Method for modulating nicotine level in nicotiana tabacum

Abstract

To provide a method for modulating the alkaloid content (e.g., the nicotine content) of a tobacco plant or part thereof, or of a tobacco plant cell.SOLUTION: A method is provided which comprises modifying the plant or cell providing at least one mutation in a Nic3 locus. The present invention provides a method for modulating (e.g., decreasing) the nicotine content of a plant (e.g., a tobacco plant) or part thereof, or of a tobacco plant cell, the method comprising modifying the plant or cell to modulate the expression or activity of at least one Nic3 gene. The present invention also provides the use of the Nic3 locus for modulating the alkaloid content of a plant, as well as tobacco cells, plants, plant propagation materials, harvested leaves, processed tobaccos or delivery systems obtainable in accordance with the invention.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods for modulating (e.g., reducing) the alkaloid content, e.g., nicotine content, of a tobacco plant or part thereof or tobacco plant cells. The present invention also relates to methods for modulating the expression and / or activity of a polypeptide encoded by a gene that modulates alkaloid content in a plant. Alternatively, the present invention provides methods for modulating the expression and / or activity of a gene encoding a polypeptide that modulates alkaloid content (e.g., nicotine content) in a plant. The present invention also relates to methods for modulating (e.g., reducing) the alkaloid content in a plant by introducing a mutation into a tobacco plant or part thereof or a tobacco plant cell. The present invention also relates to plants produced by any of the methods described herein. The present invention also relates to constructs that can be used to modulate polypeptides, tobacco plant cells transformed with such constructs, and transgenic tobacco plants themselves. The present invention also relates to leaves harvested from tobacco plants according to the present invention having modulated alkaloid content (e.g., nicotine content), and the use of delivery systems (e.g., combustible aerosol delivery systems, non-combustible aerosol delivery systems, or aerosol-free delivery systems) comprising such leaves or extracts thereof. The present invention also relates to the use of tobacco plants according to the invention having a low alkaloid content (eg nicotine content) in molecular agriculture. [Background technology]

[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. Alkaloids can be classified according to the similarity of their carbon skeletons, for example, into indole-like, isoquinoline-like, and pyridine-like. Pyridine derivatives are a class of monomeric alkaloids, including simple derivatives of pyridine, polycyclic fused and non-fused pyridine derivatives, and sesquiterpene pyridine derivatives. Examples include nicotine, nornicotine, anabasine, myosmine, and anatabine. Most of the known biological functions of alkaloids are related to defense.

[0003] Nicotine occurs naturally in several plants, but is found at the highest levels in tobacco plants. Nicotine is produced in wild and cultivated Nicotiana species and is the primary plant They play an important role in plant defense against predators and insects (Voelckel, C., Krugel, T., Gase, K., Heidrich, N., van Dam, NM, Winz, R., and Baldwin, IT (2001). Anti-sense expression of putrescine N-methyltransferase confirms the defensive role of nicotine in Nicotiana sylvestris against Manduca sexta. Chemoecology 11, 121-126, incorporated herein by reference), and account for approximately 90% of the total alkaloid content. The remaining 10% of the alkaloid pool is composed approximately of nornicotine, anatabine, myosmine, and anabasine.

[0004] The regulation of alkaloid content in tobacco is complex. Several factors, including genotype, environment, fertilization, and agricultural practices (e.g., topping), affect the regulation of alkaloid content in tobacco plants. Affects steroid levels.

[0005] In the 1930s, certain Cuban cigar tobacco (Nicotiana tabacum) types were identified as having extremely low alkaloid content, and this trait was introduced into US tobacco breeding lines (Valleau W., incorporated herein by reference). 1949. Breeding low-nicotine tobacco. Journal of Agricultural Research 78: 171-181). Subsequently, the low-alkaloid trait was introduced into cultivars through multiple generations of backcrossing. The strains were introduced into the La Burley-21 (B21) genetic background (Legg PD, Collins GB, Litton CC. 1970. Registration of La Burley-21 Tobacco Germplasm. Crop Science 10(2): 212, incorporated herein by reference).

[0006] Genetic studies using low-alkaloid burley 21 (LA-B21) have suggested two unlinked loci, originally designated loci A and B (incorporated by reference, Legg P, Chaplin J, Collins G. (1969). Inheritance of percent total alkaloids in Nicotiana tabacum L.: populations derived from crosses of low alkaloid lines with burley and flue-cured varieties. Journal of Heredity 60: 213-217), which later became known as Nic1 and Nic2, contribute to nicotine levels in tobacco leaves as regulatory loci for nicotine biosynthesis (Legg P., and G., C. (1971). Inheritance of percent total alkaloids in Nicotiana tabacum L. II. Genetic effects of two loci in Burley21 X LA Burley 21 populations. Canadian Journal of Genetics and Cytology 13, 287-291; Hibi, N., Higashiguchi, S., Hashimoto, T., and Yamada, Y. (1994). Gene-Expression in Tobacco Low-Nicotine Mutants. Plant Cell 6, 723-735, incorporated by reference). LAB21 was reported to be more susceptible to insect damage, consistent with the role of alkaloids in plant defense. It has also been reported that isogenic lines of flue-cured tobacco with low total alkaloids (approximately 0.2%) have lower yields.By doubling haploids of F1 progeny from a cross between wild-type or high-alkaloid B21 (HA-B21, AABB) and LA-B21 (aabb), (Collins, GB, Legg, PD, and Kasperba, Mj (1974). Use of Anther-Derived Haploids in Nicotiana. 1. Isolation of breeding lines differing in total alkaloid content. Crop Science 14, 77-80, incorporated by reference), two other isogenic lines (NILs) of B21 were developed, one with high-intermediate alkaloids (HI-B21, AAbb) and the other with low-intermediate alkaloids (LI-B21, aaBB), which were later registered as varieties in 1988 (Nielsen, MT, Legg, PD, and Collins, GB (1988). Registration of HI and LI burley varieties, incorporated by reference). 21 tobacco germplasms. Crop Science 28, 206-207). The near isogenic line (NIL) is referred to herein as Burley 21 (B21). , Nic1Nic2), high-intermediate (HI, Nic1nic2), low-intermediate (LI, nic1Nic2) and low alkaloid B21 (LA, nic1nic2), which were later registered as varieties in 1988.

[0007] Various studies have confirmed the downregulation of nicotine biosynthesis genes in nic mutants (Hibi, N., Higashiguchi, S., Hashimoto, T., and Yamada, Y. (1994). Gene-Expression in Tobacco Low-Nicotine Mutants. Plant Cell 6, 723-735;Reed, DG, and Jelesko, JG (2004). The A and B loci of Nicotiana tabacum have non-equivalent effects on the mRNA levels of four alkaloid biosynthetic genes. Plant Science 167 1123 - 1130), Nic1 and Nic2 are These findings suggest that these two loci are regulatory loci that specifically control the expression of nicotine-related structural genes. Subsequent studies have shown that these two loci also control the expression of numerous genes unrelated to nicotine biosynthesis, such as stress response genes (Kidd, SK, Melillo, AA, Lu, RH, Reed, DG, Kuno, N., Uchida, K., Furuya, M., and Jelesko, JG (2006). The A and B loci in tobacco regulate a network of stress response genes, few of which are associated with nicotine biosynthesis. Plant Mol Biol 60, 699-716, incorporated by reference).

