Method for regulating alkaloids in tobacco by modifying WAT1-related protein expression or activity

Abstract

The present invention relates to a method for reducing the content of at least one tobacco-specific nitrosamine (TSNA) precursor in tobacco, the method comprising modulating the activity or expression of at least one gene encoding a wall thin 1-related (WAT1-related) protein.

Description

Technical Field

[0001] The present invention relates to methods for regulating the alkaloid content of plants or parts thereof, such as pseudooxynicotine content. The present invention also extends to methods for regulating the expression and / or activity of polypeptides that regulate the alkaloid content in plants. Alternatively, the present invention provides methods for regulating the expression and / or activity of genes encoding polypeptides that regulate the alkaloid content in plants. The present invention also extends to constructs that can be used to regulate polypeptides. The present invention further relates to plant cells and plants that have been modified to achieve regulation of alkaloid content. The present invention also relates to processed and harvested leaves from such regulated plants and their use in delivery systems (including combustible smoking products). Background Art

[0002] Alkaloids are a group of naturally occurring compounds that contain primarily basic nitrogen atoms and are produced by a wide variety of organisms including bacteria, fungi, plants, and animals.

[0003] Alkaloids can be classified based on similarities in their carbon skeletons, such as indole, isoquinoline, and pyridine. Pyridine derivatives are a class of monomeric alkaloids; this category includes simple pyridine derivatives, polycyclic condensed and non-condensed pyridine derivatives, and sesquiterpenoid pyridine derivatives. Examples include nicotine, nornicotine, pseudonicotine oxide, anabasine, mithosamine, and anatabine.

[0004] Most of the known biological functions of alkaloids are related to protection. Neuroactive molecules such as caffeine and nicotine serve as defense compounds against invading predators. The accumulation of these alkaloids is the result of a signal transduction cascade that monitors gene expression, enzyme activity, and alkaloid concentration. The fine regulation of alkaloid content in plants involves a negative feedback loop and degradation pathways.

[0005] Nicotine occurs naturally in several plant species, but is found at its highest levels in tobacco plants. Cultivated tobacco produces 2-4% of the alkaloid by weight. Nicotine is present in both wild and cultivated Nicotiana species ( Nicotiana ) species and plays an important role in plant defense 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 mainly composed of the structurally related compounds nornicotine, anatabine, anabasine, and pseudonicotine oxide (PON).

[0006] 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, such as putrescine N-methyltransferase (PMT), which plays a key role in this pathway, is activated by members of the ethylene response factor (ERF) superfamily, the largest family of transcription factors 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 directly regulate alkaloid levels through the binding and activation of Gbox-mediated alkaloid structural genes, and indirectly regulate alkaloid levels through the activation of ERFs.

[0007] Tobacco pyridine alkaloids are precursors of tobacco-specific nitrosamines (TSNAs), which are formed during the post-harvest leaf curing period. The four main TSNAs found in curing tobacco leaves are N'-nitrosonornicotine (NNN), N'-nitrosonatabine (NAT), N'-nitrosonatabine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). During the post-harvest leaf curing period, reactions between pyridine alkaloids and nitrosating substances lead to the formation of tobacco-specific nitrosamines (TSNAs). PON may function as a direct precursor in the synthesis of the TSNA NNK (Bush et al., 2001, which is incorporated herein by reference). Reducing the production and accumulation of nornicotine and PON is very important. The CYP82E family of nicotine demethylase genes is one of the main regulators of the conversion of nicotine to nornicotine, and altering its activity or accumulation may lead to reduced NNN levels. However, to date, the enzyme or gene responsible for the production of PON has not been identified.

[0008] As described in the examples, the inventors sought to investigate the genes responsible for the synthesis of alkaloids and / or TSNA precursors with the aim of modulating the alkaloid content in plants, for example reducing the content of pseudo-oxidized nicotine in tobacco plants. Summary of the Invention

[0009] Surprisingly, it has been discovered that by modulating the activity or expression of genes encoding Walls are thin 1-related (WAT1-related) proteins, the alkaloid content and / or TSNA content or TSNA precursor content of a plant can be modulated. WAT1-related genes, such as Nitab4.5_0010919g0010.2, as taught herein, are regulators of alkaloid and TSNA precursor content in cultivated tobacco. Specifically, WAT1-related genes, such as Nitab4.5_0010919g0010.2, as taught herein, are regulators of PON in cultivated tobacco. Nitab4.5_0010919g0010.2 encodes a WAT1-related protein according to the present invention. Nitab4.5_0001086g0030.2, Nitab4.5_0003292g0030.2, Nitab4.5_0004697g0010.2, Nitab4.5_0005998g0010.2, Nitab4.5_0011862g0010.2, Nitab4.5_0000062g0210.2, Nitab4.5_0008142g0020.2, Nitab4.5_0000062g0220.2 and Nitab4.5_0001674g0010.2 are homologues of Nitab4.5_0010919g0010.2 according to the present invention. The WAT1-related proteins according to the present invention contain a conserved domain known as the EamA domain.

[0010] According to the present invention, tobacco products can be produced with regulated alkaloid content and commercially desirable traits sought by consumers of tobacco products. In some cases, consumers may desire products with low levels of alkaloid content, such as low levels of TSNA precursors.

[0011] The present invention may be particularly useful in the field of plant molecular farming, where plants (such as tobacco and other Nicotiana species) are used to produce proteins, peptides and metabolites, for example for the production of therapeutics and drugs such as antibiotics, virus-like particles, or neutraceuticals or small molecules. In an EU-funded project called PharmPlant, tobacco has been used to develop HIV-neutralizing antibodies, and Medicago Inc. in Canada has participated in a tobacco-based platform for the production of virus-like particles for influenza vaccine manufacturing.

[0012] Therefore, plants according to the present invention can be used in molecular farming to reduce or eliminate the presence of PON and / or other nicotine alkaloids. The use of low-nicotine (or low-alkaloid, such as low-PON) plants or roots is beneficial in molecular farming and will reduce downstream processing costs associated with purification.

[0013] The present inventors have surprisingly identified a method for regulating the alkaloid content (e.g., pseudo-oxidized nicotine content) of a plant (e.g., a tobacco plant) by regulating the activity or expression of a gene encoding a WAT1-related protein. The alkaloid content (e.g., the content of one or more of PON, nicotine, nornicotine, anabasine, miosamine, or anatabine) of a plant (e.g., a tobacco plant) can be reduced by reducing the activity or expression of a gene encoding a WAT1-related protein or can be increased by increasing the activity or expression of a gene encoding a WAT1-related protein. Prior to the present invention, it was not known that regulating the activity or expression of a gene encoding a WAT1-related protein as described herein could be used to regulate alkaloid content, or in particular, PON content.

[0014] In one aspect, a method of modulating (e.g., reducing) the alkaloid content of a plant or part thereof or a cell or cell culture is provided, the method comprising modifying the plant or cell (or cell culture) by modulating the activity or expression of at least one gene encoding a WAT1-related protein.

[0015] In one aspect, a method of modulating (eg, reducing) the alkaloid content of a plant or part thereof or a cell or cell culture is provided, the method comprising modifying the plant or cell or cell culture by modulating the activity or expression of at least one WAT1-related protein.

[0016] In another aspect, a method is provided for modulating (e.g., reducing) the level of tobacco-specific nitrosamine (TSNA) precursors in a tobacco plant, or a plant part thereof, or a cell or cell culture thereof, the method comprising modifying the plant or cell culture by modulating the activity or expression of at least one gene encoding a WAT1-related protein.

[0017] In another aspect, a method is provided for modulating (e.g., reducing) the level of tobacco-specific nitrosamine (TSNA) precursors in a tobacco plant, or a plant part thereof, or a cell or cell culture thereof, the method comprising modifying the plant, cell or cell culture by modulating the activity or expression of at least one WAT1-related protein.

[0018] In a further aspect, there is provided the use of at least one gene encoding a WAT1-related protein for modulating the alkaloid content of a cell or a plant or a part thereof or a cell culture.

[0019] In yet another aspect, a method is provided for producing a plant or part thereof, cell culture, plant propagation material, leaves, cut harvested leaves, processed leaves or cut and processed leaves having a modulated (e.g., reduced) alkaloid content, the method comprising modifying the plant or cell culture to modulate the activity or expression of at least one gene encoding a WAT1-related protein.

[0020] In a further aspect, there is provided the use of at least one gene encoding a WAT1-related protein for modulating the alkaloid content of a cell or a plant or a part thereof or a cell culture.

[0021] In yet another aspect, a method is provided for producing a plant or part thereof, cell culture, plant propagation material, leaves, cut harvested leaves, processed leaves or cut and processed leaves having a modulated (e.g., reduced) alkaloid content, the method comprising modifying the plant or cell culture to modulate the activity or expression of at least one WAT1-related protein.

[0022] Suitably, the cell may be a plant cell. Suitably, the cell may be a tobacco plant cell. Suitably, the alkaloid content may be modulated (e.g., reduced) compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a WAT1-related protein.

[0023] In a further aspect, there is provided a plant or part thereof or cell culture which has been modified to achieve modulation (e.g., reduction) of alkaloid content compared to an unmodified plant or unmodified cell culture, wherein the modulation is a reduction in the activity or expression of at least one gene encoding a WAT1-related protein.

[0024] In a further aspect, there is provided plant propagation material obtainable from a plant according to the invention, or a plant or a cell or cell culture produced by a method according to the invention.

[0025] In a further aspect, there is provided a method or use according to the invention, or a plant or part thereof or cell culture according to the invention, or a plant propagation material according to the invention, wherein the alkaloid content of the plant is reduced compared to a plant or cell culture which has not been modified to modulate the activity or expression of at least one gene encoding a WAT1-related protein.

[0026] Suitably, the activity or expression of at least one gene encoding a WAT1-related protein may be reduced compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a WAT1-related protein.

[0027] Suitably, the plant may be modified to reduce the activity or expression of at least one gene encoding a WAT1-related protein, and the plant or cell culture exhibits a reduced alkaloid content compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a WAT1-related protein.

[0028] Suitably, the alkaloid content of the plant or cell culture may be increased compared to a plant or cell culture which has not been modified to modulate the activity or expression of at least one gene encoding a WAT1 -related protein.

[0029] Suitably, the plant may be modified to increase the activity or expression of at least one gene encoding a WAT1-related protein, and the plant or cell culture exhibits increased alkaloid content compared to a plant or cell culture that has not been modified to modulate the activity or expression of at least one gene encoding a WAT1-related protein.

[0030] Suitably, the total alkaloid content of the plant or cell culture may be modulated (eg reduced).

[0031] Suitably, the content of one or more alkaloids selected from PON, nicotine, nornicotine, anabasine, mithosamine, and anatabine is regulated (e.g., reduced); preferably, the content of PON, nicotine, and / or nornicotine is regulated (e.g., reduced); preferably, the content of PON is regulated (e.g., reduced). In some embodiments, two or more (or three or more) alkaloids selected from nicotine, nornicotine, PON, anabasine, mithosamine, and anatabine may be regulated (e.g., reduced).

[0032] In one aspect, the plant or plant cell is from the Solanaceae family.

[0033] Suitably, the plant or plant cell may be from the genus Nicotiana.

[0034] Suitably, the PON content may be adjusted. Suitably, the PON content may be reduced.

[0035] In one aspect, a method or use according to the invention, a plant or part thereof or cell culture according to the invention, or a plant propagation material according to the invention is provided, wherein the at least one gene encoding a WAT1-related protein encodes a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof; or

[0036] wherein the at least one gene encoding a WAT1-related protein encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or

[0037] wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof; or

[0038] wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0039] In a further aspect, there is provided a method or use according to the invention, a plant or part thereof or cell culture according to the invention, or a plant propagation material according to the invention, wherein an additional gene encoding a WAT1-related protein is also regulated, wherein the additional gene encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID No. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28 or a functional variant or functional fragment or orthologue thereof; or

[0040] wherein the additional gene encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or

[0041] wherein the additional gene comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof; or

[0042] wherein the additional gene comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0043] In one aspect, there is provided use of a plant or part or cell culture thereof according to the invention, or a plant produced by a method according to the invention, for growing plants.

[0044] In another aspect, there is provided use of a plant or part or cell culture thereof according to the invention, or a plant produced by a method according to the invention, for producing a product.

[0045] In yet another aspect, there is provided use of a plant according to the invention or a part thereof, or a plant produced by a method according to the invention, for growing crops.

[0046] In another aspect, there is provided use of a plant according to the invention or a part thereof, or a plant produced by a method according to the invention, to produce leaves.

[0047] In another aspect, there is provided harvested leaves of a plant according to the invention, or of a plant obtainable from a propagation material according to the invention, or of a plant obtainable from a use according to the invention, or of a plant produced by a method according to the invention.

[0048] Suitably, the harvested leaves may be cut harvested leaves.

[0049] In another aspect, there is provided a processed leaf, preferably a processed tobacco leaf, preferably a non-viable processed tobacco leaf, which:

[0050] obtainable from plants obtainable for use according to the invention;

[0051] Obtainable by processing the plant according to the invention;

[0052] obtainable from a plant propagated from the plant propagation material according to the invention; or

[0053] Obtainable by processing harvested leaves of a plant according to the invention; or

[0054] Obtainable from plants produced by the method according to the invention.

[0055] Suitably, the leaves may be processed by curing, fermenting, pasteurising or a combination thereof.

[0056] Suitably, the processed leaves may have a reduced content of one or more TSNAs selected from NNK, NNN, NAT and NAB, preferably a reduced content of NNK. Suitably, the content of NNK may be reduced relative to processed leaves from a comparable plant not modified according to the invention.

[0057] Suitably, the tooling blade may be a cut tooling blade.

[0058] In another aspect, there is provided a cured tobacco material made from a plant or part thereof or extract thereof according to the present invention.

[0059] In another aspect, there is provided a tobacco blend comprising the cured tobacco material according to the present invention.

[0060] In another aspect, a delivery system is provided, which is prepared by:

[0061] a tobacco plant according to the invention or a part thereof, or a tobacco cell culture according to the invention;

[0062] Tobacco plants or parts thereof propagated from tobacco plant propagation material according to the invention;

[0063] harvested leaves of a plant according to the present invention, wherein the plant is tobacco;

[0064] The processed leaf according to the present invention, wherein the plant is tobacco;

[0065] or

[0066] Plants produced by the method according to the present invention.

[0067] Suitably, the tobacco product may be a combustible smoking article.

[0068] Suitably, the tobacco product may be a smokeless tobacco product.

[0069] Suitably, the tobacco product may be a non-combustible gas aerosol delivery system, such as a tobacco heating device or an aerosol generating device.

[0070] In one aspect, there is provided use of tobacco cells according to the present invention for modulating alkaloid content in cell culture.

[0071] In another aspect, a combustible smoking article, a non-combustible gas aerosol delivery system, a smokeless tobacco product or a tobacco heating device is provided, which comprises a plant or part thereof or an extract thereof (e.g., a tobacco extract) according to the invention, or a tobacco cell culture according to the invention; or a cured tobacco material according to the invention; or a tobacco blend according to the invention.

[0072] In one aspect, the present invention provides a method for selecting plants having a modulated (e.g., reduced) alkaloid content and / or a modulated (e.g., reduced) tobacco-specific nitrosamine (TSNA) precursor content by using a nucleotide sequence of at least one gene encoding a WAT1-related protein, preferably wherein the sequence of the WAT1-related protein is selected from SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28 or a functional variant or functional fragment or ortholog thereof; or wherein the sequence of the WAT1-related protein is a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one gene encoding a WAT1-related protein comprises a sequence of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof; or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0073] In another aspect, a mutant of a plant is provided that carries a heritable mutation in the nucleotide sequence of at least one gene encoding a WAT1-related protein, preferably wherein the gene is selected from SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 or a functional variant or functional fragment or ortholog thereof; or wherein the gene is selected from sequences having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30; wherein the heritable mutation modulates (e.g., reduces) the activity or expression of the at least one gene encoding a WAT1-related protein, and wherein the mutant plant has a modulated (e.g., reduced) alkaloid content and / or a modulated tobacco-specific nitrosamine (TSNA) precursor content relative to a comparable plant not carrying the heritable mutation.

[0074] In another aspect, progeny or seeds of a mutant plant carrying a heritable mutation according to the present invention are provided.

[0075] In another aspect, there is provided a harvested leaf, processed leaf or cured tobacco material produced from a plant comprising a modification in the nucleotide sequence of at least one gene encoding a WAT1-related protein, wherein the at least one gene is selected from SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 or a functional variant or functional fragment or ortholog thereof; or wherein the at least one gene is selected from the group consisting of SEQ ID NO. NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 having a sequence with at least 80% identity; wherein the modification modulates (e.g., reduces) the activity or expression of the at least one gene encoding a WAT1-related protein, and wherein the plant has modulated (e.g., reduced) alkaloid content and / or modulated tobacco-specific nitrosamine (TSNA) precursor content relative to a comparable plant not carrying the modification in the at least one gene encoding a WAT1-related protein.

[0076] In another aspect, there is provided a method, leaf, plant, plant propagation material, harvested leaf, processed tobacco, tobacco product, use, or combination thereof as herein described with reference to the specification and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0078] Figure 1 Shown are PON content in leaves of 5-week-old TN90 plants overexpressing Nitab4.5_0010919g0010.2 (SEQ ID NO. 3). PON content is expressed relative to the control and includes two biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.001.

[0079] Figure 2 PON content is shown in 5-week-old TN90 leaves expressing antisense RNA targeting Nitab4.5_0010919g0010.2 (SEQ ID NO. 3). PON content is expressed relative to the control and includes two biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.001.

[0080] Figure 3Shown are PON levels in 5-week-old TN90 leaves expressing an artificial miRNA (SEQ ID NO. 31) targeting Nitab4.5_0010919g0010.2 (SEQ ID NO. 3). PON levels are expressed relative to controls and include two biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.001.

[0081] Figure 4 The amino acid sequence of Nitab4.5_0010919g0010.2 is shown - SEQ ID NO. 1 - from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0082] Figure 5 The coding sequence of Nitab4.5_0010919g0010.2 is shown - SEQ ID NO. 2 - encoding the protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0083] Figure 6 The genomic sequence of Nitab4.5_0010919g0010.2 is shown – SEQ ID NO. 3 – encoding the protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0084] Figure 7 The amino acid sequence of Nitab4.5_0001086g0030.2 is shown - SEQ ID NO. 4 - from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0085] Figure 8 The coding sequence of Nitab4.5_0001086g0030.2 is shown - SEQ ID NO. 5 - encoding a protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0086] Figure 9 The genomic sequence of Nitab4.5_0001086g0030.2 is shown – SEQ ID NO. 6 – encoding the protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0087] Figure 10 The amino acid sequence of Nitab4.5_0003292g0030.2 is shown - SEQ ID NO. 7 - from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0088] Figure 11 The coding sequence of Nitab4.5_0003292g0030.2 is shown - SEQ ID NO. 8 - encoding a protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0089] Figure 12 The genomic sequence of Nitab4.5_0003292g0030.2 – SEQ ID NO. 9 – is shown – encoding the protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0090] Figure 13 The amino acid sequence of Nitab4.5_0004697g0010.2 is shown - SEQ ID NO. 10 - from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0091] Figure 14 The coding sequence of Nitab4.5_0004697g0010.2 - SEQ ID NO. 11 - is shown, encoding a protein derived from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0092] Figure 15 The genomic sequence of Nitab4.5_0004697g0010.2 – SEQ ID NO. 12 – is shown, encoding the protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0093] Figure 16 The amino acid sequence of Nitab4.5_0005998g0010.2 is shown – SEQ ID NO. 13 – from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0094] Figure 17 The coding sequence of Nitab4.5_0005998g0010.2 - SEQ ID NO. 14 - is shown, encoding a protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0095] Figure 18 The genomic sequence of Nitab4.5_0005998g0010.2 – SEQ ID NO. 15 – is shown, encoding the protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0096] Figure 19 The amino acid sequence of Nitab4.5_0011862g0010.2 is shown – SEQ ID NO. 16 – from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0097] Figure 20 The coding sequence of Nitab4.5_0011862g0010.2 is shown - SEQ ID NO. 17 - encoding a protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0098] Figure 21 The genomic sequence of Nitab4.5_0011862g0010.2 – SEQ ID NO. 18 – is shown, encoding the protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0099] Figure 22 The amino acid sequence of Nitab4.5_0000062g0210.2 is shown - SEQ ID NO. 19 - from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0100] Figure 23 The coding sequence of Nitab4.5_0000062g0210.2 is shown - SEQ ID NO. 20 - encoding a protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0101] Figure 24 The genomic sequence of Nitab4.5_0000062g0210.2 – SEQ ID NO. 21 – is shown – encoding the protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0102] Figure 25 The amino acid sequence of Nitab4.5_0008142g0020.2 is shown - SEQ ID NO. 22 - from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0103] Figure 26 The coding sequence of Nitab4.5_0008142g0020.2 is shown - SEQ ID NO. 23 - encoding a protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0104] Figure 27 The coding sequence of Nitab4.5_0008142g0020.2 is shown - SEQ ID NO. 24 - encoding a protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0105] Figure 28 The amino acid sequence of Nitab4.5_0000062g0220.2 is shown - SEQ ID NO. 25 - from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0106] Figure 29 The coding sequence of Nitab4.5_0000062g0220.2 is shown - SEQ ID NO. 26 - encoding a protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0107] Figure 30 The coding sequence of Nitab4.5_0000062g0220.2 is shown - SEQ ID NO. 27 - encoding a protein from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0108] Figure 31 The amino acid sequence of Nitab4.5_0001674g0010.2 is shown – SEQ ID NO. 28 – from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0109] Figure 32 The coding sequence of Nitab4.5_0001674g0010.2 - SEQ ID NO. 29 - is shown, encoding a protein derived from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0110] Figure 33 The coding sequence of Nitab4.5_0001674g0010.2 - SEQ ID NO. 30 - is shown, encoding a protein derived from Nicotiana tabacum ( Nicotiana tabacum ) of WAT1-related proteins.

