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US-12624365-B2 - Method for modulating the alkaloid content of a tobacco plant

US12624365B2US 12624365 B2US12624365 B2US 12624365B2US-12624365-B2

Abstract

Disclosed are methods of modulating the alkaloid content of a plant or a part thereof by modulating the activity or expression of at least one gene encoding a BTB/POZ NPH3 domain-containing protein. Methods of reducing the content of at least one tobacco specific nitrosamine (TSNA) precursor in tobacco are also provided.

Inventors

  • Sara Ben Khaled
  • Francisco Anastacio De Abreu E Lima

Assignees

  • BRITISH AMERICAN TOBACCO (INVESTMENTS) LIMITED

Dates

Publication Date
20260512
Application Date
20200703
Priority Date
20190703

Claims (20)

  1. 1 . A method of modulating the alkaloid content of a plant or a part thereof, or a cell, the method comprising modifying said plant or cell by modulating the expression or decreasing the activity of at least one gene encoding a BTB/POZ NPH3 domain-containing protein, wherein i) the content of one or more alkaloids selected from nicotine, nornicotine, pseudooxynicotine (PON), anabasine, and anatabine is modulated; ii) the plant or part thereof, or cell is from the genus Nicotiana, iii) modulation of the expression of said at least one gene is achieved by overexpression, mutation, knock out, gene silencing or RNA silencing of said at least one gene; iv) the BTB/POZ NPH3 domain-containing protein comprises an amino acid sequence: a) as set out in SEQ ID NO: 1, or b) a functional variant of SEQ ID NO: 1 comprising a BTB/POZ domain with a sequence set forth in SEQ ID NO: 31 or a sequence with at least 98% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO: 32 or a sequence with at least 98% identity thereof, or c) a sequence which has at least 98% identity to SEQ ID NO: 1; or the at least one gene encoding the BTB/POZ NPH3 domain-containing protein encodes the BTB/POZ NPH3 domain-containing protein as defined in a), b) or c) above and comprises a nucleotide sequence: a) as set out in SEQ ID NO: 2 or SEQ ID NO: 3, b) a functional variant of SEQ ID NO: 2 or SEQ ID NO: 3 comprising a nucleotide sequence encoding a BTB/POZ domain with a sequence set forth in SEQ ID NO: 31 or a sequence with at least 98% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO: 32 or a sequence with at least 98% identity thereof, or c) a sequence which has at least 98% identity to SEQ ID NO: 2 or 3; and v) when the plant or cell is modified to increase the expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein, the plant or cell exhibits increased alkaloid content in comparison to a plant or cell which has not been modified to modulate the expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein, or when the plant or cell is modified to decrease the activity or expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein, the plant or cell exhibits decreased alkaloid content in comparison to a plant or cell which has not been modified to modulate the expression or decrease the activity of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein.
  2. 2 . The method according to claim 1 , wherein the alkaloid content is modulated in comparison to a plant or cell which has not been modified to modulate the expression or decrease the activity of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein.
  3. 3 . A plant or part thereof or cell, or plant propagation material obtained therefrom, which has been modified to achieve a modulation in alkaloid content in comparison to an unmodified plant, wherein the modification is the modulation of the expression or decrease the activity of at least one gene encoding a BTB/POZ NPH3 domain-containing protein from tobacco, wherein i) the content of one or more alkaloids selected from nicotine, nornicotine, pseudooxynicotine (PON), anabasine, and anatabine is modulated; ii) the plant or part thereof, or cell, or plant propagation material obtained therefrom is from the genus Nicotiana; iii) modulation of the expression of said at least one gene is achieved by overexpression, mutation, knock out, gene silencing or RNA silencing of said at least one gene; iv) the BTB/POZ NPH3 domain-containing protein comprises an amino acid sequence: a) as set out in SEQ ID NO: 1, or b) a functional variant of SEQ ID NO: 1 comprising a BTB/POZ domain with a sequence set forth in SEQ ID NO: 31 or a sequence with at least 98% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO: 32 or a sequence with at least 98% identity thereof, or c) a sequence which has at least 98% identity to SEQ ID NO: 1; or the at least one gene encoding the BTB/POZ NPH3 domain-containing protein encodes the BTB/POZ NPH3 domain-containing protein as defined in a), b) or c) above and comprises a nucleotide sequence: a) as set out in SEQ ID NO: 2 or SEQ ID NO: 3, b) a functional variant of SEQ ID NO: 2 or SEQ ID NO: 3 comprising a nucleotide sequence encoding a BTB/POZ domain with a sequence set forth in SEQ ID NO: 31 or a sequence with at least 98% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO: 32 or a sequence with at least 98% identity thereof, or c) a sequence which has at least 98% identity to SEQ ID NO: 2 or 3; and v) when the plant or part thereof or cell, or plant propagation material obtained therefrom is modified to increase the expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein, the plant or part thereof or cell, or plant propagation material obtained therefrom exhibits increased alkaloid content in comparison to a plant or part thereof or cell, or plant propagation material obtained therefrom which has not been modified to modulate the expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein, or when the plant or part thereof or cell, or plant propagation material obtained therefrom is modified to decrease the activity or expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein, the plant or part thereof or cell, or plant propagation material obtained therefrom exhibits decreased alkaloid content in comparison to a plant or part thereof or cell, or plant propagation material obtained therefrom which has not been modified to modulate the expression or decrease the activity of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein.
  4. 4 . The plant or part thereof or cell, or plant propagation material obtained therefrom, according to claim 3 , wherein the alkaloid content of the plant or cell is decreased in comparison to a plant or cell which has not been modified to modulate the expression or decrease the activity of at least one gene encoding a BTB/POZ NPH3 domain-containing protein.
  5. 5 . The plant or part thereof or cell, or plant propagation material obtained therefrom, according to claim 4 , wherein the activity or expression of at least one gene encoding a BTB/POZ NPH3 domain-containing protein is decreased in comparison to a plant or cell which has not been modified to modulate the expression or decrease the activity of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein.
  6. 6 . The plant or part thereof or cell, or plant propagation material obtained therefrom, according to claim 3 , wherein the alkaloid content of the plant or cell is increased in comparison to a plant or cell which has not been modified to modulate the expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein.
  7. 7 . The plant or part thereof or cell, or plant propagation material obtained therefrom, according to claim 6 , wherein the plant or cell is modified to increase the expression of at least one gene encoding a BTB/POZ NPH3 domain-containing protein and the plant or cell exhibits increased alkaloid content in comparison to a plant or cell which has not been modified to modulate the expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein.
  8. 8 . The plant or part thereof or cell, or plant propagation material obtained therefrom, according to claim 3 , wherein the content of one or more alkaloids selected from nornicotine and/or PON and/or anabasine is modulated.
  9. 9 . The plant or part thereof or cell, or plant propagation material obtained therefrom, according to claim 3 , wherein the at least one BTB/POZ NPH3 domain-containing protein comprises an amino acid sequence as set out in SEQ ID NO: 1 or a functional variant of SEQ ID NO: 1 comprising a BTB/POZ domain with a sequence set forth in SEQ ID NO: 31 and a NPH3 domain with a sequence as set forth in SEQ ID NO: 32; or the at least one gene encoding the BTB/POZ NPH3 domain-containing protein comprises a nucleotide sequence as set out in SEQ ID NO: 2 or SEQ ID NO: 3, or a functional variant of SEQ ID NO: 2 or SEQ ID NO: 3 comprising a nucleotide sequence encoding a BTB/POZ domain with a sequence set forth in SEQ ID NO: 31 and a NPH3 domain with a sequence as set forth in SEQ ID NO: 32.
