Visualizing protein-protein interactions in plants by rapamycin-dependent delocalization

Abstract

One-sentence summary: rapamycin-dependent delocalization allows quantifying proteinprotein interactions inside plant cells. Author contributions: DVD initiated the project and designed experiments. JW, EM, ADM and PG designed and performed experiments. BP wrote the script for quantification of the data. JM performed experiments. JW, PG and DVD wrote the paper. AbstractIdentifying protein-protein interactions (PPI) is crucial to understand any type of biological process. Many PPI tools are available, yet only some function within the context of a plant cell.Narrowing down even further, only few PPI tools allow visualizing higher order interactions.Here, we present a novel and conditional in vivo PPI tool for plant research. Knocksideways in plants (KSP) uses the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations, which other PPI systems hold. It is an in vivo tool, it is flexible concerning the orientation of protein tagging as long as this does not interfere with the interaction and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore does not require additional controls. The interactions can be quantified and in high throughput by the scripts that we provide. Finally, we demonstrate that KSP can visualize higher-order interactions. It is therefore a versatile tool, complementing the PPI methods field with unique characteristics and applications.

The TPLATE subunit is essential for structural assembly of the endocytic TSET complex

“…To address the TML-TPLATE interaction, we utilized the recently developed knocksideway assay in plants (KSP) (Winkler et al, 2020). KSP uses the ability of rapamycin to change the localization of a bait protein and its interacting partner via hetero-dimerization of the FK506-binding protein (FKBP) and the rapamycin-binding domain of mTOR (FRB).…”

“…Using KSP, it was previously shown that full-length TPLATE can re-localize together with full-length TML. Furthermore, this tool allowed visualization of the ternary interaction between LOLITA, TASH3 and TPLATE ( 21 ). To this end, we transiently co-expressed various full-length and domain constructs of TML and TPLATE fused either to FKBP-mCherry or GFP, together with mitochondria-targeted FRB.…”

“…Expression constructs used for KSP were cloned by combining a 35S promoter, entry vectors used in this study as well as FKBP and FRB entry vectors ( 21 ) from full length pDONR plasmids. Expression constructs were verified by restriction digest.…”

“…Leaves were imaged in a 20-45min time window. Autophagosomal recruitment as well as KSP images were analysed based on the signal in mitochondria versus the cytoplasm ( 21 ).…”

“…48 hours after infiltration, N. benthamiana leaves were infiltrated with 1 μM rapamycin (Sigma-Aldrich) and imaged in a window between 20-45min. Autophagosomal as well as KSP images were analysed based on the signal in mitochondria versus the cytoplasm (Winkler et al, 2020).…”