Author ORCID Identifier

https://orcid.org/0000-0002-2445-8927

Date Available

12-20-2020

Year of Publication

2019

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Plant and Soil Sciences

First Advisor

Dr. Jason M. Unrine

Abstract

RNA interference has enormous potential as a potent, specific, and environmentally friendly alternative to small molecule pesticides for crop protection. The use of exogenous double-stranded RNA offers flexibility in targeting and use in crops in which transgenic manipulation is not an option. The combination of RNAi with nanotechnology offers further advantages that are not available with dsRNA alone. In this work, I have evaluated several different combinations of nanomaterials and polymers for use in RNAi-based pest control systems. First, I have characterized the use of chitosan/dsRNA polyplex nanoparticles for gene knockdown using the model nematode Caenorhabditis elegans. Though chitosan/dsRNA polyplexes are equally as effective as naked dsRNA for gene knockdown on a concentration basis, these materials are assimilated into cells in a manner independent of dsRNA specific transport proteins. The mechanism of uptake is likely clathrin-mediated endocytosis. In addition, I identify a significant and yet unreported side-effect associated with chitosan exposure, the dysregulation of a major myosin isoform. Next, I have determined the efficacy of chitosan/dsRNA polyplex nanoparticles under different environmental conditions. The presence of inorganic ions (phosphate and nitrate) at realistic environmental concentrations does not alter the efficacy of the nanoparticles for gene knockdown, nor do they inhibit knockdown by naked dsRNA. These conditions did not cause any significant changes to the hydrodynamic diameter or zeta potential of the particles themselves between treatments. By contrast, a pH higher than six and the presence of natural organic matter significantly reduce the efficacy of the nanomaterials at gene knockdown but leave knockdown by naked dsRNA unaffected. Though some changes in polyplex size are observed in the pH treatments, these changes are comparatively small, and particles remain well within the size that can be ingested by C. elegans. At pH 8, the charge of the particles is effectively neutral. Similarly, concentrations of natural organic matter >2.5 mg/L cause a charge reversal of the particles, from strongly cationic to strongly anionic. Large aggregates are also visible in each of these treatments. Lastly, I characterize the efficacy of a suite of different polymer and solid core nanomaterials for dsRNA delivery, similar to the above. Poly-L-lysine, poly-L-arginine, Ge-doped imogolite, and poly-L-arginine-citrate coated Au nanoparticles all fail to cause any appreciable knockdown in the same C. elegans reporter system. Uptake of the polymers was exceedingly poor, and though the Au particles appear to have been ingested, there is no evidence of significant gene knockdown. Furthermore, poly-L-arginine caused significant injury to the mouthparts of C. elegans exposed to these materials. Layered double-hydroxide nanoparticles were successful at gene knockdown, and appear to function slightly better than naked dsRNA alone, and were translocated in C. elegans in a similar fashion to naked dsRNA. Taken together, these findings aid in the development of safe and effective RNAi biological control agents.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2019.469

Funding Information

This material is based upon work supported by the National Science Foundation under Grant No. CBET-1712323 and Cooperative Agreement EF-0830093. Granted 2017.

Portions of the work were also supported by the U.S.-Israel Agricultural Research and Development Fund through grant number IS-4964-16 R. Granted 2019.

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