  10. 10 . The plant or part thereof or cell, or plant propagation material obtained therefrom, according to claim 3 , wherein an additional gene encoding a BTB/POZ NPH3 domain-containing protein is modulated wherein the additional gene is at least one selected from SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28; or the at least one gene encoding the BTB/POZ NPH3 domain-containing protein comprises a nucleotide sequence as set out in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 30.
  11. 11 . A harvested leaf, a cut harvested leaf, a processed leaf, a processed tobacco leaf, a non-viable processed tobacco leaf, or a cut processed leaf of the plant according to claim 3 or a plant propagated from a plant propagation material obtained therefrom, wherein said harvested leaf, a cut harvested leaf, a processed leaf, a processed tobacco leaf, a non-viable processed tobacco leaf, or a cut processed leaf has a modulation of the expression or a decrease in the activity of at least one gene encoding a BTB/POZ NPH3 domain-containing protein from tobacco to achieve a modulation in alkaloid content in comparison to an unmodified harvested leaf, a cut harvested leaf, a processed leaf, a processed tobacco leaf, a non-viable processed tobacco leaf, or a cut processed leaf, wherein the modulation is as defined in claim 6 .
  12. 12 . The processed leaf, a processed tobacco leaf, a non-viable processed tobacco leaf, or cut processed leaf according to claim 11 , wherein the leaf is processed by curing, fermenting, pasteurising or a combination thereof.
  13. 13 . A cured tobacco material, or a tobacco blend comprising the cured tobacco material, made from the plant or part thereof of claim 3 or a plant propagated from a plant propagation material obtained therefrom, wherein said cured tobacco material or tobacco blend has a modulation of the expression or a decrease in the activity of at least one gene encoding a BTB/POZ NPH3 domain-containing protein from tobacco to achieve a modulation in alkaloid content in comparison to an unmodified cured tobacco material or tobacco blend, wherein the modulation is as defined in claim 3 .
  14. 14 . A mutant of a plant carrying a heritable mutation in a nucleotide sequence of at least one gene encoding the BTB/POZ NPH3 domain-containing protein, wherein the BTB/POZ NPH3 domain-containing protein comprises an amino acid sequence: a) as set out in SEQ ID NO: 1, or b) a functional variant of SEQ ID NO: 1 comprising a BTB/POZ domain with a sequence set forth in SEQ ID NO:31 or a sequence with at least 98% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO:32 or a sequence with at least 98% identity thereof, or c) a sequence which has at least 98% identity to SEQ ID NO: 1; or the at least one gene encoding the BTB/POZ NPH3 domain-containing protein encodes the BTB/POZ NPH3 domain-containing protein as defined in a), b) or c) above and comprises a nucleotide sequence: a) as set out in SEQ ID NO: 2 or SEQ ID NO: 3, b) a functional variant of SEQ ID NO: 2 or SEQ ID NO: 3 comprising a nucleotide sequence encoding a BTB/POZ domain with a sequence set forth in SEQ ID NO:31 or a sequence with at least 98% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO:32 or a sequence with at least 98% identity thereof, or c) a sequence which has at least 98% identity to SEQ ID NO: 2 or 3; wherein said heritable mutation modulates the expression or decrease the activity of the at least one gene encoding the BTB/POZ NPH3 domain-containing protein by overexpression, mutation, knock out, gene silencing or RNA silencing of said at least one gene; and wherein the mutant plant has modulated alkaloid content relative to a comparable plant which does not carry said heritable mutation, wherein i) the content of one or more alkaloids selected from nicotine, nornicotine, pseudooxynicotine (PON), anabasine, and anatabine is modulated; ii) the plant is from the genus Nicotiana , and iii) when said heritable mutation increases the expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein the plant exhibits increased alkaloid content in comparison to a plant which does not carry said heritable mutation, or when said heritable mutation decreases the activity or expression of the at least one gene encoding a BTB/POZ NPH3 domain-containing protein the plant exhibits decreased alkaloid content in comparison to a plant which does not carry said heritable mutation.
