(276g) Discovery of Plant Derived Cell-Penetrating Peptides for Crop Bioengineering and Agricultural Biotechnology

Bioengineering of plants is critical for agriculture, molecular pharming, bio-commodities, and fundamental plant biology. However, current biotechnology tools for plant bioengineering are low in throughput, applicable to a narrow set of plant species, or automatic triggers for extensive regulatory oversight.¹ In particular, biotechnology tools for directly delivering protein to mature plant cells are currently limited to particle bombardment, a low efficiency technique which causes significant tissue damage to plants. In contrast, a plethora of biotechnology tools are available to efficiently deliver protein to mammalian cells for clinical and fundamental applications. My research in this space draws inspiration from mammalian biotechnology tools, specifically cell penetrating peptides, and investigates application of these tools to plants.

Cell penetrating peptides (CPPs) were discovered over three decades ago and now serve as biotechnology tools for delivering a variety of cargoes to mammalian cells. To date, investigation of CPPs as biotechnology tools for plants is scant, in part because of the challenge of investigating biotechnology tools in plants in general. Due to fundamental anatomical differences, considerations for development, validation, and optimization of biotechnology tools in plant and mammalian cells are different.² Exploration of CPPs in plants in particular was limited by the lack of methods for unambiguously and quantitatively identifying the biological location of CPPs in planta; methods designed to track the location of CPPs in mammalian cells were not applicable to plants. To solve this challenge, we developed a fluorescence complementation platform termed DCIP for quantitatively tracking the biological location of CPPs in planta.³ The DCIP platform provided the first strong evidence that CPPs function in plant cells in a manner similar to mammalian cells and provided proof of concept for further investigation of protein delivery to plant cells using CPPs.

After demonstrating CPPs function in plants, we leveraged the DCIP platform to investigate CPP mediated delivery of protein cargoes to plant cells. During this work, a control experiment unexpectedly demonstrated certain plant proteins, specifically plant homeoproteins, internalize into plant cells without a CPP delivery tool. While initially surprising, animal homeoproteins are known to be cell penetrating to mammalian cells and in fact were the source of the second ever discovered CPP, 30 year ago. Thus we hypothesized, plant homeoproteins may similarly be cell penetrating but specifically to plants. I computationally searched tens of thousands of plant homeodomain proteins, identifying a conserved domain within plant homeoproteins with homology to animal homeoproteins. Once again leveraging the DCIP platform, I experimentally validated the ability of this conserved domain to penetrate plant cells, identifying the first plant based CPPs (pCPPs).⁴ We demonstrated these pCPPs were capable of delivering protein cargoes including transcription factors and recombinases at efficiencies an order of magnitude greater than current particle bombardment approaches.

Our discovery of pCPPs has drawn interest from key industry players in agriculture as a solution towards direct delivery of genome editing nucleases to plants to reduce the time, cost, and regulatory burden of bioengineering crop species. Generally, we anticipate direct delivery of proteins to plants could enable a variety of currently inaccessible applications of plant biotechnology, mirroring the advances enabled by direct delivery of proteins in mammalian cells.

References

1. Squire, H.J., Tomatz, S., Voke, E., González-Grandío, E., and Landry, M. (2023). The emerging role of nanotechnology in plant genetic engineering. Nat Rev Bioeng 1, 314–328. https://doi.org/10.1038/s44222-023-00037-5.
2. Squire, H.J., Tomatz, S., Wang, J.W.-T., González-Grandío, E., and Landry, M.P. (2025). Best Practices and Pitfalls in Developing Nanomaterial Delivery Tools for Plants. ACS Nano 19, 7–12. https://doi.org/10.1021/acsnano.4c12116.
3. Wang, J.W., Squire, H.J., Goh, N.S., Ni, M.N., Lien, E., Wong, C., González-Grandío, E., and Landry, M.P. (2023). Delivered complementation in planta (DCIP) enables measurement of peptide-mediated protein delivery efficiency in plants. Communications Biology 6, 840. https://doi.org/10.1038/s42003-023-05191-5.
4. Squire, H.J., Wang, J.W., and Landry, M.P. (2025). The third alpha helix of plant homeoproteins are generally cell-penetrating to plant cells. Preprint, https://doi.org/10.1101/2025.04.01.646723 https://doi.org/10.1101/2025.04.01.646723.