Visualization of in vivo protein–protein interactions in plants

Abstract

Molecular processes depend on the concerted and dynamic interactions of proteins, either by one-on-one interactions of the same or different proteins or by the assembly of larger protein complexes consisting of many different proteins. Here, not only the protein-protein interaction (PPI) itself, but also the localization and activity of the protein of interest (POI) within the cell is essential. Therefore, in all cell biological experiments, preserving the spatio-temporal state of one POI relative to another is key to understand the underlying complex and dynamic regulatory mechanisms in vivo. In this review we examine some of the applicable techniques to measure PPI in planta as well as recent combinatorial advances of PPI methods to measure the formation of higher order complexes with an emphasis on in vivo imaging techniques. We compare the different methods and discuss their benefits and potential pitfalls to facilitate the selection of appropriate techniques by giving a comprehensive overview about how to measure in vivo PPI in plants.

The Ti‐TAN plasmid toolbox for TurboID‐based proximity labeling assays in Nicotiana benthamiana

“…Several methods to experimentally detect protein–protein interactions in planta have been developed in the past decades; some of them, like affinity purification followed by mass spectrometry (AP‐MS), can be implemented to identify proteins associated with a protein of interest, while others, like bimolecular fluorescence complementation (BiFC), split‐luciferase, co‐immunoprecipitation, or Förster resonance energy transfer by fluorescence lifetime imaging (FRET‐FLIM) assays, are used to test the interaction between two proteins of choice (Strotmann and Stahl, 2022). These experiments can be carried out in a fast, simple, and inexpensive manner in leaves of the model plant Nicotiana benthamiana , which is amenable to Agrobacterium tumefaciens ‐mediated transient transformation, an approach that results in high and robust accumulation of the desired protein(s).…”

One Pattern Analysis (OPA) for the quantitative determination of protein interactions in plant cells

“…Compared to BiFC, FRET assays have the advantage of having higher spatial resolution and, more importantly, are more specific, as complementing FP fragments show a tendency for self-assembly resulting in false positive interactions (Horstman et al 2014;Shyu and Hu 2008). FLIM in contrast to intensity-or spectral-based FRET methods, is more gentle on cells and tissues due to lower required laser intensity and is generally considered the more accurate method to detect FRET (Strotmann and Stahl 2022;Long et al 2017;Long et al 2018;Stahl et al 2013;Russinova et al 2004;Glockner et al 2022).…”

“…(iii) Donor and acceptor molecules must be in close proximity to each other (< 10 nm or 100 Å distance). FRET is considered the more accurate method and less susceptible to false positive interactions when compared to BiFC (Strotmann and Stahl 2022;Horstman et al 2014;Bucherl et al 2014). Commonly, FRET is measured either by fluorescence intensity-based techniques such as FRET-Acceptor Photo Bleaching (APB) and recording of sensitized emission, or by the analysis of the fluorescence lifetime of donor fluorescence using FLIM.…”

“…Commonly, FRET is measured either by fluorescence intensity-based techniques such as FRET-Acceptor Photo Bleaching (APB) and recording of sensitized emission, or by the analysis of the fluorescence lifetime of donor fluorescence using FLIM. Intensity-based FRET usually requires strict controls and correction for spectral bleed-through, whereas lifetime acquisition by FLIM is more robust (Strotmann and Stahl 2022;Godet and Mely 2019;Bucherl et al 2014;Spatola Rossi et al 2022;Piston and Kremers 2007). Additionally, FLIM-FRET is independent from local Donor and Acceptor concentrations and requires only relatively low irradiation of cells, and is thus considered to be superior compared to intensity-based techniques (Long et al 2017;Glockner et al 2022;Long et al 2018;Stahl et al 2013;Russinova et al 2004;Denay et al 2019).…”

Protein-protein interactions in plant antioxidant defense

“…However, classical FRET analysis suffers from the presence of high background caused by spectral bleed-through and autofluorescence ( Xing et al., 2016 ). Therefore, several FRET modifications suitable for in vivo studies of PPIs have been developed, such as FRET-FLIM (fluorescence lifetime imaging microscopy), FRET-APB (acceptor photobleaching), Triple-FRET, Homo-FRET and others ( Xing et al., 2016 ; Cui et al., 2019 ; Strotmann and Stahl, 2022 ).…”