Visualizing protein-protein interactions in plants by rapamycin-dependent delocalization

• Published in: The Plant cell 2021-05, Vol.33 (4), p.1101-1117
• Main Authors: Winkler, Joanna, Mylle, Evelien, De Meyer, Andreas, Pavie, Benjamin, Merchie, Julie, Grones, Peter, Van Damme, Dani L
• Format: Article
• Language: English
• Subjects: Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Mitochondria - drug effects; Mitochondria - metabolism; Nicotiana - drug effects; Nicotiana - genetics; Nicotiana - metabolism; Plant Cells - drug effects; Plant Cells - metabolism; Plant Leaves - drug effects; Plant Leaves - genetics; Plant Proteins - genetics; Plant Proteins - metabolism; Protein Interaction Mapping - methods; Protein Multimerization; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Red Fluorescent Protein; Sirolimus - pharmacology; Tacrolimus Binding Proteins - genetics; Tacrolimus Binding Proteins - metabolism
• ISSN: 1040-4651; 1532-298X; 1532-298X
• DOI: 10.1093/plcell/koab004

Identifying protein-protein interactions (PPIs) is crucial for understanding biological processes. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only a few tools allow complex multi-protein interactions to be visualized. Here, we present a conditional in vivo PPI tool for plant research that meets these criteria. Knocksideways in plants (KSP) is based on the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations of other PPI systems. This in vivo tool does not require spatial proximity of the bait and prey fluorophores and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore the visualization of the proteins in the absence of rapamycin acts as an internal control. We used KSP to confirm previously identified interactions in Nicotiana benthamiana leaf epidermal cells. Furthermore, the scripts that we generated allow the interactions to be quantified at high throughput. Finally, we demonstrate that KSP can easily be used to visualize complex multi-protein interactions. KSP is therefore a versatile tool with unique characteristics and applications that complements other plant PPI methods.