Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium.
Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium.
Plant Cell. 2023 Sep 1;35(9):3280-3302. doi: 10.1093/plcell/koad188.
Protein activities depend heavily on protein complex formation and dynamic posttranslational modifications, such as phosphorylation. The dynamic nature of protein complex formation and posttranslational modifications is notoriously difficult to monitor in planta at cellular resolution, often requiring extensive optimization. Here, we generated and exploited the SYnthetic Multivalency in PLants (SYMPL)-vector set to assay protein-protein interactions (PPIs) (separation of phases-based protein interaction reporter) and kinase activities (separation of phases-based activity reporter of kinase) in planta, based on phase separation. This technology enabled easy detection of inducible, binary and ternary PPIs among cytoplasmic and nuclear proteins in plant cells via a robust image-based readout. Moreover, we applied the SYMPL toolbox to develop an in vivo reporter for SNF1-related kinase 1 activity, allowing us to visualize tissue-specific, dynamic SnRK1 activity in stable transgenic Arabidopsis (Arabidopsis thaliana) plants. The SYMPL cloning toolbox provides a means to explore PPIs, phosphorylation, and other posttranslational modifications with unprecedented ease and sensitivity.
蛋白质的活性在很大程度上依赖于蛋白质复合物的形成和动态的翻译后修饰，如磷酸化。蛋白质复合物形成和翻译后修饰的动态性质在植物细胞分辨率下进行监测非常困难，通常需要大量的优化。在这里，我们生成并利用了 SYnthetic Multivalency in PLants（SYMPL）载体系统，基于相分离，在植物体内测定蛋白质-蛋白质相互作用（PPIs）（基于相分离的蛋白相互作用报告）和激酶活性（基于相分离的激酶活性报告）。该技术通过强大的基于图像的读数，可轻松检测植物细胞质和核蛋白之间的诱导型、二聚体和三聚体 PPIs。此外，我们应用 SYMPL 工具盒开发了 SNF1 相关激酶 1 活性的体内报告，使我们能够在稳定的转基因拟南芥（Arabidopsis thaliana）植物中可视化组织特异性、动态的 SnRK1 活性。SYMPL 克隆工具盒为探索蛋白质相互作用、磷酸化和其他翻译后修饰提供了一种前所未有的简便和敏感的方法。
