Fluorescence complementation enables quantitative imaging of cell penetrating peptide-mediated protein delivery in plants including WUSCHEL transcription factor

Abstract

Protein delivery to plants offers many opportunities for plant bioengineering via gene editing and through direction of protein-protein interactions. However, the delivery of proteins to plants presents both practical and analytical challenges. We present a GFP bimolecular fluorescence complementation-based tool, delivered complementation in planta (DCIP), which allows for unambiguous and quantitative measurement of protein delivery in leaves. Using DCIP, we demonstrate cell-penetrating peptide mediated cytosolic delivery of peptides and recombinant proteins in Nicotiana benthamiana. We show that DCIP enables quantitative measurement of delivery efficiency and enables functional screening of cell penetrating peptide sequences. We also use DCIP to evidence an endocytosis independent mechanism of nona-arginine cell penetrating peptide delivery. In addition to the importance cell penetrating peptide sequence, we show that cargo stability may play an important role in delivery effectiveness. Finally, we demonstrate that DCIP detects cell penetrating peptide mediated delivery of recombinantly expressed proteins into intact leaves. As a proof of concept, we also show that ectopic protein-protein interactions can be formed using delivered recombinant proteins. By using a cell penetrating peptide to deliver the actin binding peptide, Lifeact, fused to GFP11, we enable fluorescence complementation-based scaffolding of a GFP1-10 fusion protein to endogenous f-actin in plant leaves. DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and outlines new techniques for engineering plant biology.