[0111] Figure 34 The amino acid sequence of Arabidopsis thaliana WAT1 – SEQ ID NO. 31 is shown.

[0112] Figure 35 SEQ ID NO. 32, the amino acid sequence of the first EamA domain of Arabidopsis thaliana WAT1 (residues 32-161 of SEQ ID NO. 31), is shown.

[0113] Figure 36 SEQ ID NO. 33, the amino acid sequence of the second EamA domain of Arabidopsis thaliana WAT1 (residues 210-339 of SEQ ID NO. 31), is shown.

[0114] Figure 37 SEQ ID NO. 34, the amino acid sequence of the first EamA domain of Nitab4.5_0010919g0010.2 (residues 31-160 of SEQ ID NO. 1), is shown.

[0115] Figure 38 SEQ ID NO. 35, the amino acid sequence of the second EamA domain of Nitab4.5_0010919g0010.2 (residues 196-329 of SEQ ID NO. 1), is shown.

[0116] Figure 39 SEQ ID NO. 36, the artificial miRNA sequence targeting Nitab4.5_0010919g0010.2 used in Example 3, is shown.

[0117] Sequence Listing

[0118] A summary of sequence identifiers used throughout this specification and the corresponding sequence listing is provided, wherein:

[0119] SEQ ID NO. 1 corresponds to the amino acid sequence of Nitab4.5_0010919g0010.2.

[0120] SEQ ID NO. 2 corresponds to the coding sequence of Nitab4.5_0010919g0010.2.

[0121] SEQ ID NO. 3 corresponds to the genomic sequence of Nitab4.5_0010919g0010.2.

[0122] SEQ ID NO. 4 corresponds to the amino acid sequence of Nitab4.5_0001086g0030.2.

[0123] SEQ ID NO. 5 corresponds to the coding sequence of Nitab4.5_0001086g0030.2.

[0124] SEQ ID NO. 6 corresponds to the genomic sequence of Nitab4.5_0001086g0030.2.

[0125] SEQ ID NO. 7 corresponds to the amino acid sequence of Nitab4.5_0003292g0030.2.

[0126] SEQ ID NO. 8 corresponds to the coding sequence of Nitab4.5_0003292g0030.2.

[0127] SEQ ID NO. 9 corresponds to the genomic sequence of Nitab4.5_0003292g0030.2.

[0128] SEQ ID NO. 10 corresponds to the amino acid sequence of Nitab4.5_0004697g0010.2.

[0129] SEQ ID NO. 11 corresponds to the coding sequence of Nitab4.5_0004697g0010.2.

[0130] SEQ ID NO. 12 corresponds to the genomic sequence of Nitab4.5_0004697g0010.2.

[0131] SEQ ID NO. 13 corresponds to the amino acid sequence of Nitab4.5_0005998g0010.2.

[0132] SEQ ID NO. 14 corresponds to the coding sequence of Nitab4.5_0005998g0010.2.

[0133] SEQ ID NO. 15 corresponds to the genomic sequence of Nitab4.5_0005998g0010.2.

[0134] SEQ ID NO. 16 corresponds to the amino acid sequence of Nitab4.5_0011862g0010.2.

[0135] SEQ ID NO. 17 corresponds to the coding sequence of Nitab4.5_0011862g0010.2.

[0136] SEQ ID NO. 18 corresponds to the genomic sequence of Nitab4.5_0011862g0010.2.

[0137] SEQ ID NO. 19 corresponds to the amino acid sequence of Nitab4.5_0000062g0210.2.

[0138] SEQ ID NO. 20 corresponds to the coding sequence of Nitab4.5_0000062g0210.2.

[0139] SEQ ID NO. 21 corresponds to the genomic sequence of Nitab4.5_0000062g0210.2.

[0140] SEQ ID NO. 22 corresponds to the amino acid sequence of Nitab4.5_0008142g0020.2.

[0141] SEQ ID NO. 23 corresponds to the coding sequence of Nitab4.5_0008142g0020.2.

[0142] SEQ ID NO. 24 corresponds to the genomic sequence of Nitab4.5_0008142g0020.2.

[0143] SEQ ID NO. 25 corresponds to the amino acid sequence of Nitab4.5_0000062g0220.2.

[0144] SEQ ID NO. 26 corresponds to the coding sequence of Nitab4.5_0000062g0220.2.

[0145] SEQ ID NO. 27 corresponds to the genomic sequence of Nitab4.5_0000062g0220.2.

[0146] SEQ ID NO. 28 corresponds to the amino acid sequence of Nitab4.5_0001674g0010.2.

[0147] SEQ ID NO. 29 corresponds to the coding sequence of Nitab4.5_0001674g0010.2.

[0148] SEQ ID NO. 30 corresponds to the genomic sequence of Nitab4.5_0001674g0010.2.

[0149] SEQ ID NO. 31 corresponds to the amino acid sequence of the WAT1 protein from Arabidopsis thaliana.

[0150] SEQ ID NO. 32 corresponds to the amino acid sequence of the first EamA domain of Arabidopsis thaliana WAT1 (residues 32-161 of SEQ ID NO. 31).

[0151] SEQ ID NO. 33 corresponds to the amino acid sequence of the first EamA domain of Arabidopsis thaliana WAT1 (residues 210-339 of SEQ ID NO. 31).

[0152] SEQ ID NO. 34 corresponds to the amino acid sequence of the first EamA domain of Nitab4.5_0010919g0010.2 (residues 31-160 of SEQ ID NO. 1).

[0153] SEQ ID NO. 35 corresponds to the amino acid sequence of the second EamA domain of Nitab4.5_0010919g0010.2 (residues 196-329 of SEQ ID NO. 1).

[0154] SEQ ID NO. 36 is the artificial miRNA sequence targeting Nitab4.5_0010919g0010.2 used in Example 3.

[0155] Some of the sequences disclosed herein contain "X" or "N" in the nucleotide sequence. An "X" or "N" can be a deletion or insertion of any nucleotide or one or more nucleotides. For example, in some cases, a string of "X"s or "N"s is shown. The number of "X"s or "N"s does not necessarily correlate with the actual number of nucleotides at that position. More or fewer nucleotides than indicated by "X"s or "Ns" may be present in the sequence. DETAILED DESCRIPTION

[0156] The present inventors have shown for the first time that by modulating the activity or expression of at least one gene encoding a WAT1-related protein in a plant (eg a tobacco plant), the alkaloid and / or TSNA precursor content of the plant (or processed plant) can be modulated.

[0157] The present invention provides a method for modulating (eg, reducing) the alkaloid content of a plant or a part thereof, the method comprising modifying the plant by modulating (eg, reducing) the activity or expression of at least one gene encoding a WAT1-related protein.

[0158] Also provided are methods of modulating (e.g., reducing) the level of tobacco-specific nitrosamine (TSNA) precursors in a tobacco plant or plant part thereof, the method comprising modifying the plant by modulating (e.g., reducing) the activity or expression of at least one gene encoding a WAT1-related protein.

[0159] The at least one gene encoding a WAT1-related protein may be selected from at least one gene encoding a WAT1-related protein, the WAT1-related protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleic acid sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30. Suitably, the WAT1-associated protein may comprise one or more EamA domains. Suitably, the WAT1-associated protein may comprise two EamA domains.

[0160] Suitably, more than one WAT1-related protein may be modified. In one embodiment, at least one WAT1-related protein gene is modified and is selected from a gene encoding a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one WAT1-related protein encoding gene comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a sequence having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO.

[0161] In one embodiment, at least two WAT1-related protein genes are modified, which are selected from genes encoding polypeptides comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one WAT1-related protein encoding gene comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO.

[0162] In one embodiment, at least three, such as at least four, such as at least five, such as at least six, such as at least seven, such as at least eight, such as at least nine, such as ten WAT1-related protein genes are modified, which are selected from genes encoding polypeptides comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleic acid sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30. Suitably, the WAT1-associated protein may comprise one or more EamA domains. Suitably, the WAT1-associated protein may comprise two EamA domains.

[0163] In one aspect, the at least one WAT1-related protein gene encodes a polypeptide comprising the amino acid sequence as shown in SEQ ID NO. 1, or a functional variant, functional fragment, or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1; or wherein the at least one gene encoding a WAT1-related protein comprises the nucleotide sequence as shown in SEQ ID NO. 2 or 3, or a functional variant, functional fragment, or ortholog thereof, or a nucleic acid sequence having at least 80% identity with SEQ ID NO. 2 or 3. Suitably, the WAT1-related protein may comprise one or more EamA domains. Suitably, the WAT1-related protein may comprise two EamA domains.

[0164] In one aspect, the activity or expression of at least one further gene is modulated. Suitably, at least two (or at least three or at least four or at least five or at least six or at least seven or at least eight or at least nine) further genes selected from SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30, or a nucleic acid sequence having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 27 or 30 may also be modulated. Suitably, the WAT1-associated protein may comprise one or more EamA domains. Suitably, the WAT1-associated protein may comprise two EamA domains.

[0165] The "expression" of a gene encoding a WAT1-related protein may refer to the level of transcription, translation, ie, protein expression.

[0166] Measuring the level or amount of a gene product can be performed by any suitable method, such as comparison of mRNA transcript levels, protein or peptide levels and / or plant phenotype between modified plants and comparable plants not modified according to the invention.

[0167] As defined herein, the term "comparable product" will be a product derived from a plant (e.g., a tobacco plant) that has not been modified according to the present invention, but in which all other relevant characteristics are the same (e.g., plant species, growing conditions, methods of processing the plant, such as tobacco, etc.). A comparable product according to the present invention may mean a plant (e.g., a tobacco plant) or part thereof, such as a leaf (e.g., a tobacco leaf), a harvested leaf (e.g., a harvested tobacco leaf), a cut harvested leaf (e.g., a cut harvested tobacco leaf), a processed leaf (e.g., a processed tobacco leaf), or a plant propagation material (e.g., a tobacco plant propagation material), or a product, such as a tobacco product, or a combination thereof, that is obtainable or derived from a plant that has not been modified according to the present invention, e.g., to modulate the activity or expression of a gene encoding a WAT1-related protein. In one embodiment, a comparable product is a product that does not comprise a gene encoding a WAT1-related protein whose activity or expression has been modulated.

[0168] As used herein, the term "modification" or "modified" refers to a plant (e.g., a tobacco plant) or nucleic acid sequence that has been altered or changed. The present invention encompasses plant modifications using techniques for genetic modification of plants or non-genetic modification of plants. Such methods are well known in the art, and examples of genetic modification techniques include transformation, transgenic, cisgenics, and gene editing methods. Examples of non-genetic modification techniques include fast neutron mutagenesis, chemical mutagenesis such as ethyl methanesulfonate (EMS) mutagenesis, and modern population analysis methods.

[0169] In one embodiment, a natural variant with a modified WAT-1 gene is selected and this trait or gene is bred into a second plant that may have a commercially desirable trait.

[0170] In one embodiment, the plant according to the invention is a transgenic plant.In one embodiment, the plant according to the invention is a non-transgenic plant.

[0171] As defined herein, the term "unmodified plant" refers to a plant (e.g., a tobacco plant) that has not been modified according to the present invention, for example, to modulate the activity or expression of a gene encoding a WAT1-related protein or to modify the nucleic acid sequence of at least one gene encoding a WAT1-related protein; and wherein all other relevant characteristics are the same (e.g., plant species, growth conditions, method of processing tobacco, etc.). In one embodiment, an unmodified plant is a plant that does not contain a gene encoding a WAT1-related protein whose activity or expression has been modulated. In one embodiment, an unmodified plant is a plant that does not contain a modified nucleic acid sequence encoding at least one gene encoding a WAT1-related protein.

[0172] WAT1-associated proteins

[0173] As used herein, "WAT1-related protein" has its ordinary meaning in the art and refers to a protein expressed as Simulation Arabidopsis thaliana gene Wall Thin 1 (WAT1) Arabidopsis WAT1 protein (i.e., WAT1 The illustrative sequence of the protein encoded by the gene is provided by UniProtKB database entry Q94AP3-1. Figure 35 31).

[0174] Arabidopsis WAT1 protein is a predicted intrinsic membrane protein with 10 predicted transmembrane domains and is a member of the plant drug / metabolite export protein (P-DME) family (transporter classification number: TC 2.A.7.4). WAT1 protein is ubiquitously expressed, but is preferentially associated with vascular tissues (such as developing xylem vessels and fibers). At the cellular level, WAT1 protein is localized to the tonoplast (the membrane surrounding the main vacuole). WAT1 participates in cell wall formation (Ranocha et al. (2010) The Plant Journal 63, 469-483) and exports auxins (auxin) from the vacuole (Ranocha et al. (2013) Nature Communications 4: 2625).

[0175] Arabidopsis WAT1 contains two EamA domains, specifically at residues 32-161 and 210-339 of SEQ ID NO. 31. The sequences of the first and second EamA domains from WAT1 are respectively Figure 36 and 37 The EamA domain is presented as SEQ ID NO. 32 and SEQ ID NO. 33 in WAT1. The EamA domain is found in a wide range of proteins in bacteria and plants, many of which are predicted integral membrane proteins and have two copies of the EamA domain. The EamA domain was previously called DUF6 (domain of unknown function 6). Each EamA domain in WAT1 spans five predicted transmembrane domains.

[0176] WAT1-associated proteins can be identified by comparing the protein in question with the Arabidopsis WAT1 protein. The amino acid sequences of the proteins can be compared, where sequence identity or similarity to the Arabidopsis WAT1 amino acid sequence (SEQ ID NO.31) will indicate that the protein is a WAT1-associated protein. Alternatively or alternatively, the domain structure of the protein can be compared. Domain prediction software known in the art can be used to identify domains within the amino acid sequence of the protein. Domains are also described in protein databases such as UniprotKB. Proteins with domain structures similar to those of WAT1 will be considered WAT1-associated proteins. Specifically, WAT1 contains two EamA domains. Proteins containing two predicted EamA domains with configurations similar to those found in WAT1 can be considered WAT1-associated proteins.

[0177] An example of a WAT1-related protein is the protein encoded by the tobacco gene Nitab4.5_0010919g0010.2. The protein, coding, and gene sequences of Nitab4.5_0010919g0010.2 are located in Figure 5 、 6 and 7 are presented as SEQ ID NOs. 1, 2, and 3. The Nitab4.5_0010919g0010.2 protein contains two EamA domains, the first at residues 31-160 of SEQ ID NO. 1, and the second at residues 196-329 of SEQ ID NO. 1. The sequences of the first and second EamA domains from the Nitab4.5_0010919g0010.2 WAT1-related protein are respectively Figure 38 and 39 Presented as SEQ ID NO. 34 and SEQ ID NO. 35.

[0178] In one embodiment, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains. As used herein, the term "EamA domain" refers to a conserved domain. The EamA domain comprises one or more transmembrane domains, such as two or more transmembrane domains, such as three or more transmembrane domains. The EamA domain can be identified by comparing the sequence and / or predicted structure of a portion of the amino acid sequence of the protein with the first and second EamA domains of WAT1 and / or Nitab4.5_0010919g0010.2 protein (SEQ ID NO.32, 33, 34 or 35). In one embodiment, the EamA domain comprises the amino acid sequence shown in SEQ ID NO. 32, 33, 34 or 35, or a sequence having at least 80% identity to SEQ ID NO. 32, 33, 34 or 35.

[0179] In one embodiment, the WAT1-related protein is a predicted integral membrane protein comprising five or more transmembrane domains. Suitably, the WAT1-related protein may comprise ten transmembrane domains.

[0180] In one embodiment, the WAT1-related protein is a transporter. As used herein, the term "transporter" refers to an intrinsic membrane protein that facilitates the transport of molecules (such as metabolites or plant hormones) across a membrane. In one embodiment, the WAT1-related protein is an auxin transporter. In one embodiment, the WAT1-related protein is a member of the P-DME family.

[0181] Without wishing to be bound by theory, it is hypothesized that regulating the content of WAT1-related proteins in plant cells or regulating the activity of WAT1-related proteins in plants, such as transporter activity, will regulate the transport of molecules, such as metabolites, between specific compartments of the cell and thereby change the distribution of metabolites within the cell. These changes in the distribution of metabolites alter the metabolic pathways that produce alkaloids and TSNA precursors (such as PON), resulting in the regulation of alkaloid content.

[0182] In one embodiment, the WAT1-related protein comprises the amino acid sequence of SEQ ID NO. 1, or a sequence having at least 80% identity thereto, or a homologue thereof. Suitably, the WAT1-related protein comprises one or more EamA domains. Suitably, the WAT1-related protein comprises two EamA domains. Suitably, the homologue of SEQ ID NO. 1 may be selected from the group consisting of SEQ ID NOs. 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% identity thereto. Suitably, the homologue of SEQ ID NO. 1 may be selected from: SEQ ID NO. 4, 7, 10, 13, 16, 19, 22, 25 or 28, wherein the sequence comprises one or more EamA domains, such as two EamA domains, or a sequence that is at least 80% identical to SEQ ID NO. 4, 7, 10, 13, 16, 19, 22, 25 or 28 and comprises one or more EamA domains, such as two EamA domains.

[0183] In one embodiment, the WAT1-related protein comprises an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% identity thereto (preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto). In one embodiment, the WAT1-related protein comprises an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% identity thereto (preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto) and comprising one or more EamA domains, such as two EamA domains.

[0184] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence as shown in SEQ ID NO. 1, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0185] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence shown in SEQ ID NO. 4, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0186] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence shown in SEQ ID NO. 7, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0187] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence shown in SEQ ID NO. 10, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0188] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence shown in SEQ ID NO. 13, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0189] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence shown in SEQ ID NO. 16, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0190] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence shown in SEQ ID NO. 19, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0191] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence shown in SEQ ID NO. 22, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0192] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence shown in SEQ ID NO. 25, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0193] Suitably, the WAT1-associated protein according to the present invention may comprise the amino acid sequence as shown in SEQ ID NO. 28, or a sequence having at least 80% identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0194] In one embodiment, the WAT1-related protein according to the present invention comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28.

[0195] Suitably, the protein may be derived from tobacco.

[0196] In one embodiment, the WAT1-related protein is encoded by a polynucleotide sequence, wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30; or a sequence having at least 80% sequence identity thereto. Suitably, the WAT1-related protein comprises one or more EamA domains. Suitably, the WAT1-related protein comprises two EamA domains.

