Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 71, 9052 Ghent, Belgium.
VIB Center for Plant Systems Biology, Technologiepark 71, 9052 Ghent, Belgium.
Plant Cell. 2021 May 31;33(4):1101-1117. 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.
鉴定蛋白质-蛋白质相互作用（PPIs）对于理解生物过程至关重要。有许多 PPI 工具可用，但只有一些在植物细胞的背景下发挥作用。更进一步，只有少数工具可以可视化复杂的多蛋白相互作用。在这里，我们提出了一种满足这些标准的植物研究条件性体内 PPI 工具。植物中的 Knocksideways（KSP）基于雷帕霉素通过 FKBP 和 FRB 结构域的异二聚化改变诱饵蛋白及其相互作用物定位的能力。KSP 固有地避免了其他 PPI 系统的许多限制。这种体内工具不需要诱饵和猎物荧光团的空间接近性，并且与广泛的荧光团兼容。KSP 也是一种条件性工具，因此在没有雷帕霉素的情况下观察蛋白质的可视化可以作为内部对照。我们使用 KSP 来确认在 Nicotiana benthamiana 叶表皮细胞中先前鉴定的相互作用。此外，我们生成的脚本允许以高通量定量这些相互作用。最后，我们证明 KSP 可以轻松用于可视化复杂的多蛋白相互作用。因此，KSP 是一种具有独特特点和应用的多功能工具，可补充其他植物 PPI 方法。
