Ralph Dewey

The formation of undesirable tobacco-specific nitrosamines (TSNAs) occurs during the curing, storage and processing of the tobacco leaf and requires two distinct components, an alkaloid substrate and a nitrosating agent. The prevailing consensus for the air-cured burley tobaccos is that the reduction of free nitrate pools in the leaf by surface microbes accounts for the formation of the highly reactive nitrite molecules that are believed to interact with plant alkaloids to form TSNAs. Our recent results demonstrated that it is possible to alter the nitrogen assimilation pathway in a manner that greatly decreases both nitrate levels and TSNAs. The experiments outlined in this proposal build on these results and will further define the optimal means by which the nitrate and TSNA contents of the cured leaf can be reduced.

There are several circumstances where it would be desirable to utilize low nicotine tobacco varieties that are of higher quality that those that are currently available. Although it has been shown that the down-regulation of various steps in the alkaloid biosynthetic pathway can result in reduced nicotine accumulation, most of these perturbations are accompanied by undesirable side-effects, such as reduced growth, or the concomitant increase in the levels of the typically minor alkaloid anatabine. In tobacco, enzymes referred to as berberine bridge enzyme-like (BBL) proteins catalyze one of last steps of alkaloid biosynthesis. We believe this step of the alkaloid pathway is a particularly attractive target for the development of low alkaloid tobacco lines. We have previously identified knockout mutations in the three BBL genes that are the most highly expressed in the tobacco genome. The pyramiding of these three BBL mutations resulted in plants with greatly reduced alkaloid content. Nevertheless, a low level of alkaloid remained. To reduce the levels of nicotine and other alkaloids even further, we propose to use custom-designed nucleases to knock out the activity of the remaining ����������������minor��������������� BBL genes.

Hybrid seed production systems are critical for maximizing yield in crop species that show a high degree of hybrid vigor. The most widely used hybrid seed systems exploit a naturally occurring trait known as cytoplasmic male sterility (CMS) to make directional crosses between two inbred lines that display high heterosis when combined. Examples of crops that have robust CMS-based systems used by the seed industry to produce hybrid seed include: sorghum, rice, sunflower and sugar beets. There are numerous additional crops species where hybrid vigor could be more efficiently exploited (and thereby yield increased) if an efficacious CMS-based hybrid seed production were to exist. AgBiotech companies have invested hundreds of millions of dollars in developing biotechnology-based systems to produce hybrid seed, yet few of these systems have been deployed commercially and their impact on the industry is currently minimal. None of the biotech-based systems of hybrid seed production developed to date have the simplicity of use, and minimization of costs as the standard CMS-based systems. The key to the success of these systems is the strict maternal inheritance of the CMS trait owing to the fact that where they exist, CMS genes are encoded by the mitochondrial genome. Using tobacco as a model system, we have tested whether current genome editing technologies could be strategically deployed to generate novel sources of CMS, along with all other components needed for producing hybrid seeds. These results were very positive, not only in demonstrating the feasibility of our original goal of creating new CMS/hybrid seeds systems, but also through unexpectedly pointing us to a novel means for producing seedless fruits, a trait enjoyed by consumers in many commodities (e.g. watermelons, common grapes, bananas and oranges), but lacking in others such as blackberries, muscadine grapes and pomegranates. This project will further advance the potential applications of mitochondrial genome editing by: (1) optimizating critical elements of the CMS/hybrid seed technology (still using tobacco as the model); and (2) the introduction of our seedless fruit technology into tomato as a proof-of-concept within a true fruit species.

Adjuvants have a key role in promotion of vaccine efficacy through eliciting a stronger immune response and reducing the volumetric need of active ingredient. Squalene as an adjuvant is a major component of many SARS-CoV-2 vaccines in development. Because squalene is made in human livers and transferred to the skin through the circulatory system, it is an ideal molecule.the development of a novel plant-based sourcing technology to provide for controlled environment cultivation/propagation of vaccine adjuvant squalene tobacco. At the time of this application, GSK������������������s AS03 squalene adjuvant for protein recombinant vaccines and Novartis������������������ MF59 influenza vaccine squalene adjuvant were in human trials for SARS-CoV-2 vaccines in the United States. In the last US virus scare, influenza A virus subtype H1N1, the US government purchased the same two squalene adjuvants in trial today for the possible scenario where H1N1 mutated and became more dangerous. Both of these products are sourced from deep-water shark livers. This carries the risk of associating persistent organic pollutants like PCB, PBDE, organochlorine pesticides, polycyclic aromatic hydrocarbons, dioxin, and heavy metals; and the possible presence of various pathogens with which sharks could be infected.Further, a movement to end shark livering is gaining ground during the current pandemic which is likely to result in its regulation in some regions. This proposal describes the development of a novel plant-based sourcing technology to provide for controlled environment cultivation/propagation of vaccine adjuvant squalene tobacco.

Squalene is a high value terpene with important applications in the cosmetic and pharmaceutical sectors. Currently, the majority of squalene is obtained through the hunting of sharks and the harvesting of their livers which are enriched in this compound. This practice is threatening shark populations, of which 198 species have already been declared as either extinct, endangered or vulnerable. The SBIR Phase II proposal by the company SynShark promises to alleviate this problem through the genetic engineering of tobacco plants as an alternative source of squalene. NCSU������������������s role in this project involves the optimization of squalene production in tobacco through: (1) combining individual squalene enhancing technologies within the same plant; (2) introducing the most promising squalene enhancing transgenes into a high yielding commercial tobacco variety; and (3) testing the efficacy of the optimized squalene producing genetic materials in the field. An additional activity involves determining the concentrations of other tobacco compounds that can be purified simultaneous with squalene extraction, as these may ultimately provide additional revenues streams and further enhance the commercial viability of the new tobacco varieties developed in this initiative. Finally, in addition to providing a novel source of squalene, and possibly other high value compounds produced within the tobacco plant, the commercial implementation of the materials produced herein promises to provide an alternative crop for farmers, particularly those who have the expertise and infrastructure for tobacco production but are facing challenges as the demand for traditional tobacco continues to decline.