[0197] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 2, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0198] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 3, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0199] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 5, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0200] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 6, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0201] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 8, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0202] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 9, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0203] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises a sequence as shown in SEQ ID NO. 11, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0204] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 12, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0205] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 14, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0206] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 15, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0207] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 17, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0208] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 18, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0209] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 20, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0210] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 21, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0211] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 23, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0212] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 24, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0213] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 26, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0214] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 27, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0215] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 29, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0216] Suitably, the WAT1-associated protein for use according to the present invention may be encoded by a polynucleotide sequence wherein the gene (before mutation) comprises the sequence shown in SEQ ID NO. 30, or a sequence having at least 80% sequence identity thereto (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto). Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0217] In one embodiment, the WAT1-related protein is encoded by a polynucleotide sequence, wherein the gene (before mutation) is selected from: SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0218] Suitably, the protein for use according to the present invention may be encoded by a polynucleotide sequence from Nicotiana tabacum.

[0219] In one aspect, the present invention provides a method for reducing the alkaloid content of a plant or a part or cell thereof (eg, a plant cell), the method comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding a WAT1-related protein.

[0220] In one aspect, the present invention provides a method for reducing the alkaloid content of a plant or a part thereof or a plant cell, the method comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding a WAT1-related protein, the WAT1-related protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30. Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0221] In one aspect, the present invention provides a method for reducing the level of tobacco-specific nitrosamine (TSNA) precursors in a plant or part thereof (e.g., leaves), the method comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding a WAT1-related protein.

[0222] In one aspect, the present invention provides a method for reducing the content of tobacco-specific nitrosamine (TSNA) precursors in a plant or a part thereof (e.g., a leaf), the method comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding a WAT1-related protein, the WAT1-related protein comprising an amino acid sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto. NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30. A functional variant or functional fragment or ortholog, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0223] In one aspect, the present invention provides a method of reducing the TSNA content in processed foliage, such as cured foliage, the method comprising:

[0224] Modifying a plant by reducing or inhibiting the activity or expression of at least one gene encoding a WAT1-related protein;

[0225] harvesting leaves from the plant;

[0226] and conditioning the harvested leaves.

[0227] Suitably, the method for reducing the content of TSNAs in processed leaves may comprise modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding a WAT1-related protein comprising an amino acid sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0228] As used herein, the term "reduce" or "inhibit" (e.g., inhibit the activity or expression of a gene encoding a WAT1-related protein) means that the activity or expression of a gene encoding a WAT1-related protein is lower or reduced compared to the activity or expression of the gene in a comparable product.

[0229] In one aspect, the present invention provides a method for increasing the alkaloid content of a plant or a part or cell thereof (eg, a plant cell), the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding a WAT1-related protein.

[0230] In one aspect, the present invention provides a method for increasing the alkaloid content of a plant or a part thereof or a plant cell, the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding a WAT1-related protein, the WAT1-related protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30. Suitably, the WAT1-associated protein comprises one or more EamA domains. Suitably, the WAT1-associated protein comprises two EamA domains.

[0231] In one aspect, the present invention provides a method for increasing the content of tobacco-specific nitrosamine (TSNA) precursors in a plant or part thereof (e.g., leaves), the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding a WAT1-related protein.

[0232] In one aspect, the present invention provides a method for increasing the content of tobacco-specific nitrosamine (TSNA) precursors in a plant or a part thereof (e.g., a leaf), the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding a WAT1-related protein, the WAT1-related protein comprising an amino acid sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto, or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence having at least 80% identity thereto. NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30. A functional variant or functional fragment or ortholog, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0233] As used herein, the term "increase" or "enhance" (e.g., increase the activity or expression of a gene encoding a WAT1-related protein) means that the activity or expression of a gene encoding a WAT1-related protein is higher or increased compared to the activity or expression of the gene in a comparable product.

[0234] According to the present invention, the activity or expression of a gene encoding a WAT1-related protein is modulated.

[0235] In one aspect, the present invention provides a method for modulating (i.e., increasing or decreasing) the alkaloid content of a plant or a part or cell thereof (e.g., a plant cell), the method comprising modifying the plant by modulating (i.e., increasing or decreasing) the activity of at least one gene encoding a WAT1-related protein.

[0236] The term "activity" refers to any function of a WAT1-related protein encoded by at least one gene. Examples of activity include enzymatic activity or localization of a WAT1-related protein.

[0237] Suitably, the activity may be the ability of a WAT1-associated protein to interact with another molecule or molecules. In some embodiments, the present invention provides a method for modulating (i.e., increasing or decreasing) the alkaloid content of a plant, or a part or cell thereof (e.g., a plant cell), the method comprising modifying the plant by modulating (i.e., increasing or decreasing) the ability of a WAT1-associated protein to interact with another molecule.

[0238] Suitably, the ability of the WAT1-associated protein to interact with other molecules is the ability to bind to other molecules. The other molecules may be proteins. Suitably, the other molecules are more than one molecule, such as one or more molecules, such as two or more molecules, such as three or more molecules. When the other molecules are more than one molecule, the other molecules may be the same molecule or may be different molecules.

[0239] Suitably, the activity is the ability of one or more other molecules of WAT1-related protein transmembrane transport. In some embodiments, the invention provides a method for regulating (i.e., increasing or decreasing) the alkaloid content of a plant or its part or cell (e.g., a plant cell), the method comprising modifying the plant by regulating (i.e., increasing or decreasing) the ability of another molecule of WAT1-related protein transmembrane transport. Other molecules can be proteins. Other molecules can be metabolites. Other molecules can be plant hormones. Suitably, other molecules can be auxins. Suitably, other molecules are more than one molecule, such as one or more molecules, such as two or more molecules, such as three or more molecules. In the case where other molecules are more than one molecule, other molecules can be the same molecule or can be different molecules. Suitably, the membrane to be transported across by other molecules is the tonoplast.

[0240] Modulation of the activity of a gene encoding a WAT1-related protein may require increasing or decreasing the activity of the WAT1-related protein.

[0241] Increasing the activity of a WAT1-related protein refers to enhancing or improving the ability of the WAT1-related protein to perform a specific function compared to the WAT1-related protein in a plant that has not been modified according to the present invention.

[0242] Reducing the activity of a WAT1-related protein means reducing, inhibiting, or destroying the ability of a WAT1-related protein to perform a specific function compared to a WAT1-related protein in a plant that has not been modified according to the present invention. The activity of a WAT1-related protein can be reduced to such an extent that the activity is prevented or eliminated.

[0243] In some embodiments, the activity of a WAT1-related protein may be modulated (i.e., increased or decreased) by at least about 10%, 20%, 30% or 40%, suitably at least about 50%, 60%, 70%, more suitably at least about 80%, 90%, 95% or 100%, compared to the activity of a gene encoding a WAT1-related protein in a plant (e.g., a tobacco plant) not modified according to the invention.

[0244] In some embodiments, the regulated WAT1-related protein exhibits an increased or decreased activity compared to an unmodified WAT1-related protein. Compared to an unmodified WAT1-related protein, the regulated WAT1-related protein may exhibit an activity that is increased or decreased by at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%.

[0245] Suitably, modulation of activity is an increase or decrease in the ability of a WAT1 -related protein to interact with (such as bind to) another molecule or molecules.

[0246] Suitably, the modulation of activity is an increase or decrease in the ability of a WAT1-related protein to transport one or more other molecules across a membrane. For example, the ability of a WAT-1-related protein to transport one or more other molecules across a membrane can be altered by disrupting one or more transmembrane domains. The transmembrane domain can be disrupted by deletion or mutation. Suitably, a mutation in a transmembrane domain may not prevent the protein from crossing the domain but may prevent transport by the protein. Suitably, a mutation in a transmembrane domain may prevent the protein from crossing the membrane.

[0247] Techniques for measuring protein activity are known in the art. For example, assays for measuring the enzymatic activity of proteins are known, and microscopy techniques can be used to identify the localization of proteins.

[0248] Specifically, the ability of a WAT1-related protein to bind to another molecule can be measured using techniques known in the art. Examples of such techniques include immunoprecipitation, isothermal calorimetry, surface plasmon resonance, and microlayer thermophoresis. For example, the ability of a regulated or mutated WAT1-related protein to bind to another molecule can be determined by performing a co-immunoprecipitation experiment using a regulated or mutated WAT1-related protein and a corresponding unmodified or unmutated WAT1-related protein. If the regulation or mutation of a WAT1-related protein reduces, inhibits, or eliminates the ability of the WAT1-related protein to bind to another molecule, the co-immunoprecipitation will show that the regulated or mutated WAT1-related protein binds less to the other molecule.

[0249] The ability of a WAT-1-associated protein to transport one or more additional molecules across a membrane can be measured using techniques known in the art. Examples of such techniques include surface plasmon resonance, plasmon-waveguide resonance, combinatorial chip-spectrometry, fluorescence measurement, scintillation proximity assay, electrophysiological assay, or stopped-flow assay. According to the present invention, the activity or expression of a WAT1-associated protein is modulated.

[0250] In one aspect, the present invention provides a method for modulating (i.e., increasing or decreasing) the alkaloid content of a plant or a part or cell thereof (e.g., a plant cell), the method comprising modifying the plant by modulating (i.e., increasing or decreasing) the expression of at least one gene encoding a WAT1-related protein.

[0251] "Expression" of a gene refers to the degree to which the information encoded in the gene is converted into function. The expression level of a gene can be equated with the amount of the gene product present in a cell or organism. Modifications that modulate (i.e., increase or decrease) gene expression are modifications that increase the amount of the gene product in a plant or cell compared to an unmodified plant or cell.

[0252] In some embodiments, expression of a WAT1-related protein is modulated (ie, increased or decreased) compared to expression of a gene encoding the WAT1-related protein in a plant (eg, a tobacco plant) that has not been modified according to the present invention.

[0253] In some embodiments, the expression of a WAT1-related protein may be modulated (i.e., increased or decreased) by at least about 10%, 20%, 30% or 40%, suitably at least about 50%, 60%, 70%, more suitably at least about 80%, 90%, 95% or 100%, compared to the expression of a gene encoding a WAT1-related protein in a plant (e.g., a tobacco plant) not modified according to the invention.

[0254] In some embodiments, the regulated WAT1-related protein exhibits increased or decreased expression compared to unmodified WAT1-related proteins. Compared to unmodified WAT1-related proteins, the regulated WAT1-related proteins can exhibit at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% increased or decreased expression.

[0255] Typically, a gene is transcribed into mRNA, which is translated into protein, the final gene product. Proteins can be sequestered in cells for storage and / or degraded. Gene expression can be regulated by modulating any or all of these steps. Thus, in some embodiments, the modification modulates expression of at least one WAT1-related protein gene in one of the following ways:

[0256] Regulates transcription from at least one WAT1-related protein gene;

[0257] regulating translation of mRNA from at least one WAT1-related protein gene;

[0258] Regulating the release of WAT1-related proteins from intracellular stores; and / or

[0259] Regulates the degradation rate of WAT1-related proteins.

[0260] The expression of a specific gene encoding a WAT1-associated protein can be measured by measuring the transcription and / or translation of the gene. Methods for measuring transcription are well known in the art and include, in particular, northern blotting, RNA-Seq, in situ hybridization, DNA microarrays, and RT-PCR. Alternatively, the expression of a gene can be measured indirectly by measuring the level of a protein encoded by the gene product, for example. For example, the expression of a WAT1-associated protein can be determined by measuring the presence of the protein using an antibody specific to the WAT1-associated protein (e.g., an antibody specific to the EamA domain) by western blotting.

[0261] Modification

[0262] The plant or cell may be modified in any manner that modulates the activity or expression of at least one gene encoding a WAT1-related protein. The types of modifications to plants and cells that modulate the activity or expression of genes and the techniques for achieving these modifications are known in the art.

[0263] In some embodiments, the present invention provides a method of reducing the alkaloid content of a plant or a part or cell thereof (e.g., a plant cell), comprising modifying the plant by reducing or inhibiting the activity or expression of at least one gene encoding a WAT1-related protein.

[0264] In some embodiments, the present invention provides a method for reducing the level of tobacco-specific nitrosamines (TSNAs) or TSNA precursors in a tobacco plant or plant part thereof, the method comprising modifying the plant or cell culture by reducing the activity or expression of at least one gene encoding a WAT1-related protein.

[0265] Any method known in the art for reducing or inhibiting the activity or expression of a gene can be used in the methods according to the present invention.

[0266] Suitably, the activity or expression of the WAT1-related protein gene may be reduced, partially inactivated, inhibited, eliminated, knocked out or lost such that the protein activity, expression or function of the WAT1-related protein gene is undetectable.

[0267] In one aspect, at least one WAT1-related protein gene is knocked out. In other words, the WAT1-related protein has been rendered completely ineffective.

[0268] For example, the method may include:

[0269] ● providing a mutation in a nucleic acid sequence encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto;

[0270] ● providing a mutation in a regulatory region (e.g., a promoter or enhancer) that helps control the expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25, or 28, or an amino acid sequence having at least 80% sequence identity thereto;

[0271] ● Provide antisense RNA, siRNA or miRNA that reduces the level of a nucleic acid sequence encoding a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto.

[0272] Each of the above methods results in a reduction or inhibition of the activity or expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, or wherein at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID No. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 nucleic acid sequences that are at least 80% identical.

[0273] As used herein, the term "mutation" includes natural genetic variants or engineered variants. Specifically, the term "mutation" refers to a variation in a nucleotide sequence encoding an amino acid sequence or an amino acid sequence compared to a sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% (preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, preferably at least 98%, preferably at least 99%) sequence identity thereto.

[0274] In one embodiment, the mutation reduces the alkaloid content of the plant. In another embodiment, the mutation reduces the content of at least one TSNA precursor in the plant, or part or leaf thereof, such as harvested or processed leaf. In one embodiment, the mutation reduces the content of one or more TSNAs selected from NNK, NNN, NAT, NAB, preferably, the NNK content is reduced in processed leaf. Suitably, the TSNA content is reduced relative to a comparable product.

[0275] In one embodiment, the method according to the invention may comprise providing a nucleic acid sequence to a plant or part thereof or a plant cell, wherein said nucleic acid results in a reduction or elimination of the activity or expression of at least one gene encoding a WAT1-related protein.

[0276] In one embodiment, the method according to the invention may comprise providing a plant or part thereof or a plant cell with a nucleic acid sequence, wherein the nucleic acid results in the modification of the nucleic acid sequence of at least one gene encoding a WAT1-related protein.

[0277] Suitably, the nucleic acid sequence may be introduced into a plant or part or cell thereof. Suitably, an endogenous nucleic acid sequence in a plant or part or cell thereof may be modified to encode a polypeptide according to the present invention (e.g., by gene editing). For example, an endogenous nucleotide sequence may be modified to reduce the activity or expression of at least one gene encoding a WAT1-related protein.

[0278] In a preferred embodiment, each copy comprises a nucleic acid sequence encoding a protein comprising a sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% sequence identity thereto, or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 nucleic acid sequences with at least 80% identity (which are present in the plant) are modified, e.g., mutated as defined herein (e.g., each genomic copy of the gene encoding the protein in the plant is mutated). For example, each copy of the gene in the allotetraploid genome of tobacco may be mutated.

[0279] In a preferred embodiment, some or all of the homologs of the WAT1-related proteins described herein are modified, e.g., inhibited or mutated. Suitably, some or all of SEQ ID NOs. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or corresponding sequences thereof having at least 80% sequence identity thereto, are modified, e.g., inhibited or mutated.

[0280] In some embodiments, the plant or plant cell according to the present invention is homozygous. Suitably, the plant or plant cell may be homozygous for a modification such as an inhibition or mutation.

[0281] In some embodiments, the plants or plant cells according to the present invention express only modified, e.g., mutant, nucleic acids encoding WAT1-related proteins. In other words, in some embodiments, no endogenous (or endogenous and functional) proteins are present in the plants according to the present invention. In other words, if any endogenous protein is present, it is preferably in an inactive form.

[0282] In one embodiment, the method can include providing a mutation in a nucleic acid sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence having at least 80% identity thereto.

[0283] The mutation can change the plant genome so that the nucleic acid sequence encoding the protein comprising the amino acid sequence as shown in SEQ ID NO.1, 4, 7, 10, 13, 16, 19, 22, 25 or 28 or an amino acid sequence having at least 80% sequence identity thereto is completely or partially deleted or otherwise modified so as to inhibit or eliminate the ability of WAT1-related proteins to transport other molecules across the membrane compared to the protein shown in SEQ ID NO.1, 4, 7, 10, 13, 16, 19, 22, 25 or 28 or a sequence having at least 80% sequence identity thereto. In some embodiments, the mutation does not change the level or expression of the protein, but reduces, inhibits or eliminates the ability of WAT1-related proteins to transport other molecules across the membrane compared to the protein shown in SEQ ID No.1, 4, 7, 10, 13, 16, 19, 22, 25 or 28 or a sequence having at least 80% sequence identity thereto. Suitably, the mutation inhibits or eliminates the ability of WAT1-related proteins to transport auxins across the membrane. The expression "inhibit or eliminate" means that the amount of other molecules that move across the membrane via the WAT1-related protein is reduced, suitably to the extent that none of the other molecules move across the membrane.

[0284] Suitably, the mutation may be in the transmembrane domain of the WAT1-associated protein. Suitably, the WAT1-associated protein may comprise multiple mutations, each mutation being in a different transmembrane domain. Suitably, the WAT1-associated protein comprises mutations in at least two transmembrane domains, such as at least three transmembrane domains, such as at least four transmembrane domains, such as at least five transmembrane domains, such as at least six transmembrane domains, such as at least seven transmembrane domains, such as at least eight transmembrane domains, such as at least nine transmembrane domains, such as ten transmembrane domains. In some embodiments, the mutation in the transmembrane domain changes the ability of the WAT1-associated protein to localize to the membrane. Suitably, the mutation prevents the WAT1-associated protein from localizing to the membrane. For example, the transmembrane domain typically comprises hydrophobic amino acids that are suitable for the hydrophobic environment of the membrane. Replacing a hydrophobic amino acid with a hydrophilic amino acid means that the transmembrane domain cannot be located in the hydrophobic environment of the membrane, and therefore the ability of the WAT1-associated protein to localize to the membrane is reduced. Suitably, the mutation comprises replacing a hydrophobic acid in the transmembrane domain with a hydrophilic amino acid. Suitably, the mutation comprises deletion of one or more transmembrane domains.

[0285] The mutation may be in one or more EamA domains, such as two EamA domains, of the WAT1-associated protein. In some embodiments, one or more EamA domains, such as two EamA domains, may be mutated, thereby altering the ability of the WAT1-associated protein to localize to the membrane. In some embodiments, one or more EamA domains, such as two EamA domains, are deleted from the WAT1-associated protein.

[0286] The mutation may interrupt the nucleic acid sequence encoding a protein comprising the amino acid sequence shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto.

[0287] The interruption may result in the nucleic acid sequence not being transcribed and / or translated.

[0288] The nucleic acid sequence can be interrupted, for example, by deleting or otherwise modifying the ATG start codon of the nucleic acid sequence such that translation of the protein is reduced or prevented.

[0289] The nucleotide sequence may comprise one or more nucleotide changes that reduce or prevent protein expression or influence protein transport. For example, protein expression may be reduced or prevented by introducing one or more premature stop codons, frameshifts, splice mutations, or non-tolerant amino acid substitutions into the open reading frame.

[0290] A premature stop codon refers to a mutation that introduces a stop codon into the open reading frame and prevents translation of the entire amino acid sequence. The premature stop codon can be a TAG ("amber"), TAA ("ochre"), or TGA ("opal" or "umber") codon.

[0291] Frameshift mutations (also known as framing errors or frame shifts) are mutations caused by the insertion / deletion (insertion or deletion) of multiple nucleotides in a nucleic acid sequence that cannot be divided by three. Due to the triplet nature of gene expression through codons, insertions or deletions can change the reading frame, resulting in a translation that is completely different from the original. Frameshift mutations will often cause the mutated codon to be read to encode a different amino acid. Frameshift mutations will generally result in the introduction of premature stop codons.

[0292] Splicing mutations insert, delete, or change a number of nucleotides at specific sites where splicing occurs during the processing of precursor messenger RNA into mature messenger RNA. The loss of splicing sites results in one or more introns remaining in the mature mRNA and may lead to the production of abnormal proteins.

[0293] Non-tolerant amino acid substitutions refer to mutations that cause non-synonymous amino acid substitutions in a protein, which result in reduced or eliminated function of the protein.