[0008] Microarray analysis of HA-B21 and LA-B21 revealed that the ERF (ethylene response factor) gene, which regulates nicotine biosynthesis, is located at the Nic2 locus. A cluster of genes was identified (Shoji, T., Kajik, awa, M., and Hashimoto, T. (2010). Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. The Plant Cell 22, 3390-3409). nic2 mutation In somatic lines, such as LA-B21 and HI-B21, these ERF genes are completely deleted. Recent studies have confirmed that the Nic2 region contains a cluster of ERF genes and is located on chromosome 19 (Kajikawa, M., Sierro, N., Kawaguchi, H., Bakaher, N., Ivanov, N.I., Hashimoto, T., and Shoji, T. (2017). Genomic insights into the evolution of the nicotine biosynthesis pathway in tobacco. Plant Physiology 174, 999-1011, incorporated herein by reference).

[0009] Elucidating the location of the Nic1 locus in tobacco has proven difficult due to the complex nature of quantitative traits such as alkaloid levels, which has hindered map-based cloning approaches.

[0010] Humphry et al., in WO 2018 / 237107 (incorporated herein by reference), first demonstrated that the Nic1 locus, located on chromosome 7, contains multiple homeologs of the Nic2 ERF genes and showed that these Nic1 ERF genes function in regulating nicotine levels, suggesting that Nic1 may regulate nicotine biosynthesis in a manner similar to Nic2.

[0011] Altering the alkaloid content in plants (e.g., tobacco) can have numerous commercial advantages. For example, reducing the total alkaloid content in a plant can increase the value of the plant as a biomass resource. For example, altering the alkaloid content can include reducing the alkaloid content, e.g., nicotine content, of a tobacco plant. Reduced-nicotine tobacco plants and products may be desirable in light of the potential adjustment of the "nicotine ceiling," i.e., the average upper limit of nicotine in a delivery system. Alternatively, increasing the alkaloid content in a plant, e.g., a tobacco plant, can aid in the plant's defense against insects and herbivores. There remains a need for plants with improved commercially desirable traits, such as adjusted alkaloid content, e.g., adjusted nicotine content, and methods for producing the same.

[0012] Tobacco pyridine alkaloids are precursors of tobacco-specific nitrosamines (TSNAs) formed during post-harvest leaf curing. The four major TSNAs found in cured tobacco leaves are N'-nitrosonornicotine (NNN), N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), and N'-nitrosoanatabine (NAT). , N'-nitrosoanabasine (NAB), and 4-(methyl nitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

[0013] TSNAs are formed when nitrous oxide species (e.g., NO, NO2, N2O3, and N2O4) react with tobacco alkaloids. NAT and NAB are formed through the nitrosation of the secondary alkaloids anatabine and anabasine, respectively. Early studies argued that NNN was derived from both nicotine and nornicotine, but more recent reports demonstrated that the appearance of NNN in cured tobacco leaves correlates with nornicotine content, rather than nicotine content (Bush et al., Rec. Adv. Tob. Sci. 27; 23-46 (2001); Lewis et al., Plant Biotech J. 6: 346-354 (2008)). Nornicotine is a demethylated derivative of nicotine and is the major alkaloid in tobacco, accounting for 90% of the total alkaloid content (Saitoh et al., 1985 Phytochemistry, 24 pp. 477-480, incorporated herein by reference). The product relationship is less clear. Some studies have suggested that NNK is a nitrosation product of nicotine, but that due to the slow kinetics of nicotine nitrosation, oxidized derivatives of nicotine, rather than nicotine itself, may serve as the direct precursor of NNK (Caldwell et al. Ann. NY Acad. Sci. 686, 213-228 (1993)). Identifying genes involved in the production and regulation of TSNA precursors. That is of high importance.

[0014] Although nornicotine typically accounts for only 2-4% of the total pyridine alkaloid content in tobacco plants, genetic instability leading to the spontaneous emergence of high-nornicotine-containing converter plants is a chronic problem in delivery systems. Maintaining low nornicotine levels may prevent the unpleasant flavor and aroma associated with this alkaloid and may also reduce the formation of N-nitrosonornicotine (NNN), a direct precursor of nornicotine, in delivery systems.

[0015] The gene responsible for most of the conversion of nicotine to nornicotine is the nicotine demethylase gene CYP82E4, which encodes a cytochrome P450 monooxygenase (Siminszky et al., Proc. Natl. Acad. Sci. 2002; 2003; 2004; 2005; 2006; 2007; 2008; 2009; 2010; 2011; 2012; 2013; 2014; 2015; 2016; 2017; 2018; 2019; 2020; 2021; 2022; 2023; 2024; 2025; 2026; 2027; 2028; 2030; 2031; 2032; USA, 102 (2005), pp. 14919-14924; Xu et al., Physiol. Plantarum, 129 (2007), pp. 307-319). The nicotine demethylase gene family in tobacco is widespread. Although nornicotine has been well characterized, little is known about other cellular processes that may affect nornicotine levels.

[0016] Nevertheless, there is a great need to devise methods that can further reduce the levels of TSNAs in tobacco plants and products produced from tobacco plants.

[0017] As described in the Examples, the present inventors sought to investigate genes involved in alkaloid synthesis with the aim of regulating alkaloid content in plants, for example, reducing nicotine content in tobacco plants.

[0018] A flue-cured tobacco variety (FC101) containing nic1 and nic2 was found to have lower nicotine levels than predicted based on these two loci alone, and it was hypothesized that a third locus, Nic3, controls the reduced nicotine levels in this variety.

[0019] These investigations led us to create a population segregating for Nic3, generated from a cross between FC101 (nic1 nic2 nic3) and LAFC53 (nic1 nic2 Nic3). The resulting F2 plants were analyzed for alkaloid content. The F2 was subjected to SNP genotyping to identify polymorphic markers for further analysis. We developed markers that cosegregate with the Nic3 locus. New potential regulators of alkaloid synthesis genes were identified. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] International Publication No. 2018 / 237107 Brochure [Non-patent literature]

[0021] [Non-Patent Document 1] Voelckel, C., Krugel, T., Gase, K., Heidrich, N., van Dam, NM, Winz, R., and Baldwin, IT (2001). Anti-sense expression of putrescine N-methyltransferase confirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta. Chemoecology 11, 121-126 [Non-patent document 2] Valleau W. 1949. Breeding low-nicotine tobacco. Journal of Agricultural Research 78: 171-181 [Non-patent document 3] Legg PD, Collins GB, Litton CC. 1970. Registration of La Burley-21 Tobacco Germplasm. Crop Science 10(2): 212 [Non-patent document 4] Legg P, Chaplin J, Collins G. (1969). Inheritance of percent total alkaloids in Nicotiana tabacum L.: populations derived from crosses of low alkaloid lines with burley and flue-cured varieties. Journal of Heredity 60: 213-217 [Non-patent document 5] Legg P., and G., C. (1971). Inheritance of percent total alkaloids in Nicotiana tabacum L. II. genetic effects of two loci in Burley21 X LA Burley 21 populations. Canadian Journal of Genetics and Cytology 13, 287-291 [Non-patent document 6] Hibi, N., Higashiguchi, S., Hashimoto, T., and Yamada, Y. (1994). Gene-Expression in Tobacco Low-Nicotine Mutants. Plant Cell 6, 723-735 [Non-Patent Document 7] Collins , GB , Legg , PD , and Kasperba.Mj (1974). Use of Anther-Derived Haploids in Nicotiana .1. Isolation of breeding lines differing in total alkaloid content. Crop Science 14, 77-80

Outdoor Tools 8

Outdoor Tools9

Outdoor Tools 10

Outdoor Content11

[0022] Surprisingly, it has been discovered that the alkaloid content (e.g., nicotine content) of tobacco plants can be modulated by modulating the activity or expression of genes at the Nic3 locus as taught herein. This allows for the production of delivery systems with modulated alkaloid content and commercially desirable traits desired by consumers of the delivery systems. In some instances, consumers may desire products with lower levels of alkaloid content, e.g., lower levels of nicotine content.