  15. 15 . Progeny or seed of the mutant plant which carries the heritable mutation according to claim 14 .
  16. 16 . A harvested leaf, a processed leaf or cured tobacco material produced from the plant according to claim 14 , wherein the harvested leaf, processed leaf or cured tobacco material has modulated alkaloid content relative to a harvested leaf, processed leaf or cured tobacco material from a comparable plant which does not carry said modification in the at least one gene encoding the BTB/POZ NPH3 domain-containing protein.
  17. 17 . The mutant plant according to claim 14 , wherein the modulated alkaloid is nicotine.
  18. 18 . The method according to claim 1 , wherein the BTB/POZ NPH3 domain-containing protein comprises: a) a functional variant of SEQ ID NO: 1 comprising a BTB/POZ domain with a sequence set forth in SEQ ID NO:31 or a sequence with at least 99% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO:32 or a sequence with at least 99% identity thereof, or b) a sequence which has at least 99% identity to SEQ ID NO: 1; or the at least one gene encoding the BTB/POZ NPH3 domain-containing protein encodes the BTB/POZ NPH3 domain-containing protein as defined in a) or b) above and comprises: a) a functional variant of SEQ ID NO: 2 or SEQ ID NO: 3 comprising a nucleotide sequence encoding a BTB/POZ domain with a sequence set forth in SEQ ID NO:31 or a sequence with at least 99% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO:32 or a sequence with at least 99% identity thereof, or b) a sequence which has at least 99% identity to SEQ ID NO: 2 or 3.
  19. 19 . The plant or part thereof or cell, or plant propagation material obtained therefrom, according to claim 3 , wherein the BTB/POZ NPH3 domain-containing protein comprises: a) a functional variant of SEQ ID NO: 1 comprising a BTB/POZ domain with a sequence set forth in SEQ ID NO:31 or a sequence with at least 99% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO:32 or a sequence with at least 99% identity thereof, or b) a sequence which has at least 99% identity to SEQ ID NO: 1; or the at least one gene encoding the BTB/POZ NPH3 domain-containing protein encodes the BTB/POZ NPH3 domain-containing protein as defined in a) or b) above and comprises: a) a functional variant of SEQ ID NO: 2 or SEQ ID NO: 3 comprising a nucleotide sequence encoding a BTB/POZ domain with a sequence set forth in SEQ ID NO:31 or a sequence with at least 99% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO:32 or a sequence with at least 99% identity thereof, or b) a sequence which has at least 99% identity to SEQ ID NO: 2 or 3.
  20. 20 . The mutant of a plant carrying a heritable mutation in a nucleotide sequence of at least one gene encoding the BTB/POZ NPH3 domain-containing protein according to claim 14 , wherein the BTB/POZ NPH3 domain-containing protein comprises: a) a functional variant of SEQ ID NO: 1 comprising a BTB/POZ domain with a sequence set forth in SEQ ID NO:31 or a sequence with at least 99% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO:32 or a sequence with at least 99% identity thereof, or b) a sequence which has at least 99% identity to SEQ ID NO: 1; or the at least one gene encoding the BTB/POZ NPH3 domain-containing protein encodes the BTB/POZ NPH3 domain-containing protein as defined in a) or b) above and comprises: a) a functional variant of SEQ ID NO: 2 or SEQ ID NO: 3 comprising a nucleotide sequence encoding a BTB/POZ domain with a sequence set forth in SEQ ID NO:31 or a sequence with at least 99% identity thereto, and a NPH3 domain with a sequence as set forth in SEQ ID NO:32 or a sequence with at least 99% identity thereof, or b) a sequence which has at least 99% identity to SEQ ID NO: 2 or 3.