[0294] Any method known in the art for providing mutations in nucleic acid sequences can be used in the methods according to the present invention. For example, homologous recombination can be used to generate vectors in which one or more related nucleic acid sequences are mutated and used to transform plants or plant cells. Recombinant plants or plant cells expressing the mutated sequences can then be selected.

[0295] In one embodiment, the mutation introduces a non-tolerant amino acid substitution in a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a sequence having at least 80% sequence identity thereto.

[0296] In some embodiments, the EamA domain may contain a mutation that reduces expression of at least one gene encoding a WAT1-related protein.

[0297] The mutation may be a deletion, a splice mutant, or a codon encoding a non-tolerant amino acid substitution.

[0298] In one embodiment, the nucleic acid sequence encoding the WAT1-related protein can be completely or partially deleted. The deletion can be continuous or can include multiple segments of the sequence. The deletion preferably removes a sufficient amount of nucleotide sequence so that the nucleic acid sequence no longer encodes a functional WAT1-related protein. When compared with the corresponding genome of a comparable unmodified plant, the deletion can be complete, in which case 100% of the coding portion of the nucleic acid sequence is absent. The deletion can, for example, remove at least 50, 60, 70, 80, or 90% of the coding portion of the nucleic acid sequence. Suitably, at least a portion of the protein can be deleted. The deletion can, for example, remove at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the coding portion of the protein.

[0299] Deletion can remove at least a portion of one or more EamA domains, such as two EamA domains. Deletion can, for example, remove at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of one or more EamA domains, such as two EamA domains. Suitably, deletion can remove at least 5 amino acids, at least 10 amino acids, at least 15, at least 20, at least 25, or at least 30 amino acids of one or more EamA domains, such as two EamA domains. Suitably, deletion can remove at least 5 amino acids, at least 10 amino acids, at least 15, at least 20, at least 25, or at least 30 amino acids of one or more EamA domains, such as two EamA domains.

[0300] Methods for deleting nucleic acid sequences in plants are known in the art. For example, homologous recombination can be used to produce a vector in which one or more related nucleic acid sequences are lost and used to transform a plant or plant cell. Recombinant plants or plant cells expressing the new sequence portion can then be selected.

[0301] Plant cells transformed with the vectors described herein can be grown and maintained according to well-known tissue culture methods, such as by culturing the cells in a suitable medium supplied with necessary growth factors such as amino acids, plant hormones, vitamins, and the like.

[0302] Targeted mutagenesis methods (also referred to as targeted nucleotide exchange (TNE) or oligonucleotide directed mutagenesis (oligo-directed mutagenesis) (ODM)) can be used to modify nucleotide sequences. Targeted mutagenesis methods include, but are not limited to, those employing zinc finger nucleases, TALENs (see WO2011 / 072246 and WO2010 / 079430), Cas9-samples, Cas9 / crRNA / tracrRNA, Cas9 / gRNA or other CRISPR systems (see WO2014 / 071006 and WO2014 / 093622), meganucleases (see WO2007 / 047859 and WO2009 / 059195), or employing the targeted mutagenesis methods of mutagenic oligonucleotides, which may contain chemically modified nucleotides (such as Or TALENs) for enhancing mutagenesis with sequence complementarity to genes that enter plant protoplasts.

[0303] Alternatively, mutagenesis systems such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al. (2000) Nat. Biotech. 18:455, and McCallum et al. (2000) Plant Physiol. 123, 439-442, both incorporated herein by reference) can be used to generate plant lines containing genes encoding proteins with mutations. TILLING uses traditional chemical mutagenesis (e.g., ethyl methanesulfonate (EMS) mutagenesis to generate random mutations) followed by high-throughput screening of mutations. Thus, plants, seeds, cells, and tissues containing genes with desired mutations can be obtained.

[0304] Said method may comprise the following steps: mutagenesis plant seeds (for example EMS mutagenesis), the merging of plant individual or DNA, the pcr amplification of target region, heteroduplex formation and high throughput detection, the evaluation of mutant plant, the order-checking of mutant pcr product.Should be understood that other mutagenesis and selection methods can be used to generate the plant of this type of modification equally.Seed can for example be irradiated or chemically treated, and can be for the phenotypic screening plant of modification.

[0305] Fast neutron deletion mutagenesis can be used in a reverse genetics sense (i.e., using PCR) to identify plant lines carrying deletions in endogenous genes. See, e.g., Ohshima et al. (1998) Virology 213:472-481; Okubara et al. (1994) Genetics 137:867-874; and Quesada et al. (2000) Genetics 154:421-4315, which are incorporated herein by reference.

[0306] In another approach, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of duplicated loci. See, e.g., Kusaba et al. (2003) Plant Cell 15: 1455-1467 (incorporated herein by reference).

[0307] Modified plants can be distinguished from unmodified plants, i.e., wild-type plants, by molecular methods, such as one or more mutations present in the DNA, and by the phenotypic characteristics of the modification. The modified plants can be homozygous or heterozygous for the modification. Preferably, the modified plants are homozygous for the modification.

[0308] In one embodiment, the method of reducing or preventing the activity or expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, does not comprise treating the plant with a chemical (e.g., an agrochemical).

[0309] Other ways of reducing or preventing expression will be apparent to those skilled in the art and include the use of virus-induced gene silencing (VIGs), microRNA silencing, RNAi, antisense, tDNA insertion, or dominant negative constructs (or antimorphic mutations).

[0310] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by virus-induced gene silencing.

[0311] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by microRNAs.

[0312] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by RNAi.

[0313] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by antisense inhibition.

[0314] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by sense suppression.

[0315] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by tDNA insertion.

[0316] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a dominant negative construct (or a retro-allele mutation).

[0317] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a targeted mutagenesis-based system.

[0318] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by a CRISPR-based system.

[0319] In one embodiment, expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto, can be reduced or eliminated by zinc finger nucleases, TALENs, meganucleases, mutagenic oligonucleotides or TILLING.

[0320] In some embodiments, the present invention provides a method of increasing the alkaloid content of a plant or a part or cell thereof (e.g., a plant cell), the method comprising modifying the plant by increasing or enhancing the activity or expression of at least one gene encoding a WAT1-related protein.

[0321] Any method known in the art for increasing or enhancing the activity or expression of a gene can be used in the methods according to the present invention.

[0322] In some embodiments, the method may include overexpressing at least one gene encoding a WAT1-related protein. Suitably, the method may include expressing one or more additional copies of at least one gene encoding a WAT1-related protein in a plant or cell. Suitably, the method may include modifying endogenous copies of at least one gene encoding a WAT1-related protein such that its expression is increased. The method may include mutating the coding sequence of at least one gene encoding a WAT1-related protein. The method may include mutating a regulatory sequence that regulates the expression of at least one gene encoding a WAT1-related protein.

[0323] Suitably, the method may comprise transforming a cell of a plant (e.g., a tobacco plant) with a gene construct encoding at least one WAT1-related protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 functional variants or functional fragments or orthologs, or with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30 having at least 80% identity of the nucleic acid sequence; or it comprises a nucleotide sequence encoding a protein capable of promoting or enhancing at least one endogenous WAT1-related protein gene. It should be understood that each of these options will result in an increase in the activity and expression of a polypeptide encoded by at least one WAT1-related protein gene. The method may comprise regenerating a plant from the transformed cell. Provided is the use of a gene construct capable of increasing the activity and / or expression of a polypeptide encoded by at least one WAT1-related protein gene for increasing the alkaloid content (e.g., nicotine content) in a plant, or part or cell thereof, transformed with the construct.

[0324] The gene construct may encode a polypeptide comprising the amino acid sequence of SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0325] In another embodiment, the present invention relates to a method for increasing the alkaloid content of a plant or a plant part thereof, comprising modifying said plant by increasing the activity of at least one gene encoding a WAT1-related protein.

[0326] In one embodiment, the activity of at least one gene encoding a WAT1-related protein may be increased by introducing (or providing) a mutation into at least one gene encoding an EamA domain.

[0327] Suitably, the activity of at least one gene encoding a WAT1-related protein may be increased by introducing a mutation into at least one gene encoding a WAT1-related protein, the WAT1-related protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0328] In some embodiments, the modification that increases the activity or expression of at least one WAT1-related protein gene and thereby increases the alkaloid content is by one of the following:

[0329] Regulates transcription from at least one WAT1-related protein gene;

[0330] regulating translation of mRNA from at least one WAT1-related protein gene;

[0331] Regulating the release of WAT1-related proteins from intracellular stores; and / or

[0332] Regulates the degradation rate of WAT1-related proteins.

[0333] Alkaloid content

[0334] In one embodiment, the present invention provides a method of modulating the alkaloid content of a plant (eg, a tobacco plant) or a part thereof, the method comprising modifying the plant by modulating the activity or expression of at least one gene encoding a WAT1-related protein.

[0335] The term "modulate" is used herein to mean to increase or decrease.

[0336] The term "increased alkaloid content" is used herein to mean that the alkaloid content in a product of the invention (e.g. a plant, a part thereof (e.g. leaves), a processed leaf or a product prepared from the plant (e.g. a tobacco product)) is higher than in a comparable product which has not been modified according to the invention.

[0337] The term "reduced alkaloid content" is used herein to mean that the alkaloid content in the product of the invention (e.g., a plant, a part thereof (e.g., leaves), processed leaves, or a product prepared from the plant (e.g., a tobacco product)) is lower than in a comparable product which has not been modified according to the invention.

[0338] In some embodiments, modulation of alkaloid content refers to an increase in alkaloid content, wherein the activity or expression of at least one WAT1-related protein gene is increased (or in other words, the protein is overexpressed).

[0339] In some embodiments, modulation of alkaloid levels refers to a decrease in alkaloid levels, wherein the expression of at least one gene encoding a WAT1-related protein is reduced, inhibited, or eliminated.

[0340] In a further aspect, the alkaloid content is measured from leaves. In one aspect, the alkaloid content is measured from green leaves. In a further aspect, the alkaloid content is measured from conditioned leaves, such as air-cured, flue-cured, fire-cured, or sun-cured leaves. In a further aspect, the alkaloid content is measured from flue-cured leaves. In a further aspect, the alkaloid content is measured from air-cured leaves.

[0341] The term "alkaloid content" is used herein to refer to the concentration and / or total amount of the entire group of compounds classified as alkaloids or the concentration and / or total amount of one or more compounds classified as alkaloids. Alkaloids commonly present in tobacco include nicotine, nornicotine, PON, anatabine, anabasine and mithosamine. In some embodiments, one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and mithosamine, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as the content of all six alkaloids is regulated. In some embodiments, one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and mithosamine, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as the content of all six alkaloids increases. In some embodiments, the content of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine, and mithosamine, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as all six alkaloids, is reduced. In some embodiments, the total alkaloid content of the plant or cell is modulated. In some embodiments, the total alkaloid content is increased. In some embodiments, the total alkaloid content is increased.

[0342] In one embodiment, the nicotine content is not adjusted, but the content of one or more alkaloids selected from PON, nornicotine, anatabine, anabasine and miosamine is adjusted. Suitably, the nicotine content is not adjustable, but the content of PON is adjusted. Suitably, the nicotine content is not adjustable, but the content of PON is reduced.

[0343] Any method known in the art for determining the concentration and / or total amount of alkaloids can be used. A preferred method for analyzing alkaloid content involves analysis by gas chromatography-flame ionization detection (GC-FID) or by reversed-phase high performance liquid chromatography and tandem mass spectrometry (LC-MS / MS).

[0344] In one embodiment, a method is provided for producing a plant (e.g., a tobacco plant) or part thereof obtainable or obtained by a plant of the present invention, plant propagation material (e.g., tobacco plant propagation material), cell (e.g., tobacco cell), leaf (e.g., tobacco leaf), harvested leaf (e.g., harvested tobacco leaf), cut harvested leaf (e.g., cut harvested tobacco leaf), processed leaf (e.g., processed tobacco leaf), cut and processed leaf (e.g., cut and processed tobacco leaf), product (e.g., tobacco product) comprising said plant or part thereof, or a combination thereof, said plant of the present invention having a modulated alkaloid content, said method comprising modifying said plant to modulate the activity or expression of a gene encoding a WAT1-related protein. The modulated alkaloid content can be determined by comparing the alkaloid content in the following: plants (e.g., tobacco plants) or parts thereof, plant propagation material (e.g., tobacco plant propagation material), cells (e.g., tobacco cells), leaves (e.g., tobacco leaves), harvested leaves (e.g., harvested tobacco leaves), cut harvested leaves (e.g., cut harvested tobacco leaves), processed leaves (e.g., processed tobacco leaves), cut and processed leaves (e.g., cut and processed tobacco leaves), products such as tobacco products comprising plants or parts thereof of the present invention, or combinations thereof, to comparable products.

[0345] Suitably, alkaloid content can be in plant, tobacco plant for example, for example regulate in the tobacco plant of modification. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the tobacco plant of modification) regulate. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the tobacco plant of modification) regulate. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the tobacco plant of modification) regulate in the results. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the results of the cutting of the tobacco plant of modification) regulate. Suitably, alkaloid content can be in processing blade (tobacco leaf for example, for example from the processing tobacco leaf of the tobacco plant of modification) regulate. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the cutting of the tobacco plant of modification) regulate in the processing. Suitably, alkaloid content can be in blade (tobacco leaf for example, for example from the tobacco leaf of the tobacco plant of modification) regulate in the modulation. Suitably, alkaloid content can be in the extract of green leaf (for example from the green tobacco leaf of the tobacco plant of modification) regulate. Suitably, alkaloid content can be regulated in the product (for example tobacco product, for example tobacco product produced by the tobacco plant of modification or its part) comprising plant of the present invention or its part. Suitably, alkaloid content can be regulated in any one of the above-mentioned products or its combination. Suitably, the regulation of above-mentioned alkaloid content can be the increase of alkaloid content. Suitably, the regulation of above-mentioned alkaloid content can be the reduction of alkaloid content (for example the reduction of PON content).

[0346] In one embodiment, the level of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine, and mithosamine is increased. In one embodiment, the level of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, and anabasine is decreased. In one embodiment, the level of PON is decreased.

[0347] Suitably, the above-mentioned adjustment of alkaloid content may be a reduction in nicotine content. Suitably, the above-mentioned adjustment of alkaloid content may be a reduction in the content of one or more alkaloids selected from nornicotine, PON, anatabine, anabasine and mithosamine, rather than a reduction in nicotine content. Suitably, the above-mentioned adjustment of alkaloid content may be a reduction in the content of one or more alkaloids selected from nornicotine, PON, anatabine and anabasine, rather than a reduction in nicotine content.

[0348] In one embodiment, the nicotine content of the modified plant (e.g., tobacco plant), plant propagation material (e.g., tobacco plant propagation material), leaves (e.g., tobacco leaves), harvested leaves (e.g., harvested tobacco leaves), cut harvested leaves (e.g., cut harvested tobacco leaves), processed leaves (e.g., processed tobacco leaves), cut and processed leaves (e.g., cut and processed tobacco leaves), or tobacco products from the modified tobacco plant is reduced.

[0349] In one embodiment, when compared to the alkaloid content of a plant (e.g., tobacco plant) or its part that has not been modified to regulate the activity or expression of at least one gene encoding a WAT1-associated protein and has been grown under similar growth conditions, the alkaloid content of the plant (e.g., tobacco plant) or its part can be regulated by at least 0.5, 1.5, 2, 3, or 4 times. Suitably, the alkaloid content can be regulated by about 0.5 times to about 4 times. Suitably, the alkaloid content can be regulated by about 4 times. Suitably, the modification can be an increase or decrease in alkaloid content. Suitably, the regulation can be the regulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine, and mithofamine. Suitably, the regulation can be the regulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, and anabasine. Suitably, PON content is regulated.

[0350] In one embodiment of the invention, compared with plant (for example tobacco plant) or its part that are not modified according to the present invention, the alkaloid content of plant (for example tobacco plant) or its part can be regulated at least 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.In one embodiment, compared with unmodified plant or its part, alkaloid content can be regulated at least 30%.In one embodiment, compared with unmodified plant or its part, alkaloid content can be regulated at least 40%.In one embodiment, compared with unmodified plant or its part, alkaloid content can be regulated at least 50%.In one embodiment, compared with unmodified plant or its part, alkaloid content can be regulated at least 60%.When compared with unmodified plant (for example tobacco plant) or its part, described regulation can be the increase or reduction of alkaloid content.Suitably, regulation can be the regulation of total alkaloid content. Suitably, the regulation may be the regulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine, anabasine and milostramine. Suitably, the regulation may be the regulation of one or more alkaloids selected from nicotine, nornicotine, PON, anatabine and anabasine. Suitably, the regulation is the regulation of nicotine content, such as a reduction in nicotine content. Suitably, the regulation is the regulation of nornicotine content, such as a reduction in nornicotine content. Suitably, the regulation is the regulation of anabasine content, such as a reduction in anabasine content. Suitably, the regulation is the regulation of PON content, such as a reduction in PON content. Suitably, the regulation is the regulation of anatabine content, such as a reduction in anatabine content. Suitably, the modulation is of more than one alkaloid, such as two or more alkaloids, such as three or more alkaloids, such as four or more alkaloids, such as five or more alkaloids, such as all six alkaloids, selected from nicotine, nornicotine, PON, anatabine, anabasine and mithosamine.

[0351] In some embodiments, the alkaloid content of a plant can be modulated by about 5% to about 100%, about 10% to about 90%, about 20% to about 80%, about 30% to about 70%, about 40% to 60%, about 40% to 50%, or about 50% to 60%.

[0352] Tobacco-specific nitrosamine (TSNA) content

[0353] In one embodiment, the present invention provides a method for reducing the content of at least one tobacco-specific nitrosamine (TSNA) precursor in a plant (e.g., a tobacco plant) or part thereof. Suitably, the method may comprise modifying the plant by modulating the activity or expression of at least one gene encoding a WAT1-related protein. In one embodiment, the present invention provides a method for producing processed leaves having a reduced TSNA content (e.g., relative to a comparable product). The method for producing processed leaves having a reduced TSNA content may comprise:

[0354] Modifying a plant by reducing or inhibiting the activity or expression of at least one gene encoding a WAT1-related protein;

[0355] harvesting leaves from the plant;

[0356] and conditioning the harvested leaves.

[0357] TSNAs can be measured in processed tobacco, such as cured tobacco or reconstituted tobacco. In one embodiment, TSNA content is measured and / or modified (e.g., reduced) in cured tobacco plants or parts thereof (e.g., cured tobacco leaves).

[0358] As used herein, the term "tobacco-specific nitrosamine" or "TSNA" has its ordinary meaning in the art, i.e., a nitrosamine found only in tobacco products or other nicotine-containing products. Suitably, the at least one tobacco-specific nitrosamine may be 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT) or N'-nitrosoanatabine (NAB).

[0359] When used in relation to at least one tobacco-specific nitrosamine, the term "precursor thereof" refers to one or more chemicals or compounds of the tobacco plant that cause the formation of tobacco-specific nitrosamines or are associated with nitrosative reactions that lead to the production of tobacco-specific nitrosamines. Suitably, the term "precursor thereof" may refer to nitrates, nitrites or nitric oxide.

[0360] In one embodiment, the TSNA is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and / or the precursor is PON.

[0361] In one embodiment, the TSNA is N'nitrosonornicotine (NNN) and / or the precursor is nornicotine.

[0362] In one embodiment, the TSNA may be one or more selected from the group consisting of N'-nitrosonornicotine (NNN), N'nitrosonicotine (NAT), N'-nitrosoanatabine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Suitably, the at least one tobacco-specific nitrosamine may be NNK or NNN. In one embodiment, the tobacco-specific nitrosamine is NNK.

[0363] In one embodiment, the precursor of TSNA is one or more selected from the group of nornicotine, anabasine, anatabine and an oxidized derivative of nicotine such as pseudo-oxidized nicotine (PON).

[0364] In one embodiment, the precursor of TSNAs can be PON. Precursors of TSNAs (e.g., NNK, NNN, NAB, and / or NAT) can be measured in green tobacco leaves, e.g., prior to processing, e.g., prior to curing. In one embodiment, precursors of TSNAs (e.g., NNK, NNN, NAB, and / or NAT) are measured and / or modified (e.g., reduced) in green tobacco leaves, e.g., prior to processing, e.g., prior to curing.

