Application of Proximity-Labeling Techniques in Plants: A Review of Successful Cases.

Exploring protein-protein interaction (PPI) networks during cellular processes is critical for understanding the molecular mechanisms underlying these processes. PL (proximity labeling) is an emerging technique with the potential to be a powerful protein interactomics tool. It employs proximity-labeling enzymes, coupled with mass spectrometry, to covalently label, capture, and identify interacting and neighboring proteins of the bait protein. The development of numerous novel PL enzymes and the improvement of biotin ligase-based enzymes have enabled efficient spatiotemporal mapping of PPIs, especially after the establishment of TurboID in plants. Most PL-associated reviews in plants focus on the potential applications of different enzyme-based PL. Here, we focus on PL cases effectively applied in plants and dissect each case in detail from the perspectives of PL expression design, labeling, extraction, enrichment, and quantitative proteomic identification. Moreover, we compare cases using biotin ligase-based PL (such as BioID and TurboID) and PUP-IT, highlighting the advantages and limitations of each PL system. We delineated the pipeline and optimization strategies for PL experiment design to facilitate successful execution by plant researchers.