[0023] The present invention relates to a method for producing a plant or part thereof, or a plant cell (such as tobacco and other Nicotiana species) , for the production of proteins, peptides and metabolites, e.g., for the production of therapeutic agents and pharmaceuticals, e.g., antibiotics, virus-like particles or neutraceuticals or small molecules Tobacco plants can be particularly useful in the field of molecular agriculture, where they are used for the development of HIV-neutralizing antibodies in an EU-funded project called PharmPlant, and Medicago, Canada, has been working on a tobacco-based platform for the generation of virus-like particles for influenza vaccine production.

[0024] In other examples, it may be desirable to produce plants with high alkaloid levels, e.g., high levels of nicotine content, so that nicotine can be purified from tobacco plants to produce pure nicotine products, for example, for use in devices that utilize nicotine-containing liquids (e.g., electronic cigarettes) or in tobacco heating devices. For example, producing plants with leaves that contain high levels of nicotine can reduce the cost of nicotine extraction for the production of e-liquids for electronic cigarettes.

[0025] The present inventors investigated the regulation of nicotine biosynthesis in tobacco plants. The inventors identified and investigated a new locus, referred to herein as the Nic3 locus. It was hypothesized that, in addition to the regulatory loci Nic1 and Nic2, Nic3 controls the expression of nicotine-related structural genes (and possibly other unrelated genes). One goal of the inventors was to provide altered alkaloid content, particularly reduced nicotine content. Surprisingly, we identified genes in the Nic3 region that regulated alkaloid content in modified tobacco plants compared to their wild-type counterparts grown under the same conditions. In particular, the inventors further reduced alkaloid (e.g., nicotine content) and TSNA precursor content in a nic1 nic2 background (i.e., in plants already having low alkaloids, e.g., low nicotine). We identified the Nic3 locus (and the Nic3 gene) that can induce phenotypic changes in the Nic3 gene.

[0026] The present inventors have surprisingly determined a method for modulating the alkaloid content, e.g., nicotine content, of a tobacco plant or part thereof or plant cell by modulating the activity or expression of the Nic3 gene and / or by providing a mutation in the Nic3 locus. Prior to the present invention, it was not known that a third genetic locus, namely, "Nic3" as described herein, could be used to modulate alkaloid content, either alone or in combination with the Nic1 and / or Nic2 loci.

[0027] The present inventors have determined that modulation of the Nic3 locus can reduce the alkaloid content (eg, nicotine content) and / or TSNA precursor or TSNA content of the modified plant.

[0028] In particular, the inventors have determined that modulation of the Nic1, Nic2, and Nic3 loci (e.g., mutation of genes within said loci) provides plants with surprisingly low alkaloid content (e.g., low nicotine content) and / or low TSNA precursor or TSNA content.

[0029] In one aspect, the invention provides a method of modulating (e.g., decreasing) the alkaloid content (e.g., nicotine content) of a tobacco plant or part thereof, or a tobacco plant cell, comprising modifying the plant or cell by modulating the activity or expression of at least one Nic3 gene from Table 3, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or ortholog of said gene.

[0030] Suitably, in any aspect of the invention, the activity or expression of at least one Nic3 gene selected from SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 103, SEQ ID NO: 106, SEQ ID NO: 109, SEQ ID NO: 112, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 124, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 136, SEQ ID NO: 139, SEQ ID NO: 142, SEQ ID NO: 145, SEQ ID NO: 148 or SEQ ID NO: 151 may be modulated. Suitably, the activity or expression of at least one Nic3 gene selected from SEQ ID NO: 73, 118, 124 or 127 may be modulated (e.g., decreased or increased).

[0031] Suitably, the activity or expression of at least one gene selected from SEQ ID NO: 73, SEQ ID NO: 76 or SEQ ID NO: 79 may be modulated (eg decreased or increased).

[0032] In another aspect, the invention provides a method of modulating (e.g., reducing) the alkaloid content (e.g., nicotine content) of a tobacco plant or part thereof, or a tobacco plant cell, comprising modifying the plant or part thereof or cell by introducing at least one mutation into the Nic3 locus (e.g., the Nic3 gene), and optionally, at least one mutation into the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation into the Nic2 locus (e.g., the Nic2 ERF gene).

[0033] Suitably, in any aspect of the invention, the Nic3 gene may be selected from SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148 or SEQ ID NO:151.

[0034] Suitably, the activity or expression of at least one gene selected from SEQ ID NO: 73, 118, 124 or 127 may be modulated (e.g., decreased or increased). Suitably, the activity or expression of at least one gene selected from SEQ ID NO: 73, SEQ ID NO: 76 or SEQ ID NO: 79 may be modulated (e.g., decreased or increased).

[0035] In one aspect, the invention provides a method for modulating (e.g., decreasing) the alkaloid content (e.g., nicotine content) of a tobacco plant or part thereof, or a tobacco plant cell, comprising: a) at least one Nic3 gene; and optionally, b) at least one Nic1 ERF gene; and / or c) at least one Nic2 ERF gene In another aspect, a method is provided comprising modifying the plant or cell by modulating the activity or expression of

[0036] In such methods, the activity or expression of at least one Nic3 gene is altered, and optionally, the activity or expression of at least one Nic1 ERF and / or Nic2 ERF gene is altered. The term "may, optionally," as used herein, requires that the feature described thereafter is merely optional, i.e., may or may not be present. The term "and / or," as used herein, allows for the presence of one or both of the features described before and after the term. Thus, in this embodiment, i) at least one Nic3 gene; ii) at least one Nic3 gene and at least one Nic1 ERF gene; iii) at least one Nic2 ERF gene; and iv) the alternatives of modifying at least one Nic3 gene and at least one Nic2 ERF gene. Suitably, in option i), there is no modification in the Nic1 ERF or Nic2 ERF genes. Such options also apply to other methods, uses, and products described herein in which such modifications are contemplated.

