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

Protein delivery to plants offers many opportunities for plant bioengineering via gene editing and through direction of protein-protein interactions. However, the delivery and confirmation of successful protein delivery to plants presents both practical and analytical challenges. We present a GFP bimolecular fluorescence complementation-based tool, delivered complementationin 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 inNicotiana benthamiana. We show that DCIP enables measurement of delivery efficiency and enables functional screening of cell penetrating peptide efficacies for in-plant protein delivery. Finally, we demonstrate that DCIP detects cell penetrating peptide mediated delivery of recombinantly expressed proteins such as mCherry and Lifeact into intact leaves. Finally, we also demonstrate, for the first time, delivery of a recombinant plant transcription factor, WUSCHEL (AtWUS), inN. benthamiana. RT-qPCR analysis of AtWUS delivery inArabidopsisseedlings also suggests delivered WUS can recapitulate AtWUS-overexpression transcriptional changes. All combined, DCIP offers a new and powerful tool for interrogating cytosolic delivery of proteins in plants and highlights future avenues for engineering plant physiology.