Department of Biochemistry and Molecular Biology/Bioagro, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
National Institute of Science and Technology in Plant-Pest Interactions, Viçosa, MG, Brazil.
Methods Mol Biol. 2024;2724:211-223. doi: 10.1007/978-1-0716-3485-1_15.
Bimolecular fluorescence complementation (BiFC) is an assay widely used for studying protein-protein interactions and determining the subcellular localization of proteins. This technique involves fusing the proteins of interest to separate structural domains of a fluorescent protein, followed by transient expression in cells. The interaction between the proteins of interest in vivo allows the reconstitution of the fluorescence that can be visualized by fluorescence microscopy. BiFC has been particularly useful in investigating the interactions between viral and host proteins. Here, we describe the steps involved in preparing expression cassettes that allow the expression of proteins of interest fused to nonfluorescent fragments of yellow fluorescent protein (YFP), Agrobacterium transformations, and agroinfiltration of Nicotiana benthamiana leaves to facilitate virus protein-host protein interactions. Finally, high-resolution images can be obtained by analyzing the leaves under a confocal microscope.
双分子荧光互补（BiFC）是一种广泛用于研究蛋白质-蛋白质相互作用和确定蛋白质亚细胞定位的检测方法。该技术涉及将感兴趣的蛋白质融合到荧光蛋白的分离结构域中，然后在细胞中转瞬表达。感兴趣的蛋白质在体内的相互作用允许再构成荧光，通过荧光显微镜可以观察到荧光。BiFC 在研究病毒和宿主蛋白之间的相互作用方面特别有用。在这里，我们描述了制备表达载体的步骤，该载体允许表达与黄色荧光蛋白（YFP）的非荧光片段融合的感兴趣的蛋白质，农杆菌转化以及烟草原生质体的侵染，以促进病毒蛋白-宿主蛋白相互作用。最后，可以通过在共聚焦显微镜下分析叶片来获得高分辨率的图像。