[0037] In another aspect, the present invention provides a method for modulating (e.g., reducing) the alkaloid content (e.g., nicotine content) of a tobacco plant or part thereof, or a tobacco plant cell, comprising modifying the plant or part thereof or cell by introducing at least one mutation into the Nic3 locus (e.g., the Nic3 gene), and optionally, at least one mutation into the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation into the Nic2 locus (e.g., the Nic2 ERF gene). In such a method, at least one mutation is introduced into the Nic3 locus, and additionally, at least one mutation may be introduced into the Nic1 locus and / or the Nic2 locus. This may therefore include: i) at least one mutation into the Nic3 locus; ii) at least one mutation into the Nic3 locus and at least one mutation into the Nic1 locus; or iii) at least one mutation into the Nic3 locus and at least one mutation into the Nic2 locus. and iv) the introduction of at least one mutation at the Nic3 locus, at least one mutation at the Nic1 locus, and at least one mutation at the Nic2 locus. Suitably, in option i), no mutations are introduced at the Nic1 or Nic2 loci. Such options similarly apply to other methods, uses, and products described herein in which such mutations are contemplated.

[0038] In a further aspect, the present invention provides a method for modulating (e.g., reducing) the content of tobacco-specific nitrosamine (TSNA) precursors in a tobacco plant or part of the plant, or in a tobacco plant cell, comprising: i) a) at least one Nic3 gene; optionally, b) at least one Nic1 ERF gene; and / or c) at least one Nic2 ERF gene modulating the activity or expression of; or ii) introducing at least one mutation into the Nic3 locus (e.g., the Nic3 gene), and optionally, introducing at least one mutation into the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation into the Nic2 locus (e.g., the Nic2 ERF gene). modifying the plant or cell by The present invention provides a method comprising:

[0039] In a further aspect, the present invention provides a method for modulating (e.g., decreasing) the alkaloid content (e.g., nicotine content) and / or TSNA precursor content of a tobacco plant or part thereof or a tobacco plant cell, comprising: a) at least one Nic3 gene, optionally at least one Nic1 ERF gene and / or at least one Nic2 ERF gene; or b) at least one mutation in the Nic3 locus (e.g., the Nic3 gene), and optionally at least one mutation in the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation in the Nic2 locus (e.g., the Nic2 ERF gene). Provides for the use of

[0040] In another aspect, the present invention provides a method for producing a plant or part thereof, tobacco plant cell, tobacco plant propagation material, tobacco leaf, cut and harvested tobacco leaf, processed tobacco leaf, or cut and processed tobacco leaf having a modulated (e.g., reduced) alkaloid content (e.g., nicotine content), comprising: i) a) at least one Nic3 gene; and optionally, b) at least one Nic1 ERF gene; and / or c) at least one Nic2 ERF gene to regulate the activity or expression of; or ii) to introduce at least one mutation in the Nic3 locus (e.g., the Nic3 gene), and optionally at least one mutation in the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation in the Nic2 locus (e.g., the Nic2 ERF gene); The method further comprises modifying the tobacco plant or part thereof or tobacco cell.

[0041] Suitably, in any aspect of the invention, the nicotine content may be reduced compared to a tobacco plant or part thereof or tobacco cell that has not been modified to introduce at least one mutation at the Nic3 locus, and optionally at least one mutation at the Nic1 locus and / or at least one mutation at the Nic2 locus.

[0042] In another aspect, the present invention provides a tobacco plant or part thereof, or tobacco cell, that has been modified to achieve a reduced alkaloid content (e.g., nicotine content) compared to an unmodified tobacco plant or part thereof, or tobacco cell, wherein the modification comprises: i) a) at least one Nic3 gene; and optionally, b) at least one Nic1 ERF gene; and / or c) at least one Nic2 ERF gene Modulated activity or expression of; or ii) at least one mutation in the Nic3 locus (e.g., the Nic3 gene), and optionally at least one mutation in the Nic1 locus (e.g., the Nic1 gene) and / or at least one mutation in the Nic2 locus (e.g., the Nic2 ERF gene). The present invention provides a tobacco plant or part thereof, or a tobacco cell, comprising:

[0043] In a further aspect, the present invention provides tobacco plant propagation material obtainable from a tobacco plant or part thereof or tobacco cell according to the invention, or from a tobacco plant or part thereof or tobacco cell produced by a method according to the invention.

[0044] Suitably, in any aspect of the invention (e.g. a method or use according to the invention, a plant or part or cell thereof according to the invention, or a plant propagation material according to the invention): a) the activity or expression of a Nic3 gene selected from those listed in Table 3, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or ortholog of said gene, may be modulated; or the at least one mutation in the Nic3 locus may be present in a Nic3 gene selected from those listed in Table 3, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or ortholog of said gene: b) the activity or expression of a Nic1 ERF gene selected from those listed in Table 1 may be modulated; or the at least one mutation in the Nic1 locus may be in a Nic1 ERF gene selected from SEQ ID NO:8; or SEQ ID NO:12; or SEQ ID NO:16; or SEQ ID NO:20; or SEQ ID NO:24; or SEQ ID NO:28; or SEQ ID NO:32, or a sequence having at least 90% identity thereto; or a functional variant, functional fragment, or ortholog of said gene; and / or c) The activity or expression of a Nic2 ERF gene selected from those listed in Table 2 may be modulated; or the at least one mutation in the Nic2 locus may be a Nic2 ERF gene selected from SEQ ID NO:69; SEQ ID NO:37; or SEQ ID NO:41; or SEQ ID NO:45; or SEQ ID NO:49; or SEQ ID NO:53; or SEQ ID NO:57; or SEQ ID NO:61; or SEQ ID NO:65, or a sequence having at least 90% identity thereto; or a functional variant, functional fragment, or ortholog of said gene. Suitably, the Nic3 gene selected from those listed in Table 3 is selected from SEQ ID NO: 73, 118, 124 or 127, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment or orthologue of said gene, and at least one mutation in the Nic3 locus is in a Nic3 gene selected from SEQ ID NO: 73, 118, 124 or 127, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment or orthologue of said gene.

[0045] Suitably, in any aspect of the invention (e.g. a method or use according to the invention, a plant or part thereof according to the invention, or a plant propagation material according to the invention), i) the activity or expression of SEQ ID NO: 8 may be modulated (e.g., decreased or increased); or the at least one mutation in the Nic1 locus may be in SEQ ID NO: 8; and / or ii) the activity or expression of SEQ ID NO:69 may be modulated (e.g., decreased or increased); or the at least one mutation in the Nic2 locus may be in SEQ ID NO:69.

[0046] Suitably, in any aspect of the invention (e.g. a method or use according to the invention, a plant or part thereof according to the invention, or a plant propagation material according to the invention), the at least one mutation in the Nic3 locus may be in a Nic3 gene selected from Table 3, or a sequence having at least 90% identity thereto, or a functional variant or functional fragment or orthologue of said gene.

