FACS-based in planta BiFC screening

对于SPP 1212的第一轮，我们提出的研究分为两个部分。在第一部分中，我们提出了双组分信号系统（TCS）的功能性作用的探索在拟南芥-微生物相互作用。不建议为这一部分提供资金。在第二部分中，我们提出了一种新的基于双分子荧光互补（BiFC）的体内双杂交系统，用于通过荧光激活细胞分选（FACS）鉴定和表征活植物细胞中蛋白质-蛋白质相互作用。基于体内BiFC的相互作用筛选的建立比预期更具挑战性。不过，我们已经设法制定了一项工作协议。我们的方法对SPP 1212内外的大多数研究组都很感兴趣，这些研究组正在研究植物中蛋白质-蛋白质相互作用和蛋白质复合物的形成。该提案旨在进一步改进基于FACS-in planta BiFC筛选方法，并以专门的方式向SPP 1212成员提供技术和专业知识。此外，我们还提供研究支持，通过为SPP 1212社区提供除BiFC筛选外的其他应用提供专门的细胞计数和FACS专业知识，例如启动子反式激活，细胞和细胞核分选“组学”工作（例如转录组学，蛋白质组学，ChIP芯片）和监测细胞周期（例如DNA含量）。

For the first round of the SPP1212, our proposed research was divided into two parts. In the first part we proposed the exploration of the functional role of two-component signalling systems (TCS) in Arabidopsis-microbe interactions. This part was not recommended for funding. In the second part we had proposed to establish a novel Bimolecular Fluorescence Complementation (BiFC)-based in vivo two-hybrid system for the identification and characterisation of protein-protein interactions in living plant cells by using fluorescence activated cell sorting (FACS). The establishment of the in vivo BiFC based interaction screen was more challenging than expected. However we have managed to develop a working protocol. Our approach is of great interest to most groups in and also outside of the SPP1212 that are working on in planta protein-protein interaction and protein complex formation. This proposal is targeted towards further improving the FACS-based in planta BiFC screening approach and offering the technology and expertise to the members of the SPP1212 in a dedicated fashion. Furthermore, we also offer research support, by providing dedicated cytometric and FACS expertise for other applications besides the BiFC screen to the SPP1212 community such as promoter transactivation, sorting of cells and nuclei for “-omics” work (e.g. transcriptomics, proteomics, ChIP-chip) and monitoring cell cycle (e.g. DNA content).