Editing of A622 genes results in ultra-low nicotine whole tobacco plants at the expense of dramatically reduced growth and development

Burner, Nathaniel; Kernodle, Sheri P.; Steede, Tyler; Lewis, Ramsey S.

doi:10.1007/s11032-022-01293-w

Cited by 5 publications

(2 citation statements)

References 16 publications

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“…One possible answer is that nicotine, like many other specialized metabolites (47, 48), has a developmental role, in addition to its insecticidal properties (7, 18, 49). This is in line with the smaller sized of our N. benthamiana a622 mutants and similar recent observation with N. tabacum mutants (50). A more likely explanation is that nicotine in N. benthamiana serves as a “second line of defense”.…”

Section: Discussionsupporting

confidence: 93%

“…It is possible that nicotine, like many other specialized metabolites (Zhou et al ., 2018; Erb & Kliebenstein, 2020) has a developmental role, in addition to its insecticidal properties (Parr & Thurston, 1972; Steppuhn et al ., 2004; Harvey et al ., 2007). This is in line with the smaller size of our N. benthamiana a622 mutants at lower temperatures, and a similar recent observation in N. tabacum mutants (Burner et al ., 2022). By contrast targeting other parts of the nicotine biosynthesis pathway, RNAi of the putrescine methyl transferase ( PMT ) gene required for production of the methylpyrrolidine ring (Steppuhn et al ., 2004) and genome editing of the berberine bridge like protein ( BBL ) gene (Vollheyde et al ., 2023), did not inhibit growth of the nicotine-reduced plants.…”

Section: Discussionsupporting

confidence: 93%

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Knockout mutations ofNicotiana benthamianadefenses reveal the relative importance of acylsugars, nicotine, and a serine protease inhibitor in a natural setting

Negin,

Wang,

Fischer

et al. 2024

Preprint

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Plants produce an immense diversity of defensive specialized metabolites. However, despite extensive functional characterization, such experiments are seldom performed in natural settings, and the balance between different defensive compounds is rarely examined. Here, we compare the efficacy of three Nicotiana benthamiana defensive compounds, nicotine, acylsugars, and a serine protease inhibitor, by growing plants with combinations of knockout mutations in a natural setting, quantifying invertebrate interactions, and comparing relative plant performance. Among the three tested compounds, acylsugars had the greatest defensive capacity, affecting aphids, leafhoppers, spiders, and flies. Nicotine mutants displayed increased leafhopper feeding and aphid colonization. Plants lacking both nicotine and acylsugars were more susceptible to flea beetles and thrips. By contrast, knockout of the serine protease inhibitor did not affect insect herbivory in the field. Complementary experiments under controlled laboratory conditions confirmed results obtained in a natural setting. We conclude that the three metabolite groups, which provide defense against different herbivores and herbivore classes, collectively provide broad-spectrum protection to N. benthamiana. However, there is a gradient in their effectiveness, with acylsugars providing the strongest protection and nicotine providing a secondary line of defense. Furthermore, we demonstrate that, even if individual metabolites do not have a measurable defensive effect on their own, they can have an additive effect when combined with other defensive compounds.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionsupporting

confidence: 93%

Knockout mutations ofNicotiana benthamianadefenses reveal the relative importance of acylsugars, nicotine, and a serine protease inhibitor in a natural setting

Negin,

Wang,

Fischer

et al. 2024

Preprint

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Development of an RNA virus vector for non-transgenic genome editing in tobacco and generation of berberine bridge enzyme-like mutants with reduced nicotine content

Xiang,

Chen,

Wang

et al. 2024

aBIOTECH

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Tobacco (Nicotiana tabacum) plants synthesize the psychoactive pyridine alkaloid nicotine, which has sparked growing interest in reducing nicotine levels through genome editing aiming at inactivating key biosynthetic genes. Although stable transformation-mediated genome editing is effective in tobacco, its polyploid nature complicates the complete knockout of genes and the segregation of transgenes from edited plants. In this study, we developed a non-transgenic genome editing method in tobacco by delivering the CRISPR/Cas machinery via an engineered negative-strand RNA rhabdovirus vector, followed by the regeneration of mutant plants through tissue culture. Using this method, we targeted six berberine bridge enzyme-like protein (BBL) family genes for mutagenesis, which are implicated in the last steps of pyridine alkaloid biosynthesis, in the commercial tobacco cultivar Hongda. We generated a panel of 16 mutant lines that were homozygous for mutations in various combinations of BBL genes. Alkaloid profiling revealed that lines homozygous for BBLa and BBLb mutations exhibited drastically reduced nicotine levels, while other BBL members played a minor role in nicotine synthesis. The decline of nicotine content in these lines was accompanied by reductions in anatabine and cotinine levels but increases in nornicotine and its derivative myosmine. Preliminary agronomic evaluation identified two low-nicotine lines with growth phenotypes comparable to those of wild-type plants under greenhouse and field conditions. Our work provides potentially valuable genetic materials for breeding low-nicotine tobacco and enhances our understanding of alkaloid biosynthesis.

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Impact of CRISPR/Cas9-induced mutations in nicotine biosynthesis core genes A622 and BBL on tobacco: Reduction in nicotine content and developmental abnormalities

Jeong,

Jeon,

Song

et al. 2024

Current Plant Biology

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Editing of A622 genes results in ultra-low nicotine whole tobacco plants at the expense of dramatically reduced growth and development

Cited by 5 publications

References 16 publications

Knockout mutations ofNicotiana benthamianadefenses reveal the relative importance of acylsugars, nicotine, and a serine protease inhibitor in a natural setting

Knockout mutations ofNicotiana benthamianadefenses reveal the relative importance of acylsugars, nicotine, and a serine protease inhibitor in a natural setting

Development of an RNA virus vector for non-transgenic genome editing in tobacco and generation of berberine bridge enzyme-like mutants with reduced nicotine content

Impact of CRISPR/Cas9-induced mutations in nicotine biosynthesis core genes A622 and BBL on tobacco: Reduction in nicotine content and developmental abnormalities

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