[0047] Suitably, said at least one mutation in the Nic3 gene is i) a mutation in SEQ ID NO: 73, or a sequence having at least 90% identity thereto, of the Nic3 gene, or a functional variant, functional fragment, or ortholog of said gene, resulting in a mutation in amino acid residues 74 to 258 or 483 to 538 of SEQ ID NO: 75, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or ortholog of said polypeptide; ii) a mutation in SEQ ID NO: 118, or a sequence having at least 90% identity thereto, which is the Nic3 gene, or a functional variant, functional fragment, or ortholog of said gene, resulting in a mutation in amino acid residues 120 to 584 of SEQ ID NO: 120, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or ortholog of said polypeptide; iii) SEQ ID NO: 126, or a sequence having at least 90% identity thereto or a functional variant or functional fragment or ortholog of said polypeptide, resulting in a mutation at amino acid residues 166 to 406 or 483 to 970 of the Nic3 gene. or a sequence having at least 90% identity thereto, or a functional variant or functional fragment or ortholog of said gene; and iv) a mutation in SEQ ID NO: 127, or a sequence having at least 90% identity thereto, of the Nic3 gene, or a functional variant, functional fragment, or ortholog of said gene, that results in a mutation in amino acid residues 171 to 406 or 509 to 967 of SEQ ID NO: 129, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or ortholog of said polypeptide. is selected from.

[0048] In one aspect, the present invention provides the use of a plant or part thereof or a plant cell according to the present invention, or a plant produced by a method according to the present invention, for breeding plants.

[0049] In one aspect, the present invention provides the use of a plant or part thereof or a plant cell according to the present invention, or a plant produced by a method according to the present invention, for the production of a product.

[0050] In one aspect, the present invention provides the use of a plant or part thereof or a plant cell according to the present invention, or a plant produced by a method according to the present invention, for growing a crop.

[0051] In one aspect, the present invention provides the use of a plant or part thereof according to the present invention, or a plant produced by a method according to the present invention, for producing leaves.

[0052] In a further aspect, the present invention provides harvested leaves obtainable from a plant according to the invention, or from a plant propagated from propagation material according to the invention, or obtainable from a plant obtained by a use according to the invention, or obtainable from a plant produced by a method according to the invention.

[0053] The harvested leaves may be green leaves, for example, green fresh leaves or dried leaves.

[0054] Suitably, the harvested leaves according to the present invention may be cut and harvested leaves.

[0055] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: obtainable (e.g. obtained) from a plant obtainable from use according to the invention, obtainable (e.g. obtained) by processing a plant according to the present invention; obtainable (e.g., obtained) from a plant propagated from plant propagation material according to the invention; or obtainable (e.g. obtained) by processing harvested leaves of a plant according to the invention, or obtainable (e.g. obtained) from a plant produced by a method according to the invention; Processed leaf, preferably processed tobacco leaf, preferably non-viable processed tobacco leaf, is provided.

[0056] Suitably, the processed leaves according to the present invention may be processed by drying, fermenting, pasteurising or a combination thereof, and preferably have a reduced content of one or more TSNAs selected from N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB), preferably have a controlled (e.g. reduced) content of NNN and / or NNK, more preferably have a controlled (e.g. reduced) content of NNN. The amount decreases.

[0057] Suitably, the processed leaves according to the present invention may be cut and processed leaves.

[0058] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: obtainable (e.g. obtained) from a plant obtainable from the use according to the invention, obtainable (e.g. obtained) by processing a plant according to the present invention; obtainable (e.g., obtained) from a plant propagated from plant propagation material according to the invention; or obtainable (e.g. obtained) by processing harvested leaves of a plant according to the invention, or obtainable from a plant produced by the method according to the invention, A dried tobacco material made from a plant or part thereof is provided.

[0059] Suitably, the dried tobacco material, tobacco blend or delivery system may comprise an average alkaloid level or average nicotine level of about 0.01%, 0.02%, 0.05%, 0.075%, 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 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%, 3%, 4% or 5% based on dry weight. Suitably, the dried tobacco material may comprise an average alkaloid level or an average nicotine level of less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.075%, less than 0.05%, less than 0.02%, or less than 0.01%.

[0060] In another aspect, the present invention provides a tobacco blend comprising a dried tobacco material according to the present invention.

[0061] In a further aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a tobacco plant or part thereof according to the present invention; a tobacco plant or part thereof propagated from the tobacco plant propagation material according to the present invention; Harvested leaves of the plant according to the invention; processed leaves according to the present invention; or Plants produced by the method according to the present invention The present invention provides a delivery system prepared from

[0062] Suitably, the delivery system according to the present invention may be a combustible smoking article.

[0063] Suitably, the delivery system according to the present invention may be a smokeless delivery system.

[0064] Suitably, the delivery system according to the present invention may be a non-combustible aerosol delivery system, for example a tobacco heating device or an aerosol generating device.

[0065] In a further aspect, the present invention provides a combustible smoking article, a non-combustible aerosol delivery system, a smokeless delivery system, or a tobacco heating device comprising a plant or part thereof or an extract thereof (e.g., a tobacco extract) according to the present invention; or a dried tobacco material according to the present invention; or a tobacco blend according to the present invention.

[0066] In another aspect, the present invention provides use of the nucleotide sequence of the Nic3 locus (e.g., a Nic3 gene selected from Table 3, or from a gene having SEQ ID NO: 73, 118, 124, or 127, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or orthologue of said gene), optionally the Nic1 locus (e.g., the Nic1 ERF gene) and / or the Nic2 locus (e.g., the Nic2 ERF gene), to select plants with reduced alkaloid content (e.g., nicotine content) and / or reduced content of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs. Suitably, the nucleotide sequence may comprise mutations.

[0067] In a further aspect, the present invention provides mutant plants having at least one genetic variation at the Nic3 locus (e.g., in a Nic3 gene selected from Table 3, or from a gene having SEQ ID NO: 73, 118, 124, or 127, or a sequence having at least 90% identity thereto, or in a functional variant, functional fragment, or ortholog of said gene), and optionally at least one genetic variation at the Nic1 locus (e.g., in the Nic1 ERF gene) and / or at least one genetic variation at the Nic2 locus (e.g., in the Nic2 ERF gene), wherein the genetic variation confers reduced alkaloid content (e.g., nicotine content) and / or reduced content of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs in the mutant tobacco plant compared to a comparable plant not having the genetic variation.

[0068] In a further aspect, the present invention provides progeny or seeds of a mutant plant having the genetic mutation according to the present invention.

[0069] In another aspect, the invention provides harvested leaves, processed leaves, or cured tobacco material produced from a plant that includes at least one mutation at the Nic3 locus (e.g., in a Nic3 gene selected from Table 3, or from a gene having SEQ ID NO: 73, 118, 124, or 127, or a gene having a sequence with at least 90% identity thereto, or in a functional variant, functional fragment, or ortholog of said gene), and optionally at least one mutation at the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation at the Nic2 locus (e.g., the Nic2 ERF gene), wherein the harvested leaves, processed leaves, or cured tobacco material have a reduced nicotine content and / or a reduced content of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs, compared to a comparable plant that does not have the mutation at the Nic3 locus, and optionally the mutation at the Nic1 locus and / or the Nic2 locus.

