phosphoregulated signaling factors. In parallel, we will use plant receptor:co-receptor heterodimerization as a platform to scaffold endogenous human immune signaling domains from Toll-like receptors. We hypothesize that engineered pathways will allow modular tuning by diverse peptide ligands, providing an alternative to current immunoglobulin or GPCR-based synthetic tools. In summary our lab is poised to deploy tools for receptor de-orphanization and signaling pathway engineering to leverage the immense diversity driven by plant-pathogen co-evolution. (30 lines)

PROJECT NARRATIVE The project seeks to define the full ligand space monitored by the large gene family of plant cell surface receptor kinases. Findings will inform mechanisms of specificity in peptide recognition by receptor ectodomains. Plant receptor pathways will be used for orthogonal control of mammalian signaling pathways with applications in biotechnology and medicine.