Proximity-dependent biotinylation methods (BioID, TurboID, miniTurbo) enable in vivo labeling of proteins near a bait before lysis, with TurboID variants providing rapid labeling compatible with plant physiology.

While yeast two-hybrid has driven high-throughput screening, it imposes a heterologous nuclear environment and is prone to biases that inflate false positives and negatives.

Affinity purification–mass spectrometry (co-immunoprecipitation) captures protein complexes but is highly sensitive to how cells are lysed, risking loss of transient interactions and mixing organelles.

Emerging frontiers include high-throughput protein microarrays for kinase–substrate mapping and optogenetic systems (such as CRY2–CIB1) to induce interactions and test causality.

Historical methods like in vitro pull-downs and Far-Western blotting miss physiological context due to lack of plant-specific modifications and non-physiological conditions.

A dynamic plant interactome exists because many plant protein interactions are weak, transient, and compartmentalized, challenging capture by traditional approaches.

Choosing the approach depends on the biological question, protein class, and resources, with the aim of delivering a rigorous, physiologically meaningful map of plant networks that could enhance crop resilience and productivity.

The authors propose a three-tier workflow: Tier 1 TurboID for broad in vivo discovery, Tier 2 NanoLuc split-luciferase to confirm true proximity and orientation, and Tier 3 quantitative FRET–FLIM to validate interactions under physiological conditions.

Applications of TurboID include identifying regulators like UBR7 in immunity, profiling rare cell types such as guard cells, and studying complexes like the m6A methyltransferase, while noting that proximity indicates proximity, not direct binding.

Split-luciferase (NanoBiT) assays offer validated, real-time readouts of interactions with minimal steric disruption and reversible complementation in living plant cells.

FRET–FLIM provides a biophysical gold standard by delivering quantitative nanoscale proximity data and enabling in vivo confirmation of interactions at native expression levels.