Split-HaloTag® Imaging Assay for Sophisticated Microscopy of Protein-Protein Interactions in planta

Abstract An ever-increasing number of protein complexes participating in metabolic pathways and of multi-protein intracellular 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. However, fluorescence proteins entail several drawbacks for sophisticated microscopy: high photobleaching rate during long-term observations, correlation of fluorescence intensity to expression level or blinking behaviour. With the HaloTag® system these drawbacks can be overcome asthis reporter is able to form covalent irreversible bonds with several synthetic photostable fluorescence ligands,which can be used in variable concentrations for optimal fluorescence intensity. Therefore, we established the new Split-HaloTag® imaging assay to enable sophisticated fluorescence imaging of protein-protein interactions. This is demonstrated using an well-characterised interaction 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 advanced subdiffractional polarisation microscopy in a more distinctive manner as example for sophisticated imaging. Therefore, this assay is a promising new tool for continuative 3D-imaging, single molecule tracking and super-resolution microscopy of protein-protein interactions in plant cells.