Profiling FERONIA interaction proteins by Pupylation-based interacting tagging in Arabidopsis

FERONIA (FER) is a ubiquitious expressed and a plasma memberane-localized receptor-like protein kinase which modulate many biological functions. Elucidating the interacting proteins is the crucial step in understanding the molecular function of FER in regulating plant development. However, weak and transient interactions make identifying FER interacting proteins a challenge. Here, we adapted a novel proximity-based labelling technique, pupylation-based interaction tagging (PUP-IT), to label interacting proteins, subsequent enriched by affinity capture, and analyzed by liquid chromatography-coupled tandem mass spectrometery (LC-MS/MS) to profile FER interacting proteins. To perform a quick screen, a transient protoplasts expression system was conducted to identify interacting proteins in mesophyll cells. In addition, we generated a transgenic line that harboring a tagging substrate-inducible system and FER-ligase recombinant protein driven by a native promoter for surveying the interacting proteins in seedlings and flower organ. We introduce and prove the concept of PUP-IT tool designed to detect protein interactions of significance in plants.