[0070] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0071] [Figure 1] Nicotine and nornicotine contents of FC101 and LAFC53 are shown. Asterisks indicate significant differences relative to FC101 (p-value < 0.01). [Figure 2] Nicotine and nornicotine contents of the F2 population between FC101 and LAFC53 are shown. (A) Nicotine content, (B) Nornicotine content. [Figure 3] 1 shows the results of quantitative trait locus (QTL) analysis for nicotine on chromosome 5 in the F2 population from FC101 x LAFC53. [Figure 4] Figure 1 shows nicotine and nornicotine contents in the F2 population from FC101 x LAFC53 segregating for marker Nt2AG2015. [Figure 5-1] ~ [Figure 8-2] SEQ ID NOs: 571 to 574 provide TRV2 sequences for silencing genes with SEQ ID NOs: 73, 118, 124 and 127, with gene-specific sequences shown in bold and underlined. [Figure 9] 1 shows that virus-induced gene silencing of genes in the Nic3 locus results in reduced nicotine content compared to nic1nic2. DETAILED DESCRIPTION OF THE INVENTION

[0072] Sequence Listing A summary of the sequence identifiers used throughout this specification and the corresponding sequence listing is as follows:

[0073] [Table 1]

[0074] [Table 2]

[0075] [Table 3] JPEG2025169355000004.jpg188135JPEG2025169355000005.jpg195138JPEG2025169355000006.jpg36133

[0076] SEQ ID NO: 298 corresponds to marker Nt1AG1750.

[0077] SEQ ID NO: 299 corresponds to marker Nt1AC2307.

[0078] SEQ ID NO: 300 is the forward primer for SNP3.

[0079] SEQ ID NO: 301 is the reverse primer for SNP3.

[0080] SEQ ID NO: 302 is the forward primer for SNP5.

[0081] SEQ ID NO: 303 is the reverse primer for SNP5.

[0082] SEQ ID NO: 304 is the forward primer for SNP15.

[0083] SEQ ID NO: 305 is the reverse primer for SNP15.

[0084] SEQ ID NO: 306 is the forward primer for SNP18.

[0085] SEQ ID NO: 307 is the reverse primer for SNP18.

[0086] SEQ ID NO: 308 is the forward primer for SNP19.

[0087] SEQ ID NO: 309 is the reverse primer for SNP19.

[0088] SEQ ID NO: 310 corresponds to marker Nt2AG2015.

[0089] SEQ ID NO: 311 corresponds to marker Nt1AG1750.

[0090] SEQ ID NO: 312 corresponds to marker Nt1AC2307.

[0091] SEQ ID NOs: 313 to 569 are the sequences of SNPs associated with the QTLs identified in the Examples.

[0092] SEQ ID NO: 570 is TRV RNA1 used in Example 7.

[0093] SEQ ID NOs: 571 to 574 are the TRV RNA2 sequences used in Example 7.

[0094] Some sequences disclosed herein contain an "N" in the nucleotide sequence. The "N" can be any nucleotide, or a deletion or insertion of one or more nucleotides. For example, in some cases, a string of "N" is shown. The number of "N"s does not necessarily correlate to the actual number of nucleotides at that position. There may be more or fewer nucleotides than shown as "N" in the sequence.

[0095] Detailed Description The present inventors have identified, for the first time, the Nic3 locus that regulates nicotine biosynthesis in tobacco.

[0096] By modulating the activity or expression of at least one Nic3 gene in a plant (e.g., a tobacco plant) or part thereof, or tobacco cells, the alkaloid and / or TSNA content of the plant can be modulated (e.g., reduced). By introducing a mutation into the Nic3 locus, the alkaloid (e.g., nicotine) content of a plant or part thereof, or plant cell can be modulated (e.g., reduced).

[0097] As used herein, the "Nic3" locus refers to any chromosomal location or location within or closely linked to the Nic3 region.

[0098] "Nic3 region" refers to the chromosomal segment delimited by markers Nt1AG1750 (SEQ ID NO: 298) and Nt1AC2307 (SEQ ID NO: 299), which corresponds to 206 cM to 398 cM shown in Figure 3 and has alleles associated with the low-alkaloid (low-nicotine) trait.

[0099] "Nic3 mutation" refers to a mutation in the Nic3 locus.

[0100] "Nic3 gene," as used herein, refers to a gene at or near the Nic3 locus, including, for example, a gene listed in Table 3; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, fragment, or ortholog of said gene.

[0101] Suitably, the Nic3 gene may be selected from SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148 or SEQ ID NO:151; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant or functional fragment or orthologue of said gene.

[0102] Suitably, the Nic3 gene may be selected from SEQ ID NO: 73, SEQ ID NO: 76 or SEQ ID NO: 79; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, functional fragment or orthologue of said gene. Suitably, the Nic3 gene may be selected from SEQ ID NO: 73, SEQ ID NO: 118, SEQ ID NO: 124 or SEQ ID NO: 127; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, functional fragment or orthologue of said gene.

[0103] In another aspect, the Nic3 locus is selected from the group consisting of SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148, and SEQ ID NO:15 The present invention also includes a sequence or chromosomal segment of up to 50, 100, 200, 300, 400, 500, 6000, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 70000 nucleotides of a sequence selected from the group consisting of SEQ ID NO: 73, SEQ ID NO: 118, SEQ ID NO: 124 and SEQ ID NO: 127.

[0104] In one aspect, the Nic3 gene is SEQ ID NO:73, SEQ ID NO:118, SEQ ID NO:124 or SEQ ID NO:127; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant or functional fragment or ortholog of said gene.

[0105] Suitably, the Nic3 gene may encode a polypeptide comprising an amino acid sequence set out in Table 3; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, functional fragment or ortholog of said polypeptide.

[0106] Suitably, the Nic3 gene may encode a polypeptide selected from SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:141, SEQ ID NO:144, SEQ ID NO:147, SEQ ID NO:150 or SEQ ID NO:153; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, functional fragment or orthologue of said polypeptide.

[0107] Suitably, the Nic3 gene may encode a polypeptide selected from SEQ ID NO: 75, SEQ ID NO: 78 or SEQ ID NO: 81; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, functional fragment or ortholog of said polypeptide.

[0108] In one aspect, the Nic3 gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:75, SEQ ID NO:120, SEQ ID NO:126, or SEQ ID NO:129; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, functional fragment, or ortholog of said polypeptide.

[0109] In one embodiment, the Nic3 locus comprises one or more sequences selected from Table 3.

[0110] In one aspect, the Nic3 locus comprises one or more sequences selected from the group consisting of SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148, and SEQ ID NO:151, and a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), and functional variants, functional fragments, and orthologs of said genes.

[0111] Suitably, the Nic3 locus may comprise one or more sequences selected from the group consisting of SEQ ID NO: 73, SEQ ID NO: 76 and SEQ ID NO: 79, and a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), and functional variants, functional fragments and orthologues of said genes.

[0112] In one aspect, the Nic3 locus comprises at least one or more of SEQ ID NO:73, SEQ ID NO:118, SEQ ID NO:124 or SEQ ID NO:127, or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant or functional fragment or ortholog of said gene.

[0113] "Closely linked to" or "associated with," as used herein, means that a marker or locus is within about 20 cM, 10 cM, 5 cM, 1 cM, 0.5 cM, or less than 0.5 cM of another marker or locus. For example, 10 cM means recombination between the marker and locus at a frequency equal to or less than about 10%.

[0114] CentiMorgan (cM), as used herein, refers to a unit of measure for recombination frequency. A cM equals 1% of the expected events in which a marker at one locus separates from a marker at a second locus by crossing over in a single generation. A method for calculating genetic distance from recombination values ​​using the Kosambi function is They are known in the art and are described, for example, in Kosambi (Annals of Eugenics, 12:172-175 (1944), incorporated herein by reference).