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This is a U.S. National Phase application claiming priority to PCT/GB2020/051602, filed Jul. 3, 2020, which claims priority to British application No. 1909562.9, filed Jul. 3, 2019, the entire contents of which are hereby expressly incorporated by reference in their entirety including, without limitation, the specification, claims, and abstract, as well as any figures, tables, or drawings thereof. FIELD OF THE INVENTION The present invention relates to methods of modulating the alkaloid content a plant or part thereof or cell or cell culture. The invention also extends to methods of modulating the expression and/or activity of polypeptides which modulate alkaloid content within plants. Alternatively, the invention provides methods of modulating the expression and/or activity of genes which encode polypeptides which modulate alkaloid content within plants. The invention also extends to constructs, which can be used to modulate the polypeptides. The invention further relates to plant cells and plants modified to achieve a modulation in alkaloid content. The invention also relates to a processed and harvested leaf from such modulated plants and use thereof in a tobacco industry product, including combustible smoking articles. BACKGROUND Alkaloids are a group of naturally occurring compounds which mostly contain basic nitrogen atoms and are produced by a large variety of organisms including bacteria, fungi, plants and animals. Alkaloids may be classified according to the similarity of the carbon skeleton e.g. indole-, isoquinoline- and pyridine-like. Pyridine derivatives are one class of monomeric alkaloids; this class includes simple derivatives of pyridine, polycyclic condensed and noncondensing pyridine derivatives and sesquiterpene pyridine derivatives. Examples are nicotine, nornicotine, pseudooxynicotine, anabasine, myosmine and anatabine. Most of the known biological functions of alkaloids are related to protection. Neuroactive molecules, such as caffeine, cocaine, morphine, and nicotine, act as defence compounds against invading predators. The accumulation of these alkaloids is the result of signal transduction cascades that monitor gene expression, enzyme activities, and alkaloid concentrations. The fine-tuning of alkaloid content in the plant involves negative feedback loops and degradative pathways. Nicotine occurs naturally in several varieties of plants but is found at the highest level in the tobacco plant. Cultivated tobacco produces 2-4% alkaloids of total dry weight. Nicotine is produced in wild and cultivated Nicotiana species and plays an important role in plant defence against herbivores and insects (Voelckel et al. (2001) Oecologia 127(2): 274-280, incorporated herein by reference). It accounts for ˜90% of the total alkaloid content. The remaining 10% of the alkaloid pool is mostly constituted by the structurally related compounds nornicotine, anatabine, anabasine and pseudooxynicotine (PON). The regulation of alkaloid content in tobacco is complex. Several factors including genotype, environment, fertilization and agronomic practices (e.g. topping) affect alkaloid levels in tobacco plants. Some key regulators of nicotine biosynthesis are well characterized, for example putrescine N-methyltransferase (PMT), which plays a pivotal role in this pathway, is activated by members of the ethylene responsive factor (ERF) superfamily, the largest transcription factor family in the tobacco genome (Rushton et al. (2008) Plant Physiol. 147(1): 280-295 incorporated herein by reference). Other transcription factors that induce alkaloid biosynthesis belong to the MYC2-like basic helix-loop-helix (bHLH) family. MYC2-like bHLHs regulate alkaloid levels directly, through the Gbox-mediated binding and activation of alkaloid structural genes, and indirectly, through the activation of ERFs. Tobacco pyridine alkaloids are precursors of tobacco-specific nitrosamines (TSNAs) that form during the post-harvest leaf curing. The four primary TSNAs found in cured tobacco leaves are N′-nitrosonornicotine (NNN), N′nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB) and 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). During the post-harvest leaf curing, reactions between pyridine alkaloids and nitrosating species leads to the formation of tobacco-specific nitrosamines (TSNAs). PON is likely to function as the direct precursor in the synthesis of the TSNA NNK (Bush et al., 2001 incorporated herein by reference). Reducing the production and accumulation of TSNAs is of high importance. The CYP82E family of nicotine demethylase genes is one of the primary regulators of nicotine to nornicotine conversion, and altering their activity or accumulation may result in a decrease in NNN levels. However, no enzymes or genes responsible for producing PON have been identified thus far. As described in the Examples, the inventors sought to investigate genes responsible for alkaloid and/or TSNA