Split-HaloTag^®Imaging Assay for Sophisticated Microscopy of Protein-Protein Interactionsin planta

Abstract

<jats:title>Abstract</jats:title><jats:p>An ever-increasing number of protein complexes participating in metabolic pathways and of multi-protein intracellular networks is identified in plant cells. Split-GFP based protein-protein interaction assays combine the advantages of <jats:italic>in vivo</jats:italic> interaction studies in native environment with additional visualisation of protein complex localisation. However, fluorescence proteins have several drawbacks for 3D- imaging and super-resolution microscopy: high photobleaching rate during long-term observations, correlation of fluorescence intensity to expression level, blinking behaviour and the tendency to form oligomers. The HaloTag<jats:sup>®</jats:sup> system was shown to overcome these drawbacks. This reporter is able to form covalent irreversible bonds with synthetic chloralkane ligands. Several stable fluorescence ligands are available like TMR or Oregon Green, which can be used in variable concentrations for optimal fluorescence intensity. Therefore, we established the new Split-HaloTag<jats:sup>®</jats:sup> imaging assay to enable advanced fluorescence imaging such as single molecule-, subdiffractional polarisation imaging or super-resolution microscopy of protein-protein interactions. This is demonstrated via studies of molybdenum cofactor biosynthesis complex anchoring to filamentous actin. Specific interactions were visualised with 3D- and advanced subdiffractional polarisation microscopy in distinctive manner. Therefore, this assay is a promising new tool for 3D-imaging and super-resolution microscopy of protein-protein interactions in plant cells.</jats:p><jats:sec><jats:title>Highlights</jats:title><jats:p>The Split-HaloTag<jats:sup>®</jats:sup> assay combine for the first time <jats:italic>in planta</jats:italic> a flexible and photostable fluorescence system with a split-reporter system, which enables protein complex analyses with advanced microscope technologies.</jats:p></jats:sec>

Imaging plant cells and organs with light-sheet and super-resolution microscopy

“…CLIP-, HALO-, and SNAP-genetically encoded tags reduced the size of the labeling compared with standard fluorescent proteins and allowed labeling with cell-permeable fluorophores suitable for single-molecule localization ( Hoelzel and Zhang, 2020 ). Such tags have recently found their way in plant cell imaging applications ( Iwatate et al, 2020 ), while a split variant of the HALO-tag was recently developed for the detection of protein–protein interactions in plants ( Minner-Meinen et al, 2021 ). In the same direction, far-red emitting silicon-rhodamine (SiR) affinity probes with suitability for single-molecule localization studies have been developed for several subcellular structures ( Wang et al, 2019 ), and it remains to see if they are cell wall permeable and applicable to plant tissues (see Outstanding Questions).…”