[0115] "Nic1" locus, as used herein, refers to any chromosomal location or position within or closely linked to the Nic1 region.

[0116] "Nic1 region" refers to a chromosomal segment as disclosed in WO 2018 / 237107 (incorporated herein by reference), e.g., a chromosomal segment delimited by markers SNP3 and SNP5 (Nt1AB6591 and Nt1AA9777) that has alleles associated with a low-alkaloid (low-nicotine) trait. The forward primer for SNP3 is SEQ ID NO: 300; the reverse primer for SNP3 is SEQ ID NO: 301. The forward primer for SNP5 is SEQ ID NO: 302; the reverse primer for SNP5 is SEQ ID NO: 303.

[0117] "Nic1 mutation" refers to a mutation in the Nic1 locus.

[0118] In one embodiment, the Nic1 locus comprises one or more sequences selected from Table 1.

[0119] In one embodiment, the Nic1 locus comprises one or more sequences selected from the group consisting of SEQ ID NO:5, SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29, and SEQ ID NO:33, and a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), and functional variants, functional fragments, and orthologs of said genes.

[0120] In one embodiment, the Nic1 locus comprises at least SEQ ID NO: 8, or at least a sequence similar to SEQ ID NO: 9. or a sequence having 80% identity (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant or functional fragment or ortholog of said gene.

[0121] In another aspect, the Nic1 locus comprises a sequence or chromosomal segment within 50, 100, 200, 300, 400, 500, 6000, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 70000 nucleotides of a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29, and SEQ ID NO:33.

[0122] "Nic1 gene," as used herein, refers to a gene at or near the Nic1 locus, including, for example, a gene listed in Table 1; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, fragment, or ortholog of said gene.

[0123] The at least one Nic1 ERF gene may be selected from the group comprising genes encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8; or SEQ ID NO:12; or SEQ ID NO:16; or SEQ ID NO:20; or SEQ ID NO:24; or SEQ ID NO:28; or SEQ ID NO:32, or a functional variant, functional fragment, or ortholog thereof, or the ERF gene comprises the nucleotide sequence set forth in SEQ ID NO:5; or SEQ ID NO:9; or SEQ ID NO:13; or SEQ ID NO:17; or SEQ ID NO:21; or SEQ ID NO:25; or SEQ ID NO:29; or a functional variant, functional fragment, or ortholog thereof.

[0124] Suitably, the at least one Nic1 ERF gene may be one, two, three, four, five, six or seven genes selected from the group comprising genes encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8; or SEQ ID NO:12; or SEQ ID NO:16; or SEQ ID NO:20; or SEQ ID NO:24; or SEQ ID NO:28; or SEQ ID NO:32, or a functional variant, functional fragment, or orthologue thereof; or the ERF gene comprises the nucleotide sequence set forth in SEQ ID NO:5; or SEQ ID NO:9; or SEQ ID NO:13; or SEQ ID NO:17; or SEQ ID NO:21; or SEQ ID NO:25; or SEQ ID NO:29; or a functional variant, functional fragment, or orthologue thereof.

[0125] In one aspect, at least one Nic1 ERF gene encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8, or a functional variant, functional fragment, or ortholog thereof; or the Nic1 ERF gene comprises the nucleotide sequence set forth in SEQ ID NO:5, or a functional variant, functional fragment, or ortholog thereof.

[0126] In one aspect, the activity or expression of at least one additional Nic1 ERF is modulated. Suitably, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight additional Nic1 ERFs selected from Table 1 may also be modulated.

[0127] In one embodiment, at least one Nic1 ERF gene encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, or a functional variant, functional fragment, or ortholog thereof; or at least one Nic1 ERF gene encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: or a functional variant, fragment, or ortholog thereof; the activity or expression of at least one additional Nic1 ERF is modulated. Suitably, the at least one additional Nic1 ERF may be selected from a Nic1 ERF gene encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4; or SEQ ID NO:12; or SEQ ID NO:16; or SEQ ID NO:20; or SEQ ID NO:24; or SEQ ID NO:28; or SEQ ID NO:32; or SEQ ID NO:36, or a functional variant, fragment, or ortholog thereof; or the ERF gene comprises the nucleotide sequence set forth in SEQ ID NO:1; or SEQ ID NO:3; or SEQ ID NO:9; or SEQ ID NO:13; or SEQ ID NO:17; or SEQ ID NO:21; or SEQ ID NO:25; or SEQ ID NO:29; or SEQ ID NO:33, or a functional variant, fragment, or ortholog thereof. Suitably, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight additional Nic1 ERFs may be modulated.

[0128] "Nic2" locus, as used herein, refers to any chromosomal location or position within or closely linked to the Nic2 region.

[0129] "Nic2 region" refers to a chromosomal segment as disclosed in WO 2018 / 237107 (incorporated herein by reference), e.g., a chromosomal segment bounded by markers SNP15 and SNP18 / 19, and carrying alleles associated with a low-alkaloid (low-nicotine) trait. The forward primer for SNP15 is SEQ ID NO: 304; the reverse primer for SNP15 is SEQ ID NO: 305. The forward primer for SNP18 is SEQ ID NO: 306; the reverse primer for SNP18 is SEQ ID NO: 307. The forward primer for SNP19 is SEQ ID NO: 308; the reverse primer for SNP19 is SEQ ID NO: 309.

[0130] "Nic2 mutation" refers to a mutation in the Nic2 locus.

[0131] In one embodiment, the Nic1 locus comprises one or more sequences selected from Table 1.

[0132] In one embodiment, the Nic2 locus comprises one or more sequences selected from the group consisting of SEQ ID NO:69, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:3, SEQ ID NO:57, SEQ ID NO:61, and SEQ ID NO:65, and a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), and functional variants, functional fragments, and orthologs of said genes.

[0133] In one aspect, the Nic2 locus comprises at least SEQ ID NO: 69 or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, functional fragment, or ortholog of said gene.

[0134] In another aspect, the Nic2 locus comprises a sequence or chromosomal segment within 50, 100, 200, 300, 400, 500, 6000, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 30000, 40000, 50000, 60000, 70000 nucleotides of a sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:3, SEQ ID NO:57, SEQ ID NO:61, and SEQ ID NO:65.

[0135] "Nic2 gene," as used herein, refers to a gene at or near the Nic2 locus, including, for example, a gene listed in Table 2; or a sequence having at least 80% identity thereto (at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identity thereto), or a functional variant, fragment, or ortholog of said gene.

[0136] The at least one Nic2 ERF gene may be selected from the group comprising genes encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:72; or SEQ ID NO:40; or SEQ ID NO:44; or SEQ ID NO:48; or SEQ ID NO:52; or SEQ ID NO:56; or SEQ ID NO:60, SEQ ID NO:64 or SEQ ID NO:68, or a functional variant, functional fragment or ortholog thereof; or the ERF gene comprises SEQ ID NO:69; or SEQ ID NO:37; or SEQ ID NO:41; or SEQ ID NO:45; or SEQ ID NO:49; or SEQ ID NO:53; or SEQ ID NO:57; SEQ ID NO:61; the nucleotide sequence set forth in SEQ ID NO:65; or a functional variant, functional fragment or ortholog thereof.

