(188n) Nanoparticle-Mediated Transgene Expression and Silencing in Agriculturally-Relevant Plants

Food security has been threatened with decreasing crop yields and increasing consumption in the light of population growth, climate change, increasing shortage of arable land and crop usage as raw materials. Plant biotechnology is critical to address the world’s leading challenges in meeting our growing food and energy demands, and as a tool for scalable pharmaceutical manufacturing. Classical plant breeding to obtain plants with preferred genotypes requires crossing and selection of multiple plant generations, which is a laborious and coarse method for creating plants with desirable traits¹. Over the past several decades, remarkable progress has been made in biotechnology with the improvement of genome editing and sequencing tools. Owing to these recent advancements, plant synthetic biology and bioengineering now has tremendous potential to benefit many fields.

However, of the many biological systems and organisms in which transgenic biotechnologies have been implemented, plants are vastly underrepresented. In general, the molecular and cellular biology toolkit for plants is jarringly outdated: genetic manipulation in bacterial, fungal, and mammalian systems is routine, while technologies to genetically manipulate plants (two: agrobacterial transformation and particle bombardment) have not been updated since their introduction in the mid-1980s. This is because, with current technologies, it can take months to test a genetic plant variant, and inevitably some portions of transgenic DNA are integrated into the plant host genome, the trademark of a genetically modified organism (GMO). The challenge of gene delivery to plants is attributed to a transport limitation: the presence of the multilayered and rigid plant cell wall, otherwise absent in animal cells, which poses an additional physical barrier for delivery of exogenous biomolecules to plant cells, and is one of the key reasons for the slower implementation and employment of genetic engineering tools to plants². To date, plant biotechnology lacks a method that allows passive delivery of diverse biomolecules into a broad range of plant species without the aid of external force and without causing tissue damage. We posit nanotechnology as a key driver in the creation of a transformational tool to address delivery challenges and enhance the utility of plant genetic engineering.

Herein, we develop a nanoparticle-based platform that can deliver functional biomolecules into both model and crop plants with high efficiency and no toxicity³. Specifically, carbon nanotubes (CNTs) are leveraged to deliver plasmid and linear DNA into mature arugula (dicot) and wheat (monocot) leaves, from which we obtain strong transient protein expression, with efficiencies comparable to or higher than conventional gene delivery techniques. We further show that nanotube-based gene expression is transient, suggesting no transgene integration in the plant host genome. Additionally, we achieve 95% transient gene silencing in Nicotiana benthamiana leaves through CNT-mediated delivery of small interfering RNA molecules operating within RNA interference pathway. Furthermore, we discuss the implementation of DNA origami techniques for elucidating the mechanism of nanoparticle and nanostructure transport in the plant, and resulting gene expression or silencing efficacies. This study establishes efficient transient gene expression and silencing in mature plants through passive nanoparticle-mediated delivery of functional biomolecules and can enable high-throughput genetic plant transformations for a variety of plant biotechnology applications.

1. Cunningham, F.J., Goh N.S. and Demirer, G.S. et al. (2018) Nanoparticle-mediated delivery towards advancing plant genetic engineering. Trends Biotechnol. In Press
2. Demirer, G.S., Landry, M.P. Delivering Genes to Plants. AIChE SBE (2017)
3. Demirer, G.S. et al. (2018) High Aspect Ratio Nanomaterials Enable Biomolecule Delivery and Transgene Expression or Silencing in Mature Plants. bioRxiv DOI: 10.1101/179549