Phase separation-based visualization of protein--protein interactions and kinase activities in plants.

Protein activities depend heavily on protein complex formation and dynamic post-translational modifications, such as phosphorylation. The dynamic nature of protein complex formation and post-translational modifications is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to assay protein-protein interactions (PPIs) (SPPIER - Separation of Phases-based Protein Interaction Reporter) and kinase activities (SPARK - Separation of Phases-based Activity Reporter of Kinase) in planta, based on phase separation. This technology enabled easy detection of inducible, binary and ternary protein-protein interactions among cytoplasmic and nuclear proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SNF1-related kinase 1 (SnRK1) activity, allowing us to visualize tissue-specific, dynamic SnRK1 activity in stable transgenic Arabidopsis (Arabidopsis thaliana) plants. The SYMPL cloning toolbox provides a means to explore PPIs, phosphorylation, and other post-translational modifications with unprecedented ease and sensitivity.