An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualisation of protein complex localisation. Due to its simple protocols, it has become one of the most frequently used methods. However, standard fluorescent proteins entail several drawbacks for sophisticated microscopy. With the HaloTag system, these drawbacks can be overcome as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we established the Split-HaloTag imaging assay in plants which is based on the reconstitution of a functional HaloTag protein upon protein-protein interaction and subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using well-characterised interactions as an example for protein-protein interaction at cellular structures: the molybdenum cofactor biosynthesis complex anchoring to filamentous actin. Additionally, a specific interaction was visualised with subdiffractional polarisation microscopy in a more distinctive manner as example for sophisticated imaging. Split-GFP and Split-HaloTag can complement one another as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interaction with specific microscopic techniques such as 3D-imaging, single molecule tracking and super-resolution microscopy.

中文翻译：

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复杂的植物蛋白质相互作用的显微镜​​的拆分-HaloTag成像分析

在植物细胞中已经发现越来越多的细胞内多蛋白网络。基于Split-GFP的蛋白质-蛋白质相互作用测定结合了天然环境中体内相互作用研究的优势以及蛋白质复合物定位的额外可视化。由于其简单的协议，它已成为最常用的方法之一。然而，标准的荧光蛋白对于复杂的显微镜检查具有若干缺点。使用HaloTag系统，可以克服这些缺点，因为该报告基因与合成的光稳定荧光配体形成共价不可逆键。染料的浓度可调节，适用于高级显微镜方法。因此，我们在植物中建立了Split-HaloTag成像测定，该测定基于蛋白质-蛋白质相互作用和添加的荧光配体随后的共价结合后功能性HaloTag蛋白质的重构。使用良好表征的相互作用作为细胞结构上蛋白质-蛋白质相互作用的例子，证明了它的适用性和鲁棒性：钼辅因子生物合成复合物锚定在丝状肌动蛋白上。另外，用亚衍射偏振显微镜以更独特的方式可视化特定的相互作用，例如用于复杂成像。Split-GFP和Split-HaloTag可以相互补充，因为Split-HaloTag代表了体内方法的大型工具箱中的另一种选择。因此，