[0365] In one embodiment, performance of the methods and or uses of the present invention results in a reduction in at least one TSNA or precursor thereof in the modified tobacco plant (or part thereof) when compared to a tobacco plant (or part thereof) not modified according to the invention.

[0366] The term "reduction of at least one TSNA or precursor thereof" or "reduction of at least one TSNA or precursor thereof" is used herein to mean that the concentration and / or total content of at least one TSNA or precursor thereof is lower in the product, method, or use of the present invention relative to a comparable product, method, or use. For example, a comparable delivery system would be derived from a tobacco plant that has not been modified according to the present invention, but in which all other relevant characteristics are the same (e.g., plant species, growing conditions, method of processing the tobacco, etc.).

[0367] Any method known in the art for determining the concentration and / or level of at least one TSNA or its precursor may be used. In particular, a method may be used which may include the addition of a deuterium-labeled internal standard, water extraction and filtration followed by analysis using reverse phase high performance liquid chromatography with tandem mass spectrometry (LC-MS / MS). Other examples of methods for determining the concentration and / or level of tobacco-specific nitrosamine precursors include methods such as CORESTA recommended method CRM-72: Determination of Tobacco Specific Nitrosamines in Tobacco and Tobacco Products by LC-MS / MS; being developed as a CRM for ISO / DIS 21766 or the method detailed in Wagner et al. (2005) Analytical Chemistry, 77(4), 1001-1006, which are incorporated herein by reference in their entirety.

[0368] Suitably, the concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor can be reduced by implementing the methods and / or uses of the present invention. Suitably, the concentration and / or level of at least one tobacco-specific nitrosamine or its precursor can be reduced in the tobacco plant of the present invention (e.g., obtainable or obtained by the methods and / or uses of the present invention) when compared to the concentration and / or level of at least one tobacco-specific nitrosamine or its precursor in a tobacco plant not modified according to the present invention.

[0369] The concentration and / or total content of at least one tobacco-specific nitrosamine or its precursor can be reduced in tobacco leaves, harvested leaves, processed tobacco leaves, delivery systems, or combinations thereof obtainable or obtained from a tobacco plant (or part of a tobacco plant or tobacco cell culture) of the present invention when compared to tobacco leaves, harvested leaves, processed tobacco leaves, delivery systems, or combinations thereof obtainable or obtained from a tobacco plant (or part of a tobacco plant or tobacco cell culture) that has not been modified according to the present invention.

[0370] Suitably, the concentration and / or total content of at least one tobacco-specific nitrosamine or a precursor thereof may be reduced in the processed tobacco leaves.

[0371] Suitably, the concentration and / or level of at least one tobacco-specific nitrosamine or a precursor thereof may be reduced in the delivery system.

[0372] In one embodiment, at least one tobacco-specific nitrosamine or its precursor can be reduced by at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In some embodiments, at least one tobacco-specific nitrosamine or its precursor can be reduced by about 5% to about 50%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, or about 40% to 50%.

[0373] With respect to processed (e.g., cured) tobacco leaves (e.g., cured or reconstituted), at least one tobacco-specific nitrosamine or its precursor can be reduced by about 5000 ng / g to about 50 ng / g, about 4000 ng / g to about 100 ng / g, about 3000 ng / g to 500 ng / g, or 2000 ng / g to 1000 ng / g. In some embodiments, at least one tobacco-specific nitrosamine or its precursor can be reduced by at least about 5000 ng / g, at least about 4000 ng / g, at least about 3000 ng / g, at least about 2000 ng / g, at least about 1000 ng / g, at least about 500 ng / g, at least about 100 ng / g, or at least about 50 ng / g.

[0374] Biomass production

[0375] In some cases, it may be desirable to produce plants or biomass with high alkaloid levels (e.g., high levels of nicotine content) so that the nicotine can be purified to produce a pure nicotine product, such as for use in devices that utilize liquids containing nicotine (e.g., e-cigarettes) or within tobacco heating devices. For example, producing nicotine in this manner can reduce the cost of nicotine extraction for producing e-liquids for e-cigarettes.

[0376] In one aspect, the present invention provides a method of producing biomass, comprising:

[0377] Cells that have been engineered to modulate (eg, increase) the activity or expression of a gene encoding a WAT1-related protein are grown under conditions that produce biomass.

[0378] In one embodiment, the present invention provides a method for producing biomass having an altered (e.g., increased) concentration and / or total content of nicotine, comprising growing cells that have been engineered to reduce the activity or expression of at least one gene encoding a WAT1-related protein, the WAT1-related protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one gene encoding a WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

[0379] Cells can be engineered to alter the activity or expression of at least one gene encoding a WAT1-associated protein by any method known in the art. Suitably, cells can be engineered to express an exogenous gene encoding a WAT1-associated protein. Suitably, cells can be engineered to overexpress a gene encoding a WAT1-associated protein. Suitably, cells can be engineered to reduce the activity or expression of a gene encoding a WAT1-associated protein.

[0380] Suitably, the biomass may contain a lower concentration and / or total content of nicotine than biomass produced from comparable cells which have not been modified according to the invention.

[0381] Suitably, the cells used for biomass production may be plant cells, such as tobacco cells.

[0382] Suitably, the cells used for biomass production may be yeast cells.

[0383] In one embodiment, the cell (e.g., yeast cell) can be further modified to include one or more sequences that increase nicotine alkaloid biosynthesis. Suitably, these one or more sequences can be incorporated into a nucleic acid construct suitable for transformation of the cell (e.g., yeast cell). The one or more sequences can be overexpressed in the cell (e.g., yeast cell). The sequence can be selected from one or more of the following genes: MPO (or methylputrescine oxidase or MPO1 or MPO2); A622 (or isoflavone reductase-like protein or isoflavone reductase homolog (homolog) or isoflavone reductase-like protein); BBL (or berberine bridge enzyme or berberine bridge enzyme-like or BBE or NBB1); PMT (or putrescine N-methyltransferase or putrescine methyltransferase or S-adenosyl-L-methionine:putrescine N-methyltransferase or PMT or PMT1 or PMT2 or PMT3 or PMT4) and QPT (or quinolinate phosphoribosyltransferase). In one embodiment, the sequence may be selected from one or more of the following genes: BBL, A622, PMT and MPO (MPO1 or MPO2). Genes suitable for modification in this manner can be taught, for example, in US2016032299, which is incorporated herein by reference.

[0384] Commercially desirable traits

[0385] In one embodiment, the plants of the present invention have a modified (i.e., increased or decreased) total alkaloid content, and / or a modified (i.e., increased or decreased) content of one or more alkaloids, while the aroma profile and / or other commercially desirable traits are at least maintained. In one embodiment, the plants of the present invention produce leaves of similar grade and / or quality as plants that have not been modified according to the present invention.

[0386] In one embodiment, a plant of the invention has a reduced PON content without a significant change in the plant's aroma profile (eg, compared to the same plant that has not been modified according to the invention).

[0387] In one embodiment, the plants of the present invention have reduced TSNA precursor content without a significant change (e.g., a decrease) in other commercially desirable traits of the plant (e.g., compared to the same plant that has not been modified according to the present invention). In particular, the modified plant preferably does not have a reduced yield compared to the same plant that has not been modified according to the present invention.

[0388] Thus, in one embodiment, the methods and uses of the present invention are directed to reducing TSNA precursor levels while maintaining flavor profile and / or other commercially desirable traits (eg, yield).

[0389] As used herein, the term "commercially desirable traits" will include traits such as yield, mature plant height, number of harvestable leaves, average node length, cutter leaf length, cutter leaf width, quality (e.g., leaf mass, appropriately modulated leaf mass), abiotic (e.g., drought) stress tolerance, herbicide tolerance, and / or biotic (e.g., insect, bacterial, or fungal) stress tolerance.

[0390] Leaf quality can be measured based on the color, texture, and aroma of the cured leaves, for example according to United States Department of Agriculture (USDA) grades and standards.

[0391] Tobacco grades are evaluated based on factors including, but not limited to, petiole position, leaf size, leaf color, leaf uniformity and integrity, maturity, texture, elasticity, gloss (related to the intensity and depth of color and shine of the leaf), hygroscopicity (the ability of the tobacco leaf to absorb and retain moisture from the environment), and green nuance or cast.

[0392] Leaf grade can be determined using standard methods known in the art, for example, using the Official Standard Grades (7 USC §511) published by the Agricultural Marketing Service of the US Department of Agriculture. See, e.g., the official standard grades for Burley Tobacco (U.S. Type 31 and Foreign Type 93), effective November 5, 1990 (55 FR 40645); the official standard grades for Flue-cured Tobacco (U.S. Types 11, 12, 13, 14 and Foreign Type 92), effective March 27, 1989 (54 FR 7925); the official standard grades for Pennsylvania Seedleaf Tobacco (U.S. Type 41), effective January 8, 1965 (29 FR 16854); the official standard grades for Ohio Cigar-Leaf Tobacco (U.S. Types 42, 43, and 44), effective December 8, 1963 (28 F.R. 11719 and 28 FR 16854). 11926); the official standard grade for Wisconsin Cigar-Binder Tobacco (U.S. 54 and 55), effective November 20, 1969 (34 FR 17061); the official standard grade for Wisconsin Cigar-Binder Tobacco (U.S. 54 and 55), effective November 20, 1969 (34 F.R. 17061); the official standard grade for Georgia and Florida Shade Grown Cigar-Wrapper Tobacco (U.S. 62), effective April 1971. USDA grade index values ​​may be determined based on industry-recognized grade indices. See, e.g., Bowman et al. (1988) Tobacco Science, 32:39-40; Legacy Tobacco Document Library (Bates Document #523267826-523267833, July 1, 1988, Memorandum on the Proposed Burley Tobacco Grade Index); and Miller et al. (1990) Tobacco Intern., 192:55-57 (all of the foregoing references are incorporated herein in their entireties).

[0393] In one aspect, the USDA grade index is a 0-100 numerical representation of the received federal grade and is a weighted average of all shank positions. A higher grade index indicates higher quality. Alternatively, the blade grade can be determined via hyper-spectral imaging. See, for example, WO 2011 / 027315 (which is incorporated herein by reference).

[0394] In one embodiment, the tobacco plants of the present invention provide commercially acceptable grades of tobacco.

[0395] Suitably, the tobacco plants of the present invention provide commercially acceptable grades of cured tobacco.

[0396] In one embodiment, when growing under similar growth conditions, tobacco plant of the present invention can produce the leaf blade of at least about 70% the USDA grade index value with comparable plant leaves. Suitably, when growing under similar growth conditions, tobacco plant disclosed herein may be able to produce the leaf blade of at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% the USDA grade index value with the USDA grade index value of control plant. Suitably, the tobacco plants disclosed herein may be capable of producing leaves having a USDA Grade Index value of 65% to 130%, 70% to 130%, 75% to 130%, 80% to 130%, 85% to 130%, 90% to 130%, 95% to 130%, 100% to 130%, 105% to 130%, 110% to 130%, 115% to 130%, or 120% to 130% of the USDA Grade Index value of a comparable plant.

[0397] In one aspect, the tobacco plants of the present invention are capable of producing leaves having a USDA Grade Index value of at least 50. Suitably, the tobacco plants disclosed herein may be capable of producing leaves having a USDA Grade Index value of 55 or greater, 60 or greater, 65 or greater, 70 or greater, 75 or greater, 80 or greater, 85 or greater, 90 or greater, and 95 or greater.

[0398] Unless otherwise indicated, tobacco yield as used herein refers to cured leaf yield, which is calculated based on cured tobacco leaf weight per acre under standard field conditions following standard agronomic and curing practices.

[0399] In one aspect, plants of the invention (e.g., tobacco plants) have a yield that is 50% to 150%, 55% to 145%, 60% to 140%, 65% to 135%, 70% to 130%, 75% to 125%, 80% to 120%, 85% to 115%, 90% to 110%, 95% to 105%, 50% to 100%, 55% to 100%, 60% to 100% of the yield of comparable plants when grown under similar field conditions. %, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 100% to 150%, 105% to 150%, 110% to 150%, 115% to 150%, 120% to 150%, 125% to 150%, 130% to 150%, 135% to 150%, 140% to 150% or 145% to 150%.

[0400] In another aspect, the yield of plants (e.g., tobacco plants) of the invention is about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 times greater than the yield of comparable plants when grown under similar field conditions.

[0401] In another aspect, the yield of tobacco plants of the present invention is comparable to the yield of comparable flue-cured plants when grown under similar field conditions.

[0402] In one aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of about 1200 to 3500, 1300 to 3400, 1400 to 3300, 1500 to 3200, 1600 to 3100, 1700 to 3000, 1800 to 2900, 1900 to 2800, 2000 to 2700, 2100 to 2600, 2200 to 2500, and 2300 to 2400 pounds per acre.

[0403] In another aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of: about 1200 to 3500, 1300 to 3500, 1400 to 3500, 1500 to 3500, 1600 to 3500, 1700 to 3500, 1800 to 3500, 1900 to 3500, 2000 to 3500, 2100 to 3500, 2200 to 3500, 2300 to 3500, 2400 to 3500, 2500 to 3500, 2600 to 3500, 2700 to 3500, 2800 to 3500, 2900 to 3500, 3000 to 3500, and 3100 to 3500 pounds per acre.

[0404] In a further aspect, the tobacco plant of the present invention provides a yield selected from the group consisting of: about 1200 to 3500, 1200 to 3400, 1200 to 3300, 1200 to 3200, 1200 to 3100, 1200 to 3000, 1200 to 2900, 1200 to 2800, 1200 to 2700, 1200 to 2600, 1200 to 2500, 1200 to 2400, 1200 to 2300, 1200 to 2200, 1200 to 2100, 1200 to 2000, 1200 to 1900, 1200 to 1800, 1200 to 1700, 1200 to 1600, 1200 to 1500, and 1200 to 1400 pounds per acre.

[0405] Plant breeding

[0406] In one embodiment, the present invention provides a method for producing a plant having a modified alkaloid content and / or a modified tobacco-specific nitrosamine (TSNA) precursor content, comprising:

[0407] a. crossing a donor plant having a modified nicotine content and / or a modified tobacco-specific nitrosamine (TSNA) precursor content, and wherein the activity or expression of at least one gene encoding a WAT1-related protein according to the present invention has been modulated in the donor plant, with a recipient tobacco plant that does not have a modified nicotine content or a modified tobacco-specific nitrosamine (TSNA) precursor content and has commercially desirable traits;

[0408] b isolating genetic material from the offspring of the donor plant hybridized with the recipient plant; and

[0409] c. Molecular marker-assisted selection using molecular markers, comprising:

[0410] i. Identifying an introgressed region comprising a mutation in a polynucleotide sequence encoding a protein defined in a.

[0411] Suitably, the activity or expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or orthologue thereof, or a sequence having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30; or encoded by a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or orthologue thereof, or a sequence having at least 80% identity to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog of SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO.

[0412] Molecular marker-assisted selection can include performing PCR to identify a nucleic acid sequence containing a gene introgression that modulates the activity or expression of a protein comprising an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or an amino acid sequence having at least 80% sequence identity thereto.

[0413] plant

[0414] Suitable plants according to the present invention include plants of the Solanaceae family, which includes, for example, datura, eggplant, mandrake, deadly nightshade (belladonna), capsicum (chili pepper, capsicum), potato and tobacco.

[0415] In one embodiment, a suitable genus of the Solanaceae family is Nicotiana ( Nicotiana ), such as tobacco or Nicotiana rustica ( Nicotiana rustica ).

[0416] A suitable species of the genus Nicotiana may be Nicotiana tabacum. Species of the genus Nicotiana may be referred to herein as tobacco plants, or simply tobacco.

[0417] tobacco plants

[0418] The present invention provides methods, uses, and cells (eg, tobacco cells), plants (eg, tobacco plants), and plant propagation material for plants (eg, tobacco plants).

[0419] As used herein, the term "tobacco plant" refers to a plant in the genus Nicotiana used in the production of tobacco products. Non-limiting examples of suitable "tobacco" plants include Nicotiana tabacum and Nicotiana rustica (e.g., Nicotiana N. tabacum L.), LAB21, LN KY171, Tl 1406, Basma, Galpao, Perique, Beinhart 1000-1 and Petico).

[0420] In one aspect, tobacco plant according to the present invention is a non-natural tobacco plant. Suitably, the tobacco plant can be a sudden change, a non-natural tobacco plant. Suitably, the tobacco plant can be a transgenic tobacco plant.

[0421] In one aspect, the tobacco plant according to the present invention comprises a non-naturally occurring mutation that modulates (e.g., reduces) the activity or expression of at least one protein encoding wall thinning 1-related (WAT1-related). Suitably, the tobacco plant may comprise an introduced mutation.

[0422] Tobacco material can be derived from or derive from the variant of tobacco type, is commonly referred to as burley tobacco variant, flue-cured tobacco (flue) or bright color (bright) variant and dark (dark) variant.In some embodiments, tobacco material is derived from burley tobacco, Virginia (Virginia) or dark tobacco plant.Tobacco plant can be selected from burley tobacco, rare tobacco, special product tobacco (speciality tobacco), expanded tobacco etc.

[0423] This paper also contemplates the use of tobacco cultivars and original species (elite) tobacco cultivars. Therefore, the tobacco plants used for this paper can be tobacco variants or original species tobacco cultivars. Especially useful tobacco variants include flue-cured tobacco Virginia type, burley type and Oriental type.

[0424] In some embodiments, the tobacco plant can be, for example, selected from one or more of the following varieties: L. cultivar TI 1068, AA 37-1, B 13P, Xanthi (Mitchell-Mor), KT D#3 Hybrid 107, Bel-W3, 79-615, Samsun Holmes NN, F4 from the cross BU21 x Hoja Parado, line 97, KTRDC#2 Hybrid 49, KTRDC#4 Hybrid 1 10, Burley 21, PM016, KTRDC#5 KY 160 SI, KTRDC#7 FCA, KTRDC#6 TN 86SI, PM021, K 149, K 326, K 346, K 358, K 394, K 399, K 730, KY 10, KY 14, KY 160, KY 17, KY 8959, KY 9, KY 907, MD 609, McNair 373, NC2000, PG 01, PG 04, P01, P02, P03, RG 11, RG17, RG 8, Speight G-28, TN 86, TN 90, VA 509, AS44, Banket A1, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 319, Coker 347, CriolloMisionero, PM092, Delcrest, Djebel 81, DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona、PM102、Kutsage E1、KY 14 x L8, KY 171, LA BU 21, McNair944, NC 2326, NC 71, NC 297, NC 3, PVH 03, PVH 09, PVH 19, PVH 21 10, Red Russian, Samsun, Saplak, Simmaba, Talgar28, PM132, Wislica, Yayaldag, NC 4. TRMadole, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, YakaJB 125 / 3, TI-1068, KDH-960, TI-1070, TW136, PM204, PM205, Basma, TKF4028, L8, TKF2002, TN 90, GR141, Basma xanthi, GR149, GR153 and Petit Havana.

[0425] Non-limiting examples of varieties or cultivars are: BD 64, CC 101, CC 200, CC 27, CC 301, CC400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker48, CD 263, DF91 1, DT 538 LC, Galpao tobacco, GL 26H, GL350, GL 600, GL 737, GL 939, GL973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K 149, K 326, K346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY171, KY 907, KY907LC, KTY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371LC, NC 100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC71, NC 72, NC 810, NC BH129, NC 2002,Neal Smith Madole,OXFORD 207. PD 7302 LC, PD7309 LC, PD 7312 LC 'Periq'e' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51, R 610, R 630, R7-1 1, R 7-12, RG 17, RG 81, RG H51, RGH 4, RGH 51, RS 1410, Speight 168, Speight172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, Tl 1406, Tl1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TND950, TR (TomRosson) Madole, VA309, VA359, AA 37-1, B 13P, Xanthi (Mitchell-Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC No. 2 Hybrid 49, Burley 21, KY 8959, KY9, MD 609, PG 01, PG 04, P01, P02, P03, RG 1 1, RG 8, VA 509, AS44, Banket A1, Basma Drama B84 / 31, Basma IZichna ZP4 / B, BasmaXanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81. DVH 405. Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1, LA BU 21, NC2326, NC 297, PVH 21 10, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156 / 1, Prilep P12-2 / 1, Yaka JK-48, Yaka JB 125 / 3, TI-1068, KDH-960, Tl-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141, Basma xanthi, GR149, GR153, Petit Havana. Even though not specifically identified herein, the aforementioned lowconverter subvarieties are also contemplated.

