Janik Katrin, Stellmach Hagen, Mittelberger Cecilia, Hause Bettina
Functional Genomics, Laimburg Research Centre, Auer/Ora (BZ), Italy.
Department of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, Halle, Germany.
Methods Mol Biol. 2019;1875:321-331. doi: 10.1007/978-1-4939-8837-2_24.
Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves and how to transform these protoplasts with BiFC expression vectors containing the phytoplasma effector and the host interaction partner, respectively. If an interaction occurs, a fluorescent YFP-complex is reconstituted in the protoplast, which can be visualized using fluorescence microscopy.
近年来，阐明植物病害发生的分子机制已成为植原体研究的一个重要方面。特别是揭示植原体效应蛋白的功能，已在分子水平上对植原体与宿主的相互作用有了有趣的见解。在这里，我们描述了如何在本氏烟草叶肉原生质体中使用双分子荧光互补（BiFC）分析和可视化植原体效应蛋白与其蛋白质宿主伴侣的相互作用。该方案包括如何从叶片中分离原生质体以及如何用分别含有植原体效应蛋白和宿主相互作用伴侣的BiFC表达载体转化这些原生质体的描述。如果发生相互作用，荧光YFP复合物将在原生质体中重新组装，可通过荧光显微镜观察到。
