(27n) Fluorescence Complementation Enables Quantitative Imaging of Transcription Factor Delivery in Plants

Protein delivery to plants offers many opportunities for bioengineering via gene editing, transcriptional activation or repression, and direction of protein-protein interactions. However, plants possess unique biological barriers which are difficult to overcome with traditional protein delivery approaches. Furthermore, beyond the challenge of delivery itself, evaluating protein delivery success in plants presents both practical and analytical challenges. Classical approaches to evaluate the success of delivery strategies are often uninterpretable or ambiguous due to the resolution limitations of microscopy and the anatomical structure of plant cells. Here, we report a bimolecular fluorescence complementation-based tool, delivered complementation in planta (DCIP), which allows for unambiguous evaluation of protein delivery success and quantitative measurement of delivery efficiency in plant cells. First, using DCIP, we demonstrate cell-penetrating peptides can mediate cytosolic delivery of recombinant peptides in Nicotiana benthamiana. We utilize DCIP to screen a variety of cell penetrating peptide sequences for delivery efficiency and show that nona-arginine (R9) enables protein delivery with ~80% delivery efficiency. Next, we demonstrate DCIP confirms successful delivery of recombinant proteins such as mCherry and Lifeact to intact leaves via cell penetrating peptides. Finally, with DCIP we report the first instance of successful delivery of a plant transcription factor, WUSCHEL (AtWUS), to N. benthamiana. Transcriptional analysis via RT-qPCR suggests delivered AtWUS results in differential expression of WUSCHEL target genes in Arabidopsis thaliana. Taken together, DCIP is a novel, powerful tool for investigating delivery of proteins in plants and opens new avenues for genetic engineering and manipulation of plant physiology.