[0426] The tobacco plant may be Burley, Flue-cured Virginia, or Oriental.

[0427] In one embodiment, the plant propagation material may be obtainable from a plant of the invention (eg, a tobacco plant).

[0428] As used herein, "plant propagation material" refers to any plant matter taken from a plant from which further plants can be produced. Suitably, the plant propagation material may be selected from seeds, plant callus and plant pieces. Suitably, the plant propagation material may be seeds. Suitably, the plant propagation material may be plant callus. Suitably, the plant propagation material may be plant pieces.

[0429] In one embodiment, cells (eg, tobacco cells), tobacco plants and / or plant propagation material may be obtainable (eg, obtained) by a method according to the invention.

[0430] Suitably, a tobacco plant according to the present invention may have a modulated (e.g. reduced) nicotine content when compared to an unmodified tobacco plant, wherein the tobacco plant has been modified to modulate (e.g. reduce) the activity or expression of at least one gene encoding a WAT1-related protein.

[0431] Suitably, a tobacco plant according to the present invention may have a modulated (e.g. reduced) content of tobacco-specific nitrosamine (TSNA) precursors when compared to an unmodified tobacco plant, wherein the tobacco plant has been modified to modulate (e.g. increase) the activity or expression of at least one gene encoding a WAT1-related protein.

[0432] In one embodiment, a tobacco plant according to the present invention comprises a tobacco cell of the present invention.

[0433] In another embodiment, the plant propagation material may be obtainable (eg, obtained) from a tobacco plant of the present invention.

[0434] In one embodiment, there is provided use of a tobacco plant as described herein for breeding tobacco plants.

[0435] In another embodiment, the present invention also provides use of the tobacco plant of the preceding embodiment for producing a delivery system.

[0436] In another embodiment, there is provided use of the tobacco plants of the present invention to grow crops.

[0437] In one embodiment, a cell, such as a plant cell, such as a tobacco plant cell, is provided that has an activity or expression that is regulated (e.g., reduced) by at least one gene encoding a wall thin 1-related (WAT1-related) protein. Suitably, the cell can be a non-naturally occurring cell. Suitably, the cell can be a mutant cell. Suitably, the cell can be a non-naturally occurring mutant cell. For example, the cell can comprise a non-naturally occurring mutation that regulates (e.g., reduces) the activity or expression of at least one gene encoding a wall thin 1-related (WAT1-related) protein.

[0438] In one embodiment, there is provided the use of a cell as provided in the preceding embodiments for production delivery.

[0439] In one embodiment, the invention provides cell cultures (eg, in vitro cultures).

[0440] As used herein, "cell culture" refers to a plurality of cells.

[0441] For example, the cell culture according to the present invention comprises a plurality of cells according to the present invention. Suitably, the cell culture may comprise a plurality of tobacco cells according to the present invention.

[0442] The cell culture can be a cell suspension culture. These in vitro cultured cells can be incorporated into a delivery system, for example as a substitute for conventional tobacco particles, shreds, fine or long cut tobacco flakes, as an additive ingredient, or both as a substitute and as an additive. Suitably, the cell culture can produce nicotine.

[0443] In one embodiment, there is provided the use of cells or cell cultures, eg, cells or cell cultures harvested and / or processed according to the invention, for production delivery.

[0444] Tobacco cells harvested from in vitro culture can be dried, such as freeze-dried, for example to produce a powder.

[0445] In one embodiment, said cell or cell culture are tobacco cells or cell culture.Technician will know the known methods for establishing the in vitro culture of tobacco cell.Only for example, can use following method: collect from the seed of target tobacco plant, and sterilize to eliminate unwanted biology to its outside, plant said seed with the growth target tobacco plant, from tobacco plant (for example, from tobacco stem), take out tissue and supply as explant, establish callus culture from tobacco explant, establish cell suspension culture from callus culture, and gather in the crops culture material (for example comprising tobacco cell) to produce tobacco cell culture.

[0446] Tobacco cells can be harvested by various methods including filtration, such as vacuum filtration. The sample can be washed in a filter by adding water and removing the remaining liquid by filtration, such as vacuum filtration.

[0447] The harvested tobacco cell culture may be further processed, for example dried, such as air-dried and / or freeze-dried.The harvested tobacco cell culture or dried harvested tobacco cell culture or extract thereof may be incorporated into a delivery system according to the present invention.

[0448] In one embodiment, the invention provides plants (e.g., tobacco plants) or parts thereof for molecular planting. Suitably, plants modified according to the present invention or parts thereof can be used to make proteins, such as therapeutic agents, for example, antibiotics, virus-like particles, nutritional medicines or small molecules.

[0449] In one embodiment, the present invention provides a method for producing a protein (e.g., a therapeutic protein), the method comprising modifying a plant or part thereof capable of producing the protein (e.g., a therapeutic protein) by regulating the activity or expression of at least one WAT1-related protein gene, wherein the at least one WAT1-related protein gene encodes an amino acid sequence as shown in SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 1, 4, 7, 10, 13, 16, 19, 22, 25 or 28; or wherein the at least one gene encoding the WAT1-related protein comprises a nucleotide sequence as shown in SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a functional variant or functional fragment or ortholog thereof, or a sequence having at least 80% identity with SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30, or a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30; and cultivating the plant under conditions sufficient to allow production of the protein (e.g., a therapeutic protein).

[0450] product

[0451] The present invention also provides products obtainable or obtained from a plant according to the invention.Products obtainable or obtained from a plant in which the activity or expression of a gene encoding a WAT1-related protein has been modulated are provided.

[0452] In one embodiment, a product may comprise a construct of the invention that modulates the activity or expression of at least one gene encoding a WAT1-related protein as defined herein. In one embodiment, a product may comprise a construct of the invention that modifies the nucleic acid sequence of at least one gene encoding a WAT1-related protein as defined herein.

[0453] The invention also provides products obtainable or derived from tobacco according to the invention.

[0454] In one embodiment, there is provided use of the tobacco plant of the present invention to produce tobacco leaves.

[0455] Suitably, the tobacco leaves may be subjected to downstream applications, such as processing.

[0456] Therefore, in one embodiment, the use of the aforementioned embodiments can provide processed tobacco leaves. Suitably, the tobacco leaves can be subjected to conditioning, fermentation, pasteurization, or a combination thereof. In another embodiment, the tobacco leaves can be cut. In some embodiments, the tobacco leaves can be cut before or after being subjected to conditioning, fermentation, pasteurization, or a combination thereof.

[0457] In one embodiment, the present invention provides harvested leaves of the tobacco plant of the present invention.

[0458] In a further embodiment, harvested leaves may be obtainable (eg, obtained) from a tobacco plant propagated from the propagation material of the invention.

[0459] In another embodiment, there is provided harvested leaves obtainable from the methods or uses of the invention.

[0460] Suitably, the harvested leaves may be cut harvested leaves.

[0461] In some embodiments, the harvested leaves may contain living tobacco cells.In other embodiments, the harvested leaves may be subjected to further processing.

[0462] Processed tobacco leaves are also provided.

[0463] Processed tobacco leaves may be obtainable from a tobacco plant of the present invention. Suitably, processed tobacco leaves may be obtainable from a tobacco plant obtained according to any of the methods and / or uses of the present invention.

[0464] In another embodiment, processed tobacco leaves may be obtainable from tobacco plants propagated from tobacco plant propagation material according to the present invention.

[0465] The processed tobacco leaves of the present invention may be obtainable by processing the harvested leaves of the present invention.

[0466] As used herein, the term "processed tobacco leaf" refers to a tobacco leaf that has undergone one or more processing steps to which tobacco is subjected in the art."Processed tobacco leaf" contains no or substantially no living cells.

[0467] The term "living cell" refers to a cell that is capable of growth and / or metabolic activity. Thus, if a cell is said to be not living, also known as "non-viable", the cell does not exhibit the characteristics of a living cell.

[0468] The term "substantially free of living cells" means that less than about 5% of all cells are living. Preferably, less than about 3%, more preferably less than about 1%, even more preferably less than about 0.1% of all cells are living.

[0469] In one embodiment, processed tobacco leaves may be processed by one or more of: curing, fermenting, and / or pasteurizing.

[0470] Suitably, the processed tobacco leaves may be processed by curing.

[0471] The tobacco leaves may be cured by any method known in the art. In one embodiment, the tobacco leaves may be cured by one or more curing methods selected from the group consisting of air-curing, flame-curing, flue-curing, and sun-curing.

[0472] Suitably, the tobacco leaves may be air-cured.

[0473] Typically, air-curing is achieved by hanging the tobacco leaves in a well-ventilated curing barn and allowing them to dry. This is usually done over a period of four to eight weeks. Air-curing is particularly suitable for Burley tobacco.

[0474] Suitably, the tobacco leaves may be open-fire cured. Open-fire curing is typically achieved by hanging the tobacco leaves in large curing barns where a hardwood fire is kept smoldering continuously or intermittently, and typically takes from three days to ten weeks, depending on the process and the tobacco.

[0475] In another embodiment, the tobacco leaves can be flue-cured. Flue-curing can include stringing tobacco leaves onto tobacco rods and hanging them on tier-poles in a curing barn. Curing barns typically have flues that operate with an externally supplied firebox. Typically, this results in tobacco that has been heat-cured without being exposed to smoke. Typically, the temperature rises slowly during the curing process, and the entire process takes about 1 week.

[0476] Suitably, the tobacco leaves may be sun-cured. This process generally involves exposing uncovered tobacco to the sun.

[0477] Suitably, the processed tobacco leaves may be processed by fermentation.

[0478] Fermentation can be carried out in any manner known in the art. Usually, during the fermentation, tobacco leaves are piled into a tobacco pile (heap) covered with a modulation in a sack for example to retain moisture. The combination of the residual water in the leaf and the tobacco weight has generated the natural heat that makes tobacco ripe. The temperature in the center of the heap is monitored every day. In some methods, the whole heap is opened weekly. The leaves are then taken out to shake and moisten, and the heap is rotated so that the inner leaves are outside and the bottom leaves are placed on the top of the heap. This ensures the uniform fermentation of the whole heap. The extra moisture on the leaf adds the actual rotation generation heat of the leaf itself, thereby releasing the natural ammonia of the tobacco and reducing nicotine, while also deepening color and improving the aroma of the tobacco. Usually, the fermentation process continues up to 6 months, and this depends on the variety of the tobacco, the handle position on the leaf, the thickness of the leaf and the intended use.

[0479] Suitably, the processed tobacco leaves may be processed by pasteurisation.Pasteurisation may be particularly preferred when the tobacco leaves are to be used in the preparation of a smokeless delivery system, most preferably snus.

[0480] The pasteurization of tobacco leaves can be carried out by any method known in the art. For example, the pasteurization method can be as described in J Foulds, L Ramstrom, M Burke, K Fagerstrom. Effect of smokeless tobacco (snus) onsmoking and public health in Sweden. Tobacco Control (2003) 12 : 349–359 (the teachings of which are incorporated herein by reference).

[0481] During snus production, pasteurization is typically performed by a process in which the tobacco is heat-treated with steam for 24-36 hours, reaching a temperature of approximately 100° C. This results in a virtually sterile product, and without wishing to be bound by theory, one of the consequences of this is believed to be limiting further TSNA formation.

[0482] In one embodiment, the pasteurization process may be steam pasteurization.

[0483] In some embodiments, the processed tobacco leaves can be cut. The processed tobacco leaves can be cut before or after processing. Suitably, the processed tobacco leaves can be cut after processing.

[0484] In one embodiment, use of the foregoing embodiments can provide reconstituted tobacco.

[0485] In one embodiment, reconstituted tobacco is provided.

[0486] As used herein, "reconstituted" may also be referred to as reconstituted, recycled or homogenized sheet tobacco, and refers to tobacco material generated from processed tobacco leaf residues. Reconstituted tobacco allows the production of consistent, high-quality blends and allows the ratios of the various components to be adjusted.

[0487] Reconstituted tobacco can be nanofibre reconstituted (nanofibres can be extracted in solid or liquid form), paper reconstituted (which uses stems, chips and midrib as raw materials), or slurry reconstituted (which uses a mixture of finely ground material and tobacco stems mixed with water and a plant binder; the soluble residue is formed into sheets by extracting the water).

[0488] Any method known in the art can be used to prepare reconstituted tobacco, see, for example, CORESTA Congress, Sapporo, 2012, Smoke Science / Product Technology Groups, SSPT 12 (incorporated herein by reference).

[0489] In some embodiments, the leaves of the results of tobacco plants, tobacco plants and / or processed tobacco leaves can be used to extract nicotine. The extraction of nicotine can be achieved using any method known in the art. For example, the method for extracting nicotine from tobacco is taught in US 2,162,738, which is incorporated herein by reference.

[0490] In one aspect, the present invention provides a cured tobacco material made from a tobacco plant or part thereof according to the present invention.

[0491] In another aspect, the invention provides a tobacco blend comprising a tobacco material made from a tobacco plant according to the invention or its part or from a tobacco cell or cell culture according to the invention. In one aspect, the invention provides a tobacco blend comprising a tobacco material prepared according to the invention.

[0492] Suitably, tobacco blends according to the present invention can comprise about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 10% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 20% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 30% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 40% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 50% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 60% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 70% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 80% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture. Suitably, tobacco blends can comprise about 90% tobacco from tobacco plant according to the present invention or its part or from tobacco cell according to the present invention or cell culture.

[0493] In one aspect, the tobacco blend products of the present invention comprise at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95 percent by dry weight of tobacco cured from a tobacco plant or part thereof according to the present invention or a tobacco cell or cell culture according to the present invention.

[0494] Suitably, the cured tobacco material may be air-cured. Suitably, the cured tobacco material may be flue-cured. Suitably, the cured tobacco material may be sun-cured. Suitably, the cured tobacco material may be flame-cured.

[0495] Delivery systems or smoking articles according to the invention may comprise a tobacco material (eg, a cured tobacco material or a reconstituted tobacco material) according to the invention.

[0496] In another aspect, the present invention provides a delivery system.

[0497] In one embodiment, the delivery system according to the present invention may be a blended delivery system. Suitably, a tobacco blend may comprise a cured tobacco material according to the present invention.

[0498] In one embodiment, the delivery system can be prepared from the tobacco plant of the present invention or a part thereof.

[0499] Suitably, tobacco plants or parts thereof may be propagated from tobacco plant propagation material according to the present invention.

[0500] The term " part " used in the context of tobacco plant as this paper refers to a part of tobacco plant. Suitably, " part " can be a leaf, root or stem or a flower of tobacco plant. Suitably, " part " can be a leaf, root or stem of tobacco plant.

[0501] Delivery system

[0502] As used herein, the term "delivery system" is intended to encompass systems that deliver at least one substance to a user and includes:

[0503] Combustible gas aerosol delivery systems, such as cigarettes, cigarillos and cigars, and tobacco for pipe smoking or roll-your-own or homemade cigarettes (whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, tobacco substitutes or other smokable materials);

[0504] Non-combustible aerosol delivery systems that release compounds from an aerosol-generating material without burning the aerosol-generating material, such as electronic cigarettes, tobacco heating products, and hybrid systems that use a combination of aerosol-generating materials to generate an aerosol; and

[0505] Non-aerosol delivery systems that deliver at least one substance to a user orally, nasally, transdermally or otherwise without forming an aerosol, including but not limited to lozenges, gums, patches, products containing inhalable powders, and oral products such as oral tobacco including snus or moist snuff, wherein the at least one substance may or may not contain nicotine.

[0506] According to the present disclosure, a "combustible" aerosol supply system is one in which the constituent aerosol generating materials of the aerosol supply system (or components thereof) ignite or burn during use to facilitate delivery of at least one substance to a user.

[0507] In some embodiments, the delivery system is a combustible aerosol supply system, such as a system selected from the group consisting of cigarettes, cigarillos, and cigars.

[0508] In some embodiments, the present disclosure relates to components for use in a combustible gas aerosol supply system, such as a filter, a filter rod, a filter rod segment, a tobacco rod, a plug, an aerosol modifier release component, such as a capsule, a string, a bead, or a paper, such as a plug wrap, tipping paper, or cigarette paper.

[0509] According to the present disclosure, a "non-flammable" aerosol delivery system is one in which constituent aerosol-generating materials of the aerosol delivery system (or components thereof) do not ignite or burn to facilitate delivery of at least one substance to a user.

[0510] In some embodiments, the delivery system is a non-combustible gas aerosol supply system, such as a powered non-combustible gas aerosol supply system.

[0511] In some embodiments, the non-combustible aerosol delivery system is an electronic cigarette, also known as a vaping device or an electronic nicotine delivery system (END), although it should be noted that the presence of nicotine in the aerosol-generating material is not required.

[0512] In some embodiments, the non-combustible gas aerosol supply system is an aerosol generating material heating system, also known as a heat-not-burn system. An example of such a system is a tobacco heating system.

[0513] In some embodiments, the non-combustible gas aerosol supply system is a hybrid system to generate aerosol using a combination of aerosol generating materials (one or more of which can be heated). Each of the aerosol generating materials can be in the form of, for example, solid, liquid, or gel and can contain or can contain no nicotine. In some embodiments, the hybrid system comprises a liquid or gel aerosol generating material and a solid aerosol generating material. The solid aerosol generating material can include, for example, tobacco or non-tobacco products.

[0514] Generally, the non-combustible gas aerosol supply system may include a non-combustible gas aerosol supply device and consumables for use with the non-combustible gas aerosol supply device.

[0515] In some embodiments, the present disclosure relates to consumables that contain an aerosol generating material and are configured for use with a non-combustible gas aerosol supply device. These consumables are sometimes referred to as articles of manufacture throughout the disclosure.

[0516] In some embodiments, the non-combustible gas aerosol supply system, such as a non-combustible gas aerosol supply device thereof, can include a power source and a controller. For example, the power source can be an electrical source or an exothermic power source. In some embodiments, the exothermic power source includes a carbon substrate that can be energized to distribute energy in the form of heat to the aerosol generating material or a heat transfer material near the exothermic power source.

[0517] In some embodiments, the non-combustible gas aerosol supply system may include an area for receiving a consumable product, an aerosol generator, an aerosol generating area, a housing, a mouthpiece, a filter, and / or an aerosol modifier.

[0518] In some embodiments, a consumable product for use with a non-combustible gas aerosol supply device may include an aerosol generating material, an aerosol generating material storage region, an aerosol generating material transfer assembly, an aerosol generator, an aerosol generating region, a housing, a wrapper, a filter, a mouthpiece, and / or an aerosol modifier.

[0519] Suitably, the delivery system may be prepared from (eg may comprise) a tobacco plant according to the invention or a part thereof.

[0520] Suitably, the delivery system may be prepared from tobacco cell cultures according to the present invention.

[0521] Suitably, the delivery system may be prepared from (eg may comprise) a tobacco plant or part thereof propagated from tobacco plant propagation material according to the invention.

[0522] Suitably, the delivery system may be prepared from (eg may comprise) harvested leaves of a tobacco plant according to the invention.

[0523] Suitably, the delivery system may be prepared from (eg may comprise) processed tobacco leaves according to the invention.

[0524] Suitably, the delivery system may be prepared from (eg may comprise) a modulated tobacco material according to the invention.

[0525] Suitably, the delivery system may be prepared from (eg may comprise) a tobacco blend according to the invention.

[0526] In one embodiment, the delivery system is a combustible smoking article selected from the group consisting of cigarettes, cigarillos, and cigars.

[0527] In one embodiment, the delivery system comprises one or more components of a combustible smoking article, such as a filter, a filter rod, a filter rod segment, tobacco, a tobacco rod, a tobacco rod segment, a spill, an additive release component such as a capsule, a string, a bead, a paper such as a plug wrap, tipping paper or cigarette paper.

[0528] In one embodiment, the delivery system is a non-combustible gas aerosol supply system.

[0529] In one embodiment, the delivery system comprises one or more components of a non-combustible gas aerosol supply system, such as a heater and an aerosolizable substrate.

[0530] In one embodiment, the aerosol delivery system is an electronic cigarette, also known as a vaping device.

