Nanoparticles As Biomolecular Cargo Transporters in Plants and Plastids

The field of targeted plant genome editing has experienced exciting progress since the production of the first transgenic plants. Recent advances in targeted genome engineering– development of zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR/Cas systems– allow modification of DNA sequences of interest, once inside living cells.¹ To introduce genome engineering tools into plant cells and plastids, several methods have been established, such as Agrobacterium or polyethylene glycol (PEG) mediated gene delivery, electroporation, biolistic particle gun, and microinjection. However, current methods are limited by host range limitations, low transfection efficiency, toxicity, and in most cases, transfection limited to protoplast cultures where the cell wall is removed, and not to the mature whole plant.² We explore the use of high-aspect ratio nanomaterials for efficient through-cell-wall gene transfer into all desired genotypes of any plant species.

Nanomaterials, with distinct and tunable physical properties (size, shape, and surface charge), effectively deliver biological cargoes to traditionally hard-to-access intracellular locations, such as walled plant cells. We have previously shown that certain nanomaterial formulations can penetrate the mature plant cell, and also the chloroplast.^3,4 Here, we present recent results on the passive delivery of nanoparticles, loaded with plasmid DNA, into walled plant cells and protoplasts. In particular, we examine the delivery mediated by single-walled carbon nanotubes (SWCNT) functionalized with GFP encoding plasmids, and the resulting GFP expression in the transfected cells of mature arugula plants. Our work provides a promising tool for generic, targeted, and passive transport of biomolecules (genes, proteins, etc.) into mature plant cells for targeted genome editing and plastid engineering.

References

1. Li, J.-F. et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature biotechnology 31, 688-691 (2013).

2. Potrykus, I. Gene Transfer to Plants: Assessment of Published Approaches and Results. Annual Review of Plant Physiology and Plant Molecular Biology 42, 205-225, doi:10.1146/annurev.pp.42.060191.001225 (1991).

3. Giraldo, J. P. et al. Plant nanobionics approach to augment photosynthesis and biochemical sensing. Nat Mater 13, 400-408, doi:10.1038/nmat3890

4. Wong, M. H. et al. Lipid Exchange Envelope Penetration (LEEP) of Nanoparticles for Plant Engineering: A Universal Localization Mechanism. Nano Letters 16, 1161-1172, doi:10.1021/acs.nanolett.5b04467 (2016).