Abstract: 

Genetic engineering of plants is at the core of sustainability efforts, natural product synthesis, and agricultural crop engineering. Plant bioengineering requires delivery of biomolecules for genetic or post-transcriptional manipulation of plants, however, current delivery methods either suffer from host range limitations, low transformation efficiencies, tissue regenerability issues, tissue damage, or unavoidable DNA integration into the host genome. We briefly discuss prior efforts in delivering DNA plasmids and small interfering RNAs to mature plant cells [1-7], and challenges in implementing these approaches for the delivery of proteins. While there exist numerous methods for nucleic acid delivery in plants, few options exist for protein delivery: biolistics and proptoplast-based PEG transformation, each with considerable drawbacks. Here, we report the discovery that plant homeoproteins [8], specifically the 3rd alpha helix of the homeodomain, are generally cell penetrating to walled plant cells with implications for both plant biotechnology and fundamental plant biology. We catalog plant homeoproteins and explore natural variation in the 3rd alpha helix of homeodomains derived from plant homeoproteins. We select a subset of identified 3rd alpha helix peptides for their ability to penetrate mature plant cells and demonstrate these plant derived peptides internalize to plant cells in an amino acid sequence and concentration dependent manner. Finally, we demonstrate delivery of recombinant protein cargo Cre recombinase and transcription factors utilizing these plant-derived peptides, into a broad range of plant tissues ranging from single-cell zygotes through mature plant tissues. Our work provides the foundation for tagging proteins with CPPs for their seamless and biolistic-independent delivery to walled plant cells, enabling a broad range of transgene-free plant biotechnology applications.

1. Demirer, G.S., Zhang, H., Goh, N.S., Grandio, E.G., Landry, M.P.‡, Carbon nanotube-mediated DNA delivery without transgene integration in intact plants. Nature Protocols (2019)
2. Demirer, G.S., Zhang, H., Matos, J., Goh, N., Cunningham, F.J., Sung, Y., Chang, R., Aditham, A.J., , Chio, L., Cho, M.J., Staskawicz, B., Landry, M.P.‡, High Aspect Ratio Nanomaterials Enable Delivery of Functional Genetic Material Without DNA Integration in Mature Plants. Nature Nanotechnology (2019)
3. Landry, M.P.‡, Mitter, N.‡ How nanocarriers delivering cargoes in plants can change the GMO landscape. Nature Nanotechnology (2019)
4. Demirer, G.S. ‡, Silva, T.N., Jackson, C.T., Thomas, J.B., Ehrhardt, D., Rhee, S.Y. ‡, Mortimer, J.C. ‡, Landry, M.P. ‡ Nanotechnology to advance CRISPR/Cas genetic engineering of plants. Nature Nanotechnology (2021)
5. Zhang, H., Zhang, H., Demirer, G.S., Gonzales-Grandio, E., Fan, C., Landry, M.P.‡ Engineering DNA nanostructures for siRNA delivery in plants. Nature Protocols (2020)
6. Zhang, H.*, Demirer, G.S.*, Zhang, H., Ye, T., Goh, N.S., Aditham, A.J., Cunningham, F.J., Fan, C., Landry, M.P. Low-dimensional DNA Nanostructures Coordinate Gene Silencing in Mature Plants. PNAS (2019)
7. Zhang, H.*, Goh, N.S.*, Wang, J., Demirer, G.S., Butrus, S., Park, S-J, Landry, M.P.‡ Nanoparticle Cellular Internalization is Not Required for RNA Delivery to Mature Plant Leaves. Nature Nanotechnology (2022)
8. Wang, J.W., Goh, N., Lien, E., Gonzalez Grandio, E., Landry, M.P.‡ Quantification of cell penetrating peptide mediated delivery of proteins in plant leaves. Nature Communications Biology (2023)