[0531] In one embodiment, an electronic cigarette includes a heater, a power source capable of powering the heater, an aerosolizable substrate such as a liquid or gel, a housing, and optionally a mouthpiece.

[0532] In one embodiment, the aerosolizable substrate is contained in a substrate container.In one embodiment, the substrate container is associated with or includes a heater.

[0533] In one embodiment, the delivery system is a heating product that releases one or more compounds by heating rather than burning a matrix material. The matrix material is an aerosolizable material, which may be, for example, tobacco or other non-delivery system, which may or may not contain nicotine. In one embodiment, the heating product is a tobacco heating product.

[0534] In one embodiment, the heated product is an electronic device.

[0535] In one embodiment, the tobacco heating product comprises a heater, a power source capable of powering the heater, and an aerosolizable substrate such as a solid or gel material.

[0536] In one embodiment, the heating product is a non-electronic article.

[0537] In one embodiment, the heating product includes an aerosolizable substrate such as a solid or gel material and a heat source capable of supplying thermal energy to the aerosolizable substrate without any electrical means, such as by burning a combustion material such as charcoal.

[0538] In one embodiment, the heated product further comprises a filter capable of filtering the aerosol generated by heating the aerosolizable substrate.

[0539] In some embodiments, the aerosolizable base material may comprise a vapor or aerosol generating agent or a humectant, such as glycerol, propylene glycol, triacetin, or diethylene glycol.

[0540] In one embodiment, the delivery system is a hybrid system that generates an aerosol by heating without burning a combination of a matrix material. The matrix material can include, for example, a solid, a liquid, or a gel, which may or may not contain nicotine. In one embodiment, the hybrid system includes a liquid or gel matrix and a solid matrix. The solid matrix can be, for example, tobacco or other non-delivery systems, which may or may not contain nicotine. In one embodiment, the hybrid system includes a liquid or gel matrix and tobacco. In another embodiment, the product can comprise a construct of the present invention that regulates at least one of the following when expressed in a plant (e.g., a tobacco plant). WAT-1 The activity or expression of related proteins is inhibited, and thereby the alkaloid content (such as PON content) is reduced.

[0541] Polynucleotide / polypeptide / construct

[0542] In certain embodiments of the present invention, a construct that regulates the activity or expression of at least one gene encoding a WAT1-related protein may be transformed into a plant cell, suitably under the direction of a promoter.

[0543] In certain embodiments of the present invention, a construct that reduces (i.e., inhibits) the activity or expression of at least one gene encoding a WAT1-related protein can be transformed into a plant cell under the guidance of a promoter. For example, the genetic construct can be a gene editing construct or can include an RNAi molecule, which can include a small interfering RNA (siRNA) molecule or a short hairpin (shRNA) molecule.

[0544] In certain embodiments of the present invention, a construct that increases the activity or expression of a gene encoding a WAT1-related protein, such as an endogenous WAT1-related protein, can be transformed into a plant cell, suitably under the direction of a promoter.

[0545] The construct can be introduced into the plant according to the present invention by means of a suitable vector, such as a plant transformation vector. The plant transformation vector can comprise an expression cassette comprising a promoter sequence, a construct sequence targeting a gene encoding a WAT1-related protein in the transcriptional direction 5'-3', and optionally a 3' non-translated terminator sequence, including a termination signal for RNA polymerase and a polyadenylation signal for polyadenylase. The promoter sequence can exist in one or more copies, and such copies can be identical or variants of the promoter sequence described above. The terminator sequence can be derived from a plant, bacterial or viral gene. Suitable terminator sequences are, for example, pea. rbcS E9 terminator sequence, derived from Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) of the nopaline synthase gene nos terminator sequence, and from cauliflower mosaic virus 35S Terminator sequences. Other suitable terminator sequences will be readily known to those skilled in the art.

[0546] The constructs of the present invention may also contain gene expression enhancing mechanisms to increase the strength of the promoter. An example of such an enhancer element is the enhancer element derived from a portion of the promoter of the pea plastocyanin gene and is the subject of International Patent Application No. WO 97 / 20056, incorporated herein by reference. Suitable enhancer elements may be, for example, the enhancer element derived from the nopaline synthase gene of Agrobacterium tumefaciens. nos enhancer element, and from cauliflower mosaic virus 35S Enhancer element.

[0547] These regulatory regions may be derived from the same gene as the promoter DNA sequence, or may be derived from tobacco or other organisms, such as from the Solanaceae family or from the subfamily Lycopodiellae ( Cestroideae All regulatory regions should be able to function in the cells of the tissue to be transformed.

[0548] The promoter DNA sequence may be derived from the same gene as the gene of interest, e.g., the gene that the promoter is to direct, such as the gene encoding a WAT1-related protein according to the present invention, the coding sequence used in the present invention, or may be derived from a different gene from tobacco or another organism, e.g., from the Solanaceae family or from the subfamily Lycopodiellae.

[0549] The expression cassette can be incorporated into a basic plant transformation vector such as pBIN 19 Plus 、 pBI 101 , pKYLX71:35S2, pCAMBIA2300 or other suitable plant transformation vectors known in the art. In addition to the expression cassette, the plant transformation vector will contain such sequences as are necessary for the transformation process. These may include Agrobacterium ( Agrobacterium ) vir gene, one or more T-DNA border sequences, and a selectable marker or other means of identifying transgenic plant cells.

[0550] The term "expression vector or plant transformation vector" refers to a construct capable of in vivo or in vitro expression. Preferably, the expression vector is incorporated into the genome of an organism. In one embodiment, the vector of the present invention expresses a protein, such as a WAT1-related protein as described herein. The term "incorporated" preferably includes stable incorporation into the genome.

[0551] Techniques for transforming plants are well known in the art and include, for example, Agrobacterium-mediated transformation. The basic principle in the construction of genetically modified plants is to insert genetic information into the plant genome in order to obtain stable maintenance of the inserted genetic material. An overview of general techniques can be found in the literature by Potrykus ( Annu Rev Plant Physiol Plant Mol Biol

[1991] 42:205-225) and Christon (AgroFood-Industry Hi-Tech 1994 Mar / Apr 17-27), which are incorporated herein by reference.

[0552] Typically, in Agrobacterium-mediated transformation, a binary vector carrying the target exogenous DNA, i.e., a construct according to the present invention, is transferred from an appropriate Agrobacterium strain to the target plant by co-cultivation of Agrobacterium with an explant from the target plant. The transformed plant tissue is then regenerated on a selective medium comprising a selective marker and a plant growth hormone. An alternative is the floral dip method (Clough & Bent, 1998 Plant J. December 1998; 16 (6): 735-43, which is incorporated herein by reference), whereby the flower buds of the complete plant are contacted with a suspension of the Agrobacterium strain containing the chimeric gene, and after fruiting, the transformed individuals are germinated and identified by growth on the selective medium. Direct infection of plant tissue by Agrobacterium is a simple technique that has been widely adopted and has been described in Butcher et al. (1980), Tissue Culture Methods for Plant Pathologists , eds. DS Ingrams and JP Helgeson, 203-208, which is incorporated herein by reference.

[0553] Further suitable transformation methods include, for example, the use of polyethylene glycol or electroporation technology, particle bombardment, microinjection, and silicon carbide fibers to transfer genes directly into protoplasts. Ballistic transformation and the production of fertile transgenic maize plants mediated by silicon carbide whiskers are taught in Frame et al. (1994) The Plant Journal 6: (6): 941-948 (incorporated herein by reference), and viral transformation techniques are taught in, for example, Meyer et al. (1992) Mol. Gen. Genet. 231 (3): 345-352 (incorporated herein by reference). Cassava mosaic virus is taught in Meyer et al. (1992) Gene 110: 213-217 (incorporated herein by reference) as a vector system for plants. Further teachings about plant transformation can be found in EP-A-0449375, incorporated herein by reference.

[0554] In a further aspect, the present invention relates to a vector system that carries a construct and introduces it into the genome of an organism such as a plant, suitably a tobacco plant. The vector system may comprise one vector, but it may comprise two vectors. In the case of two vectors, the vector system is generally referred to as a binary vector system. Binary vector systems are described in Gynheung et al., (1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19, which is incorporated herein by reference.

[0555] A widely adopted system for transforming plant cells uses the Ti plasmid from Agrobacterium tumefaciens or the Ti plasmid from Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) of the Ri plasmid, which was described by An et al. (1986) Plant Physiol. 81, 301-305 and Butcher et al. (1980) Tissue Culture Methods for Plants Pathologistseds.: DS Ingrams and JP Helgeson, 203-208 (incorporated herein by reference). After each method of introducing the desired foreign gene in a plant according to the present invention, the presence and / or insertion of further DNA sequences may be necessary. The use of T-DNA for plant cell transformation has been well studied and is described in EP-A-120516; Hoekema (1985) The Binary Plant Vector System, Offset-drukkerij Kanters BB, Amsterdam Chapter V; Fraley et al. Crit. Rev. Plant Sci. 4: 1-46; and An et al. (1985) EMBO J 4: 277-284 (incorporated herein by reference).

[0556] Plant cells transformed with one or more constructs that modulate the activity or expression of at least one gene encoding a WAT1-related protein can be grown and maintained according to well-known tissue culture methods, such as by culturing the cells in a suitable medium supplied with necessary growth factors such as amino acids, plant hormones, vitamins, and the like.

[0557] The term "transgenic plant" in connection with the present invention includes any plant comprising a construct that modulates the activity or expression of at least one gene encoding a WAT1-related protein according to the present invention. Accordingly, a transgenic plant is a plant that has been transformed with a construct according to the present invention. Preferably, according to the present invention, the transgenic plant exhibits modulated alkaloid content and / or modulated TSNA content (or its precursors). The term "transgenic plant" does not include a native nucleotide coding sequence in its natural environment when under the control of its native promoter (which is also in its natural environment).

[0558] In one aspect, the gene, construct, plant transformation vector or plant cell encoding a WAT1-related protein according to the present invention is in isolated form. The term "isolated" means that the sequence is at least substantially free of at least one other component with which the sequence is naturally associated in nature and as found in nature.

[0559] In one aspect, the gene, construct, plant transformation vector or plant cell encoding a WAT1-related protein according to the present invention is in a purified form. The term "purified" means in a relatively pure state, for example, at least about 90% pure, or at least about 95% pure, or at least about 98% pure.

[0560] As used herein, the term "nucleotide sequence" refers to an oligonucleotide sequence or a polynucleotide sequence, as well as variants, homologues, fragments and derivatives thereof (such as portions thereof). The nucleotide sequence may be of genomic or synthetic or recombinant origin, may be double-stranded or single-stranded, and may represent the sense or antisense strand.

[0561] The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably, it means a DNA encoding the present invention, more preferably a cDNA sequence.

[0562] In a preferred embodiment, when relevant to and encompassed by the scope of the present invention itself, the nucleotide sequence, i.e., a gene encoding a WAT1-related protein, comprises a native nucleotide sequence when in its natural environment and when linked to one or more of its naturally associated sequences that are also in their natural environment. For ease of reference, we shall refer to this preferred embodiment as a "native nucleotide sequence." In this regard, the term "native nucleotide sequence" means the entire nucleotide sequence, which is in its natural environment and, when operably linked to the entire promoter with which it is naturally associated, said promoter is also in its natural environment.

[0563] Nucleotide sequence for the present invention can be present in a vector, wherein the nucleotide sequence is operably linked to a regulatory sequence that can provide the expression of the nucleotide sequence by a suitable host organism. Construct for the present invention can be transformed into a suitable host cell as described herein to provide expression of polypeptide of the present invention. The selection of carriers such as plasmids, cosmids or phage vectors often depends on the host cell to be introduced therein. Carriers can be for example used for the production of RNA in vitro, or for transfection, transformation, transduction or infection of host cells.

[0564] In some applications, the nucleotide sequences used in the present invention are operably linked to regulatory sequences that can provide for expression of the nucleotide sequence, for example, by a selected host cell. For example, the present invention includes vectors comprising a nucleotide sequence encoding a gene for a WAT1-related protein as described herein operably linked to such regulatory sequences, i.e., the vector is an expression vector.

[0565] The term "operably linked" refers to a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0566] The term "regulatory sequence" includes promoters and enhancers and other expression regulatory signals. The term "promoter" is used in the ordinary sense of the art, such as an RNA polymerase binding site. The nucleotide sequence within the construct encoding the gene encoding the WAT1-related protein can be operably linked to at least one promoter.

[0567] The term "construct," which is synonymous with terms such as "cassette" or "vector," includes a nucleotide sequence for use in accordance with the present invention attached directly or indirectly to a promoter.

[0568] An example of indirect attachment is to provide a suitable spacer group, such as an intron sequence, such as the Sh1-intron or the ADH intron, between the promoter and the nucleotide sequence of the present invention. The same is true for the term "fused" in connection with the present invention, which includes direct or indirect attachment. In some cases, the term does not include the natural combination of nucleotide sequences that are typically associated with wild-type gene promoters and the coding proteins when they are all in their natural environment. The construct can even contain or express a marker that allows selection of the genetic construct.

[0569] In some embodiments, the promoter can be operably linked to a nucleotide sequence in a construct or vector for regulating the concentration and / or total content of nicotine in a cell or cell culture or a tobacco plant or part thereof.

[0570] In some embodiments, the promoter can be selected from the group consisting of: a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, and an inducible promoter.

[0571] In one embodiment, the promoter can be a constitutive promoter.

[0572] Constitutive promoters direct the expression of a gene continuously throughout all parts of the plant during plant development, although the gene may not be expressed at the same level in all cell types. Examples of known constitutive promoters include those associated with the cauliflower mosaic virus 35S transcript (Odell JT, Nagy F, Chua NH. (1985). Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature. 313 810-2), the rice actin 1 gene (Zhang W, McElroy D, Wu R. (1991). Analysis of rice Act 1 5' region activity in transgenic rice plants. Plant Cell 3 1155-65), and the maize ubiquitin 1 gene (Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE. (1993). Activity of a maize ubiquitin promoter in transgenic rice. Plant Molec. Biol. 23 567-81). A constitutive promoter, such as the carnation etched ring virus (CERV) promoter (Hull R, Sadler J, Longstaff M (1986) (CaMV / 35S), figwort mosaic virus 35S promoter. The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses. EMBO Journal, 5(2):3083-3090).

[0573] The constitutive promoter may be selected from the group consisting of: the Carnation Etch Ring Virus (CERV) promoter, the Cauliflower Mosaic Virus (CaMV35S promoter), the promoter from the rice actin 1 gene, or the maize ubiquitin 1 gene.

[0574] The promoter can be a tissue-specific promoter. A tissue-specific promoter is a promoter that directs the expression of a gene in one (or several) parts of a plant, typically throughout the life of those plant parts. The category of tissue-specific promoters also generally includes promoters whose specificity is not absolute, i.e., they can also direct expression at lower levels in tissues other than preferred tissues. Tissue-specific promoters include the phaseolin promoter, the legumin b4 promoter, the usp promoter, the sbp promoter, the ST-LS1 promoter, and the B33 (patatin class I promoter).

[0575] In another embodiment, the promoter can be a developmentally regulated promoter.

[0576] Developmentally regulated promoters direct changes in the expression of a gene in one or more parts of a plant at specific times during plant development. The gene may be expressed at different (usually lower) levels in that plant part at other times, and may also be expressed in other plant parts.

[0577] In one embodiment, the promoter can be an inducible promoter.

[0578] Inducible promoter can respond to the expression of inducer-directed gene.In the absence of inducer, gene will not express.Inducer can act directly on promoter sequence, or can work by offsetting the effect of repressor molecule.Inducer can be chemical agent such as metabolite, albumen, growth regulator (such as auxin and salicylic acid that activates OCS promoter) or toxic element, physiological stress such as heat, light (such as soybean SSU promoter), damage (for example nos, nopaline synthase promoter), or osmotic pressure, or the indirect consequence of pathogen or pest effect. The promoter of development regulation can be described as the inducible promoter of specific type, and its response is the environmental stimulation of the specific moment of endogenous inducer produced by plant or in the life history of plant. Examples of known inducible promoters include those associated with wound response, such as described by Warner SA, Scott R, Draper J. ((1993) Plant J. 3 191-201), temperature response, as disclosed by Benfey & Chua (1989) (Benfey, PN, and Chua, NH. ((1989) Science 244 174-181), and chemical induction, such as described by Gatz ((1995) Methods in Cell Biol. 50 411-424).

[0579] Can from any cell or organism that produces described protein, identify and / or separate and / or purify nucleotide sequence, described nucleotide sequence encoding has the protein of the specific property of the gene of coding WAT1-associated protein as defined herein or is suitable for the protein of modification.The various methods for identification and / or separation and / or purification of nucleotide sequence are well known in the art.For example, once suitable sequence has been identified and / or separated and / or purified, just can use PCR amplification technology to prepare more sequence.

[0580] In a still further alternative, the nucleotide sequence encoding the WAT1-related protein may be prepared synthetically by established standard methods, for example as described by Beucage et al. (1981). Tetrahedron Letters 22, 1859-1869 (which is incorporated herein by reference), or by the phosphoramidite method described in Matthes et al., (1984) EMBO J 3, 801-805 (which is incorporated herein by reference). In the phosphoramidite method, oligonucleotides are synthesized, for example in an automatic DNA synthesizer, purified, annealed, ligated, and cloned in an appropriate vector.

[0581] As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme".

[0582] The present invention also includes the use of sequences that have a certain degree of sequence identity or sequence homology with one or more amino acid sequences of a polypeptide having the specific properties defined herein, or any nucleotide sequence, i.e., a WAT1-related protein gene encoding such a polypeptide (hereinafter referred to as "one or more homologous sequences"). Here, the term "homolog" means an entity that has a certain degree of homology with the subject amino acid sequence and the subject nucleotide sequence. Here, the term "homology" can be equivalent to "identity".

[0583] Homologous amino acid sequences and / or nucleotide sequences and / or fragments should provide and / or encode polypeptides that retain the functional activity of WAT1-related protein genes and / or enhance the activity of WAT1-related protein genes. Typically, homologous sequences will contain, for example, the same active site as the subject amino acid sequence, or will encode the same active site. Although homology can also be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of the present invention, it is preferably represented by homology in terms of sequence identity. Homologous sequences typically retain functional domains or motifs.

[0584] In one embodiment, homologous sequences are taken to include amino acid sequences or nucleotide sequences that have one, two or several additions, deletions and / or substitutions compared to the subject sequence.

[0585] Sequence identity

[0586] Sequence identity comparison can be performed by eye, or more generally, by means of readily available sequence comparison programs. These commercially available computer programs can calculate the % homology between two or more sequences. % homology or % identity can be calculated on adjacent sequences, i.e., one sequence is compared with another sequence, and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called a "no gap" comparison. Typically, this no gap comparison is performed only on a relatively short number of residues.

[0587] Although this is a very simple and consistent method, it fails to take into account that, for example, in otherwise identical sequence pairs, an insertion or deletion will cause the subsequent amino acid residues to be unable to be aligned, thereby potentially leading to a significant reduction in % homology when performing an overall alignment. Thus, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without excessively penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.

[0588] However, these more sophisticated methods assign "gap penalties" to each gap that appears in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting a higher relatedness between the two compared sequences - will receive a higher score than an alignment with many gaps. An "Affine gap cost" is commonly used, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each residue following the gap. This is the most commonly used gap scoring system. A high gap penalty will of course produce an optimized alignment with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, when using such software for sequence comparison, it is preferably to use the default values.

[0589] Therefore, calculation of maximum % homology first requires producing an optimal alignment taking into account gap penalties. A suitable computer program for performing such an alignment is Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. 1999 Short Protocols in Molecular Biology, 4th edition - Chapter 18), BLAST 2 (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ ncbi.nlm.nih.gov ), FASTA (Altschul et al. 1990 J. Mol. Biol. 403-410) and AlignX. At least BLAST, BLAST 2 and FASTA can be used for offline and online searching (see Ausubel et al. 1999, pp. 7-58 to 7-60).

[0590] Although final % homology can be measured according to identity, the alignment process itself is not usually based on all-or-no pairwise comparisons. Instead, a similarity score matrix with a varying ratio is usually used, which assigns a score to each paired comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix of the BLAST suite of programs. The Vector NTI program typically uses disclosed default values ​​or custom symbol comparison tables (if provided) (for further details, see the user manual). For some applications, the default values ​​of the Vector NTI program package are preferably used.

