@article{shi_kernodle_steede_lewis_2023, title={Modified physiology of burley tobacco plants genetically engineered to express Yb<sub>1</sub>, a functional EGY enzyme}, volume={258}, ISSN={["1432-2048"]}, DOI={10.1007/s00425-023-04235-8}, number={4}, journal={PLANTA}, author={Shi, Rui and Kernodle, Sheri P. and Steede, Tyler M. and Lewis, Ramsey S.}, year={2023}, month={Oct} }
 @article{burner_mccauley_pramod_frederick_steede_kernodle_lewis_2022, title={Analyses of diverse low alkaloid tobacco germplasm identify naturally occurring nucleotide variability contributing to reduced leaf nicotine accumulation}, volume={42}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-021-01274-5}, abstractNote={Recent suggestions for mandated lowering of nicotine content in cigarettes have prompted tobacco breeders to search for N. tabacum germplasm with allelic variability contributing to low alkaloid accumulation. In this research, we phenotyped a series of 81 selected diverse tobacco introductions (TIs) to identify a sub-group with authentic low alkaloid phenotypes. We also genotyped these materials for sequences associated with the Nic1 and Nic2 loci previously reported to influence tobacco alkaloid biosynthesis. Only five low alkaloid TIs possessed previously described deletions of Ethylene Response Factor (ERF) genes at the Nic2 locus that contribute to lower alkaloid accumulation. Eleven TIs possessed an apparent deletion of ERF199, a gene recently reported to underlie the effect at the Nic1 locus. Quantitative trait locus (QTL) mapping was performed using populations derived from three selected low alkaloid TIs to possibly identify new genomic regions affecting alkaloid accumulation. A major QTL was identified on linkage group 7 in all three populations that aligned with the Nic1 locus. A newly discovered 5 bp deletion in the gene MYC2a on linkage group 5 was found to likely partially underlie the ultra-low alkaloid phenotype of TI 313. This new information is useful for tobacco breeders attempting to assemble novel genetic combinations with the potential for meeting future levels of tolerance for nicotine concentration in cigarette tobacco.}, number={1}, journal={MOLECULAR BREEDING}, author={Burner, Nathaniel and McCauley, Abigail and Pramod, Sreepriya and Frederick, Jesse and Steede, Tyler and Kernodle, Sheri P. and Lewis, Ramsey S.}, year={2022}, month={Jan} }
 @article{kernodle_webb_steede_lewis_2022, title={Combined reduced expression of two gene families lowers nicotine content to ultra-low levels in cultivated tobacco}, ISSN={["1432-203X"]}, DOI={10.1007/s00299-022-02895-6}, abstractNote={Reduced expression of two gene families results in ultra-low nicotine accumulation in Nicotiana tabacum. The potential for mandated lowering of tobacco cigarette filler nicotine levels to below 0.4 mg g -1  is currently being discussed by regulatory and public health organizations. Commercial tobacco cultivars that would routinely meet this proposed standard do not currently exist. Inactivation or silencing of gene families corresponding to single enzymatic steps in the nicotine biosynthetic pathways have not resulted in tobacco genotypes that would meet this standard under conventional agronomic management. Here, we produced and evaluated under field conditions tobacco genotypes expressing an RNAi construct designed to reduce expression of the Methyl Putrescine Oxidase (MPO) gene family associated with nicotine biosynthesis. In a standard flue-cured genetic background, cured leaf nicotine levels were reduced to only 1.08 to 1.65 mg g -1 . When MPO RNAi was combined with reduced Berberine Bridge Like (BBL) activity conferred by induced mutations, genotypes producing cured leaf nicotine levels slightly lower than 0.4 mg g -1  were generated. Past research has suggested that MPO activity may contribute to the biosynthesis of nornicotine in a route that does not involve nicotine. However, nornicotine was not reduced to zero in MPO-silenced plants that were also homozygous for induced mutations in known Nicotine Demethylase genes that are responsible for the vast majority of nornicotine accumulation.}, journal={PLANT CELL REPORTS}, author={Kernodle, Sheri P. and Webb, Sydney and Steede, Tyler M. and Lewis, Ramsey S.}, year={2022}, month={Jul} }
 @article{burner_kernodle_steede_lewis_2022, title={Editing of A622 genes results in ultra-low nicotine whole tobacco plants at the expense of dramatically reduced growth and development}, volume={42}, ISSN={["1572-9788"]}, DOI={10.1007/s11032-022-01293-w}, abstractNote={Due to potential regulations that could affect nicotine levels in some tobacco products, there is interest in using genetic modification to reduce levels of this pyridine alkaloid in tobacco leaves. Enzymes coded by A622 genes have previously been indicated to be involved in one of the latter steps of tobacco alkaloid biosynthesis. Whole tobacco plants with reduced A622 activity have never been evaluated, however. We utilized CRISPR/Cas9–based editing to introduce deleterious mutations into the two A622 genes present in the Nicotiana tabacum genome. Double homozygous A622 mutant genotypes established in four recipient genotypes varying for the presence/absence of mutations in other alkaloid biosynthetic genes exhibited severely reduced nicotine accumulation in field and greenhouse experiments. A622 knockout lines exhibited lower nicotine levels than previously created genotypes with deleterious mutations in BBL genes also associated with one of the latter steps in tobacco alkaloid biosynthesis. Reduced A622 activity resulted in plants with drastically reduced growth and development, however. A622 mutant lines were later flowering and produced green leaf yields that were 60.6% lower, on average, than those for non-A622-mutated control lines.}, number={4}, journal={MOLECULAR BREEDING}, author={Burner, Nathaniel and Kernodle, Sheri P. and Steede, Tyler and Lewis, Ramsey S.}, year={2022}, month={Apr} }
 @article{ma_hancock_nifong_kernodle_lewis_2020, title={Identification and editing of a hybrid lethality gene expands the range of interspecific hybridization potential in Nicotiana}, volume={133}, ISSN={["1432-2242"]}, DOI={10.1007/s00122-020-03641-w}, abstractNote={Identification and inactivation of hybrid lethality genes can be used to expand the available gene pool for improvement of a cultivated crop species. Hybrid lethality is one genetic mechanism that contributes to reproductive isolation in plants and serves as a barrier to use of diverse germplasm for improvement of cultivated species. A classic example is the seedling lethality exhibited by progeny from the Nicotiana tabacum × N. africana interspecific cross. In order to increase the body of knowledge on mechanisms of hybrid lethality in plants, and to potentially develop tools to circumvent them, we utilized a transposon tagging strategy to identify a candidate gene involved in the control of this reaction. N. tabacum gene Nt6549g30 was identified to code for a class of coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) proteins, the largest class of plant defense proteins. Gene editing, along with other experiments, was used to verify that Nt6549g30 is the gene at the N. tabacum Hybrid Lethality 1 (NtHL1) locus controlling the hybrid lethality reaction in crosses with N. africana. Gene editing of Nt6549g30 was also used to reverse interspecific seedling lethality in crosses between N. tabacum and eight of nine additional tested species from section Suaveolentes. Results further implicate the role of disease resistance-like genes in the evolution of plant species and demonstrate the possibility of expanding the gene pool for a crop species through gene editing.}, number={10}, journal={THEORETICAL AND APPLIED GENETICS}, author={Ma, Justin and Hancock, Wesley G. and Nifong, Jessica M. and Kernodle, Sheri P. and Lewis, Ramsey S.}, year={2020}, month={Oct}, pages={2915–2925} }