Capturing protein-protein interactions in plants: recent advances, challenges, and opportunities

Plants rely on dynamic protein-protein interaction (PPI) networks to carry out routine functions (such as photosynthesis and respiration) and responses to environmental cues. Therefore, capturing dynamic PPIs is critical for understanding molecular processes underlying the plant life cycle. Recent technological advances have significantly expanded the experimental and computational toolkit available for studying PPIs in plants. In this review, emerging and advanced technologies are presented, including proximity labeling, yeast-3-hybrid, AlphaFold 3, and data-independent acquisition mass spectrometry (MS). How these technologies address critical limitations posed by classical techniques, and their strengths, challenges, and opportunities, are discussed. The goal is to provide an updated practical guide that informs researchers on selecting, optimizing, and combining these tools to maximize protein interactome coverage. Together, these complementary tools and approaches promise to advance mechanistic understanding of plant biological processes and enable more informed manipulation of complex biological networks toward improving crop quality and yield.