(1000-25) Proteomic Profiling of Receptor-Kinase Signaling Network in Plant Development by TurboID-based Proximity Labeling and Conventional Affinity Purification Methods
postdoctoral researcher Howard Hughes Medical Institute with University of Texas at Austin Austin, Texas
Body of Abstract: Receptor-like kinases (RLKs) play a crucial role in the hierarchical regulation of plant growth and development. ArabidopsisERECTA family genes encode three leucine-rich repeat (LRR) type RLKs, ERECTA, ERECTA-LIKE1 (ERL1), ERECTA-LIKE2 (ERL2), which synergistically specify the size, shape and pattern of aerial organs by coordinating cell proliferation and differentiation. Several EPIDERMAL PATTERNING FACTORs (EPFs) have been identified as secreted ligands for ERECTA-family proteins, which partially explain the pleiotropic function of ERECTA family receptors in terms of extracellular signal perception.Upon EPFs-ERECTA recognition, numerous known and unknown co-receptors and receptor-like cytoplasmic kinases (RLCKs) are recruited, enabling spatial and temporal regulation of intracellular signal transduction. The intriguing question arises: how do ligand-receptor pairs generate distinct output signals by utilizing the conserved signaling modules such as RLCK transphosphorylation; MAP kinase cascade activation? To explore this question, we employed TurboID-based proximity labeling (TurboID-PL) technique, which enables a proteomic profiling of ERECTA associated proteins in their native cellular environment. Specifically, we generated an Arabidopsis transgenic line expressing a fused protein of ERECTA-3xHA-TurboID in er-105 mutant background. By comparing both TurboID-PL and conventional HA-tagged affinity purification (HA-AP) methods, we discovered 715 and 278 proteins, respectively, as part of the ERECTA-associated network. Among these proteins, 50 candidates were common to both methods, including plasma membrane proton ATPases and RLKs. Notably, TurboID-PL outperformed HA-AP in capturing proteins associated with endosomal trafficking. Several candidates related to clathrin-mediated endocytosis were identified by TurboID-PL, including dynamin GTPases, clathrin adaptors, clathrin heavy chain and TPLATE. However, the extracellular co-receptor TOO MANY MOUTHS (TMM), which forms a binding pocket with ERECTA for ligand recognition, was exclusively detected in the HA-AP list, implying a limitation of TurboID-PL for C-terminal TurboID-fused plasma membrane receptors. In conclusion, our study provides a proteomic landscape of the ERECTA signaling network and highlights the distinct advantages and limitations of TurboID-PL.