[0591] Alternatively, percent homology can be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.) based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73 (1), 237-244). Once the software has generated an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically performs this as part of a sequence comparison and generates a numerical result.

[0592] For BLAST GAP OPEN 0 GAP EXTENSION 0

[0593] For CLUSTAL DNA protein Word length 2 1 K triplet Gap Penalty 15 10 Gap extension 6.66 0.1

[0594] In one embodiment, CLUSTAL can be used together with gap penalty and gap extension set as defined above.In some embodiments, the gap penalty for BLAST or CLUSTAL comparison can be different from those described in detail above.Technical staff will understand that the standard parameters for carrying out BLAST and CLUSTAL comparison may change regularly, and will be able to select appropriate parameters based on the standard parameters detailed for BLAST or CLUSTAL comparison algorithm at that time.

[0595] Suitably, the degree of identity with respect to a nucleotide sequence is determined over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 70 contiguous nucleotides, preferably over at least 80 contiguous nucleotides, preferably over at least 90 contiguous nucleotides, preferably over at least 100 contiguous nucleotides, preferably over at least 150 contiguous nucleotides, preferably over at least 200 contiguous nucleotides, preferably over at least 250 contiguous nucleotides, preferably over at least 300 contiguous nucleotides, preferably over at least 350 contiguous nucleotides, preferably over at least 400 contiguous nucleotides, preferably over at least 450 contiguous nucleotides, preferably over at least 500 contiguous nucleotides, preferably over at least 550 contiguous nucleotides, preferably over at least 600 contiguous nucleotides, preferably over at least 650 contiguous nucleotides, or preferably over at least 700 contiguous nucleotides.

[0596] Suitably, the degree of identity with respect to nucleotide, cDNA, cds or amino acid sequences may be determined over the entire sequence.

[0597] The sequences may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent substances. Deliberate amino acid substitutions may be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues, as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups of similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0598] aliphatic Non-polar GAP ILV Polarity – Neutral CSTM NQ Polarity – Charged DE KR Aromatic HFWY

[0599] The present invention also includes homologous substitutions (both substitution and replacement are used herein to mean the interchange of an existing amino acid residue with an alternative residue), i.e., like-for-like substitutions, such as basic for basic, acid for acid, polar for polar, etc. Non-homologous substitutions may also occur, i.e., from one class of residue to another, or, on the other hand, involving the inclusion of unnatural amino acids, such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine, and phenylglycine.

[0600] Substitutions can also be made with unnatural amino acids, including; α* and α-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-aminobutyric acid*, L-γ-aminobutyric acid*, L-α-aminoisobutyric acid*, L-ε-aminocaproic acid # , 7-aminoheptanoic acid*, L-methionine sulfone # *, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline # , L-thioproline (thioproline) *, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino) # , L-Tyr(methyl)*, L-Phe(4-isopropyl)*, L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid)*, L-diaminopropionic acid # and L-Phe(4-benzyl)*. The notation * has been used for the purposes discussed above (in relation to homologous or nonhomologous substitution) to indicate the hydrophobic nature of the derivative, while # has been used to indicate the hydrophilic nature of the derivative, with #* indicating amphiphilic character.

[0601] In addition to amino acid spacers such as glycine or β-alanine residues, the variant amino acid sequence may include suitable spacers that may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl. Further variant forms relate to the presence of one or more amino acid residues in a peptidomimetic form, which will be fully understood by those skilled in the art. For the avoidance of doubt, "peptidomimetic form" is used to refer to variant amino acid residues in which the α-carbon substituent is located on the nitrogen atom of the residue rather than on the α-carbon. Methods for preparing peptides in a peptidomimetic form are known in the art, for example Simon et al. (1992) PNAS 89(20), 9367-9371 and Horwell (1995) Trends Biotechnol. 13(4), 132-134.

[0602] Nucleotide sequence used for the present invention can include synthetic or modified nucleotides therein.Many different types of modifications to oligonucleotides are known in the art.These include methylphosphonate and phosphorothioate backbones and / or acridine or polylysine chain additions at the 3' and / or 5' ends of the molecule.For purposes of the present invention, it will be appreciated that nucleotide sequence described herein can be modified by any method available in this area.This type of modification can be carried out to enhance the in vivo activity or lifespan of nucleotide sequence of the present invention.

[0603] The present invention also includes sequences complementary to the nucleic acid sequences of the present invention, or sequences capable of hybridizing to the sequences of the present invention or to sequences complementary thereto. As used herein, the term "hybridization" should include "the process by which a nucleic acid strand binds to a complementary strand via base pairing," as well as amplification processes such as those performed in polymerase chain reaction (PCR) technology.

[0604] The present invention also relates to nucleotide sequences that can hybridize with the nucleotide sequences of the present invention (including the complementary sequences of those presented herein). Preferably, hybridization is determined under stringent conditions (e.g., 50°C and 0.2xSSC {1xSSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0}). More preferably, hybridization is determined under high stringency conditions (e.g., 65°C and 0.1xSSC {1xSSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0}).

[0605] An overview of general techniques for transforming plants can be found in papers such as Potrykus et al. (1991) Annu Rev Plant Physiol. Plant Mol. Biol. 42:205-225 and Christou et al. (1994) Agro-Food-Industry Hi-Tech March / April 17-27, which are incorporated herein by reference. Further teachings on plant transformation can be found in EP-A-0449375, which is incorporated herein by reference.

[0606] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide a general dictionary for those skilled in the art of many of the terms used in this disclosure.

[0607] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure. Numerical ranges are inclusive of the values ​​defining the ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino to carboxyl orientation, respectively.

[0608] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be obtained by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0609] Amino acids are referred to herein using the name, three-letter abbreviation, or single-letter abbreviation of the amino acid. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide." In some cases, the term "amino acid sequence" is synonymous with the term "enzyme."

[0610] In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues can be used. The 3-letter code for amino acid residues is as defined according to the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It should also be understood that due to the degeneracy of the genetic code, polypeptides can be encoded by more than one nucleotide sequence.

[0611] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, as these may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0612] Where a range of values ​​is provided, it will be understood that, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of the range, based on one-tenth of the lower limit unit, is also specifically disclosed. Each smaller range between any specified value or intermediate value in a prescribed range and any other specified value or intermediate value in the prescribed range is included in this disclosure. Depending on any specifically excluded limits in the prescribed range, the upper and lower limits of these smaller ranges may be independently included in or excluded from the described range, and each range wherein any one, no, or both of the limits are included in the smaller range is also included in this disclosure. Where a prescribed range includes one or two limits, the range excluding any one or both of these included limits is also included in this disclosure.

[0613] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" or "nitrate reductase" includes a plurality of such candidate agents and equivalents thereof known to those skilled in the art, and so forth.

[0614] advantage

[0615] It has surprisingly been discovered that by modulating the activity or expression of at least one gene encoding a WAT1-related protein that acts as a positive regulator of PON in tobacco as taught herein, the TSNA precursor content of a plant can be modulated. Thus, tobacco products can be produced that have modulated alkaloid (e.g., PON content or total alkaloid content) and / or TSNA precursor content, as well as commercially desirable traits sought by tobacco product consumers.

[0616] The present inventors have surprisingly identified methods for regulating the PON content, or total alkaloid content, and / or TSNA precursor content, of plants (e.g., tobacco plants) by modulating the activity or expression of genes encoding WAT1-related proteins. The alkaloid (e.g., PON) or TSNA precursor content of plants (e.g., tobacco plants) can be reduced by reducing or inhibiting the activity or expression of genes encoding WAT1-related proteins. Prior to the present invention, it was not known that modulation of the activity or expression of genes encoding WAT1-related proteins, as described herein, could be used to modulate the alkaloid (e.g., PON content or total alkaloid content) and / or TSNA precursor content of plants (e.g., tobacco plants).

[0617] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publication constitutes prior art for the claims appended hereto. Example

[0618] Example 1 - Transient overexpression of WAT1-related proteins increases alkaloid content in leaves

[0619] Methods and Materials

[0620] Cloning of WAT1-related protein expression vectors

[0621] The Nitab4.5_0010919g0010.2 WAT1-related protein gene sequence (SEQ ID NO. 3) was amplified from a Gateway™ compatible cDNA library using primers located outside of the restriction sites flanking the gene sequence. The gene sequence was then transferred to an expression vector, depicted in Figure 1 middle.

[0622] The resulting plasmid was sequenced and transformed into Agrobacterium tumefaciens GV3101 pMP90 by heat shock and transiently expressed in TN90 leaves.

[0623] transient gene expression

[0624] Agrobacterium tumefaciens GV3101 strains carrying the construct of interest were grown overnight in Luria-Bertani (LB) medium supplemented with appropriate antibiotics. The culture was quickly spun down and resuspended to an OD600 of 0.6 in a buffer containing 10 mM MgCl2, 10 mM MES pH 5.6, and 100 μM acetosyringone, and incubated at room temperature for 1 hour. Infiltration into TN90 leaves was performed using a needleless syringe. Samples were obtained 5 days after infiltration.

[0625] The test was performed with two biological replicates.

[0626] Alkaloid measurement

[0627] The relative content of PON was determined by reverse-phase high-performance liquid chromatography with tandem mass spectrometry (LC-MS / MS). Chromatographic separation was achieved using a Gemini-NX column (100 mm × 3.0 mm, 3 μm particle size, Phenomenex) and a gradient of 6.5 mM ammonium acetate buffer (aq) (pH 10) and methanol.

[0628] Analytes Precursor ions Product ion (quantity / confirmation) PON 176.1 106.0 / 148 PON d4 183.1 110.0

[0629] result

[0630] The PON content of 5-week-old TN90 leaves expressing the Nitab4.5_0010919g0010.2 construct is shown in Figure 2 PON content is expressed relative to the control and includes two biological replicates analyzed by one-way t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value ≤ 0.001.

[0631] Overexpression of Nitab4.5_0010919g0010.2 resulted in a significant increase in PON content in leaves.

[0632] in conclusion

[0633] Nitab4.5_0010919g0010.2 is a positive regulator of alkaloid content in leaves, particularly PON content, and is a regulator of pyridine alkaloids in tobacco.

[0634] Example 2 - Transient expression of antisense RNA targeting Nitab4.5_0010919g0010.2 reduces alkaloid content in leaves

[0635] Materials and methods

[0636] The Nitab4.5_0010919g0010.2 coding sequence was reverse cloned into a plant expression vector driven by the CERV promoter.

[0637] result

[0638] The PON content of 5-week-old TN90 leaves expressing Nitab4.5_0010919g0010.2 antisense RNA is shown in Figure 3 PON content is expressed relative to the control and includes two biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.001.

[0639] Inhibition of Nitab4.5_0013616g0010.2 expression using antisense RNA resulted in a decrease in PON content in leaves.

[0640] in conclusion

[0641] Nitab4.5_0010919g0010.2 is a positive regulator of alkaloid content in leaves, particularly PON content, and is a regulator of pyridine alkaloids in tobacco.

[0642] Example 3 - Transient expression of an artificial miRNA targeting Nitab4.5_0010919g0010.2 reduces alkaloid content in leaves

[0643] Materials and methods

[0644] Nitab4.5_0010919g0010.2 silencing was performed using a specific artificial microRNA designed based on the methods and scripts described in microRNA Designer (http: / / wmd3.weigelworld.org and http: / / p-sams.carringtonlab.org) and then validated against tobacco transcripts to check for off-targets.

[0645] SEQ ID NO. 36 was synthesized and cloned into a plant expression vector driven by the CERV promoter. The sequence of the miRNA targeting Nitab4.5_0010919g0010.2 (SEQ ID NO. 36) is shown in Figure 39 middle.

[0646] result

[0647] The PON content of 5-week-old TN90 leaves expressing artificial miRNA targeting Nitab4.5_0010919g0010.2 is shown in Figure 4 PON content is expressed relative to the control and includes two biological replicates analyzed by t-test. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.001.

[0648] Inhibition of Nitab4.5_0013616g0010.2 expression using miRNA resulted in a decrease in PON content in leaves.

[0649] in conclusion

[0650] Nitab4.5_0010919g0010.2 is a positive regulator of alkaloid content in leaves, particularly PON content, and is a regulator of pyridine alkaloids in tobacco.

Claims

1. A method for reducing the alkaloid content of a tobacco plant or part thereof, or a tobacco plant cell or a tobacco plant cell culture, said method comprising modifying said plant or plant cell by reducing the activity or expression of a gene encoding a WAT1-related protein, wherein the gene encoding the WAT1-related protein encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; or The gene encoding the WAT1-related protein consists of the nucleotide sequence shown in SEQ ID NO. 2 or 3, and the alkaloid whose content is reduced is pseudo-oxidized nicotine.

2. Use of a gene encoding a WAT1-related protein for reducing the alkaloid content of tobacco plants or parts thereof or tobacco plant cells or tobacco plant cell cultures, wherein the gene encoding the WAT1-related protein encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; or The gene encoding the WAT1-related protein consists of the nucleotide sequence shown in SEQ ID NO. 2 or 3, wherein the alkaloid whose content is reduced is pseudo-oxidized nicotine.

3. The use according to claim 2, wherein the alkaloid content is reduced compared to a tobacco plant or tobacco plant cell that has not been modified to modulate the activity or expression of a gene encoding a WAT1-related protein.

4. A method for producing a tobacco plant or part thereof, a tobacco plant cell, or a tobacco plant cell culture having reduced alkaloid content, the method comprising modifying the plant or plant cell to reduce the activity or expression of a gene encoding a WAT1-related protein, wherein the gene encoding the WAT1-related protein encodes a polypeptide consisting of the amino acid sequence as shown in SEQ ID NO. 1; or the gene encoding the WAT1-related protein consists of the nucleotide sequence as shown in SEQ ID NO. 2 or 3, and wherein the alkaloid whose content is reduced is pseudo-oxidized nicotine.

5. The method of claim 4, wherein the tobacco plant or part thereof, or tobacco plant cell or tobacco plant cell culture is tobacco plant propagation material.

6. The method of claim 4, wherein the part of the tobacco plant is a leaf.

7. The method of claim 6, wherein the leaves are cut harvested leaves.

8. The method of claim 6, wherein the blade is a machined blade.

9. The method of claim 8, wherein the processed blade is a cut and processed blade.

10. The method of any one of claims 1, 4-9 or the use of any one of claims 2-3, wherein the alkaloid content of the plant is reduced compared to a plant or plant cell that has not been modified to reduce the activity or expression of the gene encoding the WAT1-related protein.

11. The method or use according to claim 10, wherein the activity or expression of the gene encoding the WAT1-related protein is reduced compared to a plant or cell that has not been modified to reduce the activity or expression of the gene encoding the WAT1-related protein.

12. The method according to any one of claims 1, 4-11 or the use according to any one of claims 2-3, 10-11, wherein one or more additional genes encoding WAT1-related proteins are also regulated, wherein the polypeptide encoded by the additional genes encoding WAT1-related proteins consists of the amino acid sequence shown in SEQ ID NO. 4, 7, 10, 13, 16, 19, 22, 25 or 28; or The additional gene encoding a WAT1-related protein consists of a nucleotide sequence as shown in SEQ ID NO. 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29 or 30.

13. Any of (1) to (3) for use in any of the following (a) to (c): (1) a tobacco plant part, tobacco plant cell, or tobacco plant cell culture produced by the method according to any one of claims 1 and 4 to 12; (2) A modified tobacco plant which has been modified to achieve a reduced alkaloid content compared to an unmodified plant or unmodified cell, wherein the modification is a reduction in the activity or expression of a gene encoding a WAT1-related protein, wherein the gene encoding the WAT1-related protein encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1; or The gene encoding the WAT1-related protein consists of the nucleotide sequence shown in SEQ ID NO. 2 or 3, and the alkaloid whose content is reduced is pseudo-oxidized nicotine; or (3) A tobacco plant having reduced activity or expression of a gene encoding a WAT1-related protein produced by the method of any one of claims 1, 4 to 12; (a) cultivating plants; (b) produce a product; or (c) To cause crops to grow.

14. Use according to claim 13, wherein the use is to produce leaves.

15. Non-viable processed tobacco leaves, which: Obtaining a tobacco plant having reduced activity or expression of the gene encoding a WAT1-related protein obtained from the use according to claim 2; obtained by processing a part of a tobacco plant produced by the method of any one of claims 1, 4-12; Obtained by processing a modified tobacco plant as defined in claim 13; Obtaining a tobacco plant propagated from plant propagation material obtained from a modified tobacco plant as defined in claim 13; or Obtained from a tobacco plant having reduced activity or expression of a gene encoding a WAT1-related protein produced by the method of any one of claims 1, 4-12.

16. The non-viable processed tobacco leaf of claim 15, wherein the leaf is processed by curing, fermenting, pasteurizing, or a combination thereof.

17. The non-viable processed tobacco leaf blade according to any one of claims 15-16, wherein the processed tobacco leaf blade is a cut processed tobacco leaf blade.

18. Cured tobacco material made from the tobacco plant or parts thereof, which: Obtaining a tobacco plant having reduced activity or expression of a gene encoding a WAT1-related protein obtained from the use according to claim 2; obtained by processing a part of a tobacco plant produced by the method of any one of claims 1, 4-12; Obtained by processing a modified tobacco plant as defined in claim 13; Obtaining a tobacco plant propagated from plant propagation material obtained from a modified tobacco plant as defined in claim 13; or Obtained from a tobacco plant having reduced activity or expression of a gene encoding a WAT1-related protein produced by the method of any one of claims 1, 4-12.

19. A tobacco blend comprising the cured tobacco material of claim 18.

20. Tobacco industry products prepared from: A tobacco plant having reduced activity or expression of a gene encoding a WAT1-related protein obtained from the use according to claim 2; A part of a tobacco plant produced according to the method of any one of claims 1 and 4 to 12, or a tobacco plant cell produced according to the method of any one of claims 1 and 4 to 12; A modified tobacco plant as defined in claim 13; Tobacco plants or parts thereof propagated from plant propagation material obtained from a modified tobacco plant as defined in claim 13; The non-viable processed tobacco leaf according to any one of claims 15-16.

21. The tobacco industry product according to claim 20, wherein the tobacco industry product is: a) Combustible smoking products; b) smokeless tobacco products; or c) Non-flammable aerosol supply system.

22. The tobacco industry product according to claim 21, wherein in part c), the non-combustible gas aerosol supply system is a tobacco heating device or an aerosol generating device.

23. Combustible smoking articles, non-combustible aerosol delivery systems, or smokeless tobacco products, including: (1) a tobacco plant part produced according to the method of any one of claims 1 and 4 to 12, or a tobacco extract thereof, or a tobacco plant cell culture produced according to the method of any one of claims 1 and 4 to 12; (2) a modified tobacco plant, or a part of a tobacco plant, or a tobacco extract thereof as defined in claim 13; (3) The cured tobacco material of claim 18; or the tobacco blend of claim 19.

24. A tobacco heating device comprising: (1) a tobacco plant part produced according to the method of any one of claims 1 and 4 to 12, or a tobacco extract thereof, or a tobacco plant cell culture produced according to the method of any one of claims 1 and 4 to 12; (2) a modified tobacco plant or plant part, or a tobacco extract thereof, as defined in claim 13; (3) The cured tobacco material of claim 18; or the tobacco blend of claim 19.

25. Non-viable processed tobacco leaves produced from a tobacco plant comprising a modification in the nucleotide sequence of a gene encoding a WAT1-related protein, wherein the gene is selected from SEQ ID NO. 2 or 3; wherein the modification reduces the activity or expression of the gene encoding the WAT1-related protein, and wherein the plant has a reduced alkaloid content relative to a comparable plant not carrying the modification in the gene encoding the WAT1-related protein, and wherein the alkaloid whose content is reduced is pseudo-oxidized nicotine.

26. A cured tobacco material produced from a tobacco plant comprising a modification in the nucleotide sequence of a gene encoding a WAT1-related protein, wherein the gene is selected from SEQ ID NO. 2 or 3; wherein the modification reduces the activity or expression of the gene encoding the WAT1-related protein, and wherein the plant has a reduced alkaloid content relative to a comparable plant not carrying the modification in the gene encoding the WAT1-related protein, and wherein the alkaloid whose content is reduced is pseudo-oxidized nicotine.

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