@article{dewey_selote_griffin_dickey_jantz_smith_matthiadis_strable_kestell_smith_2023, title={Cytoplasmic male sterility and abortive seed traits generated through mitochondrial genome editing coupled with allotopic expression of atp1 in tobacco}, volume={14}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2023.1253640}, abstractNote={Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Dewey, Ralph E. and Selote, Devarshi and Griffin, H. Carol and Dickey, Allison N. and Jantz, Derek and Smith, J. Jeff and Matthiadis, Anna and Strable, Josh and Kestell, Caitlin and Smith, William A.}, year={2023}, month={Sep} }
 @article{smith_matsuba_dewey_2022, title={Knockout of a key gene of the nicotine biosynthetic pathway severely affects tobacco growth under field, but not greenhouse conditions}, volume={15}, ISSN={["1756-0500"]}, DOI={10.1186/s13104-022-06188-9}, abstractNote={Abstract}, number={1}, journal={BMC RESEARCH NOTES}, author={Smith, William A. and Matsuba, Yuki and Dewey, Ralph E.}, year={2022}, month={Sep} }
 @article{lewis_drake-stowe_heim_steede_smith_dewey_2020, title={Genetic and Agronomic Analysis of Tobacco Genotypes Exhibiting Reduced Nicotine Accumulation Due to Induced Mutations in Berberine Bridge Like (BBL) Genes}, volume={11}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2020.00368}, abstractNote={Genetic methodologies for reducing nicotine accumulation in the tobacco plant (Nicotiana tabacum L.) are of interest because of potential future regulations that could mandate lowering of this alkaloid in conventional cigarettes. Inactivation of tobacco genes such as the Berberine Bridge Like (BBL) gene family believed to encode for enzymes involved in one of the latter steps of nicotine biosynthesis could be a viable strategy for producing new tobacco cultivars with ultra-low leaf nicotine accumulation. We introduced deleterious mutations generated via ethyl methanesulfonate treatment of seed or gene editing into six known members of the BBL gene family and assembled them in different combinations to assess their relative contribution to nicotine accumulation. Significant reductions (up to 17-fold) in percent leaf nicotine were observed in genotypes homozygous for combined mutations in BBL-a, BBL-b, and BBL-c. The addition of mutations in BBL-d1, BBL-d2, and BBL-e had no additional significant effect on lowering of nicotine levels in the genetic background studied. Reduced nicotine levels were associated with reductions in cured leaf yields (up to 29%) and cured leaf quality (up to 15%), evidence of physiological complexities within the tobacco plant related to the nicotine biosynthetic pathway. Further nicotine reductions were observed for a BBL mutant line cultivated under a modified production regime in which apical inflorescences were not removed, but at the expense of further yield reductions. Plants in which BBL mutations were combined with naturally occurring recessive alleles at the Nic1 and Nic2 loci exhibited further reductions in percent nicotine, but no plant produced immeasurable levels of this alkaloid. Findings may suggest the existence of a minor, alternative pathway for nicotine biosynthesis in N. tabacum. The described genetic materials may be of value for the manufacture of cigarettes with reduced nicotine levels and for future studies to better understand the molecular biology of alkaloid accumulation in tobacco.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Lewis, Ramsey S. and Drake-Stowe, Katherine E. and Heim, Crystal and Steede, Tyler and Smith, William and Dewey, Ralph E.}, year={2020}, month={Apr} }