Structural and Functional Analysis of Nucleocytoplasmic Protein O-Glycosyltransferases in Plants

Project Summary/Abstract O-linked protein glycosylation orchestrates diverse signaling events in plant development in response to the changing environment. We have discovered the first nucleocytoplasmic O-fucosyltransferase, SPINDLY (SPY) in Arabidopsis, which together with its paralog SECRET AGENT (SEC), an O-GlcNAc transferase (OGT), post- translationally modify the conserved DELLA family of transcription regulators, such as RGA, to control their ability to modulate multiple signaling pathways. SPY-catalyzed O-fucosylation of RGA enhances the RGA interaction with key transcription factors to suppress plant growth, whereas SEC-mediated O-GlcNAcylation weakens RGA activity and promotes plant development. Despite their functional significance, how these two homologous glycosyltransferases, SPY and SEC, have evolved distinct substrate specificities and how these two types of glycosylation, O-fucosylation by SPY and O-GlcNAcylation by SEC, regulate the RGA conformation to influence plant development at the molecular level have remained unknown. We propose to elucidate the molecular details of SPY, SEC, and their substrate and product complexes, as well as the distinct structural and functional consequences of RGA O-fucosylation and O-GlcNAcylation. Recent proteomic analysis has unveiled hundreds of SPY and SEC substrates beyond RGA, highlighting the broad implication of protein glycosylation in regulating cellular functions. In order to overcome the potential pleotropic effects brought by spy and sec mutations in planta and decipher the functional consequences of O-fucosylation and O-GlcNAcylation for specific target proteins, we will develop nanobody-fused SPY and SEC variants for precise target modification. Collectively, our study will fill the critical knowledge gap about the structural and functional consequences of protein glycosylation in plants. As SPY-like genes are conserved in diverse organisms, including plants, bacteria, and parasitic protists, and as SEC-like (OGT) genes are broadly distributed in plants and animals, a molecular understanding of these enzymes and their distinct structural and functional consequences will have a profound impact in plant biology, agriculture, and human health and diseases.