[0137] Suitably, the at least one Nic2 ERF gene may be one, or two, or three, or four, or five, or six, or seven, or eight, or nine genes selected from Table 2.

[0138] In one aspect, at least one Nic2 ERF gene encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 72, or a functional variant, functional fragment, or ortholog thereof; or the Nic1 ERF gene comprises the nucleotide sequence set forth in SEQ ID NO: 59, or a functional variant, functional fragment, or ortholog thereof.

[0139] The term "modulate" is used herein to mean either to increase or to decrease.

[0140] The term "increasing the alkaloid content" is used herein to mean that the concentration and / or total alkaloid content in a product of the invention (e.g., a plant, a part thereof (e.g., a leaf), a processed leaf, or a product made from the plant (e.g., a delivery system)) is higher compared to a comparable product that has not been modified according to the present invention.

[0141] The term "reducing the alkaloid content" is used herein to mean that the concentration and / or total alkaloid content in a product of the invention (e.g., a plant, a part thereof (e.g., a leaf), a processed leaf, or a product made from the plant (e.g., a delivery system)) is lower compared to a comparable product that has not been modified according to the present invention.

[0142] In one aspect, a tobacco plant or part thereof or tobacco cell according to the present invention comprises a total alkaloid level of less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, or less than 0.05%.

[0143] In one aspect, the tobacco plant or part thereof or tobacco cell according to the invention has less than 3%, less than 2.75%, less than 2.5%, less than 2.25%, less than 2%, less than 1.75%, less than 1.5%, less than 1.25%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6% %, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, or less than 0.05%. In one aspect, a tobacco plant or part thereof or tobacco cell according to the invention comprises an alkaloid or nicotine level that is less than 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the alkaloid or nicotine level of a comparable plant or part thereof or cell.

[0144] In one embodiment, the invention provides a method for modulating (e.g., reducing) the content of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs in a plant (e.g., a tobacco plant) or part thereof, comprising modifying the plant by modulating the activity or expression of at least one Nic3 gene. Suitably, the method may comprise modulating (e.g., reducing) the activity or expression of at least one Nic3 gene, and optionally at least one Nic1 ERF gene and / or at least one Nic2 ERF gene.

[0145] In one embodiment, the TSNA is N'-nitrosonornicotine (NNN) and / or the precursor is nornicotine.

[0146] In one embodiment, the TSNA can be one or more members selected from the group consisting of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

[0147] In a preferred embodiment, the TSNA is N'-nitrosonornicotine (NNN).

[0148] TSNAs can be measured in processed tobacco, such as cured tobacco or reconstituted tobacco. In one embodiment, TSNA content is measured and / or altered (e.g., reduced) in a cured tobacco plant or part thereof (e.g., in cured tobacco leaves).

[0149] The term "tobacco-specific nitrosamine" or "TSNA," as used herein, has its ordinary meaning in the art, i.e., a nitrosamine found only in a delivery system or other nicotine-containing product. Suitably, the at least one tobacco-specific nitrosamine may be N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT), or N-nitrosoanabasine (NAB).

[0150] The term "precursor thereof," when used in connection with at least one tobacco-specific nitrosamine, refers to one or more chemicals or compounds in the tobacco plant that cause the formation of, or are involved in, the nitrosation reaction that leads to the production of, the tobacco-specific nitrosamine.

[0151] In one embodiment, the precursor of the TSNA is one or more of the group selected from nornicotine, anabasine, anatabine, and an oxidized derivative of nicotine, such as pseudooxynicotine (PON).

[0152] In a preferred embodiment, the precursor of the TSNA is nornicotine.

[0153] Precursors of TSNAs (e.g., NNN, NNK, NAB, and / or NAT) can be measured, for example, in green tobacco leaves prior to processing, e.g., prior to curing. Precursors of TSNAs (eg, NNN, NNK, NAB and / or NAT) are measured and / or reduced in green tobacco leaves, for example, prior to processing, eg, prior to curing.

[0154] In one embodiment, practicing the method and / or use of the present invention results in a reduction of at least one TSNA or precursor thereof in a modified tobacco plant (or part thereof) or tobacco cell when compared to a tobacco plant (or part thereof) that has not been modified according to the present invention.

[0155] The terms "reducing at least one TSNA or precursor thereof" or "reduction of at least one TSNA or precursor thereof" are used herein to mean that the concentration and / or total content of at least one TSNA or precursor thereof in a product, method, or use of the invention is lower 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 invention, but where all other relevant characteristics are the same (e.g., plant species, growing conditions, method of processing the tobacco, etc.).

[0156] Any method known in the art for determining the concentration and / or level of at least one TSNA or its precursor can be used. In particular, such a method can include the addition of a deuterium-labeled internal standard, aqueous extraction and filtration, followed by analysis using reversed-phase high-performance liquid chromatography (LC-MS / MS) with the possibility of using tandem mass spectrometry. Other examples for determining the concentration and / or level of precursors of tobacco-specific nitrosamines include CORESTA recommended method CRM-72: Determination of Tobacco Specific Nitrosamines in Tobacco and Delivery Systems by LC-MS / MS; the CRM developed in ISO / DIS 21766; or methods such as those detailed in Wagner et al. Analytical Chemistry (2005) 77(4), 1001-1006, all of which are incorporated herein by reference.

[0157] Suitably, the concentration and / or total content of at least one tobacco-specific nitrosamine or precursor thereof can be reduced by practicing the methods and / or uses of the present invention. Suitably, the concentration and / or level of at least one tobacco-specific nitrosamine or precursor thereof can be reduced in a 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 precursor thereof in a tobacco plant that has not been modified according to the present invention.

[0158] The concentration and / or total content of at least one tobacco-specific nitrosamine or precursor thereof can be reduced in tobacco leaves, harvested leaves, processed tobacco leaves, delivery systems, or combinations thereof obtainable or derived from tobacco plants (or tobacco plant parts or tobacco cell cultures) of the present invention, when compared to tobacco leaves, harvested leaves, processed tobacco leaves, delivery systems, or combinations thereof obtainable or derived from tobacco plants (or tobacco plant parts or tobacco cell cultures) that have not been modified according to the present invention.

[0159] Suitably, the concentration and / or total content of at least one tobacco-specific nitrosamine or precursor thereof can be reduced in the processed tobacco leaves.

[0160] Suitably, the delivery system is capable of reducing the concentration and / or level of at least one tobacco-specific nitrosamine or precursor thereof.

[0161] In one embodiment, at least one tobacco-specific nitrosamine or precursor thereof 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%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some embodiments, at least one tobacco-specific nitrosamine or precursor thereof can be reduced by between about 5% and about 95%, between about 10% and about 90%, between 20% and about 80%, between 30% and about 70%, or between about 40% and 60%.

[0162] In the context of processed (e.g., cured) tobacco leaves (e.g., cured or reconstituted), at least one tobacco-specific nitrosamine or precursor thereof can be reduced by between about 5000 ng / g and about 50 ng / g, between about 4000 ng / g and about 100 ng / g, between about 3000 ng / g and 500 ng / g, or between 2000 ng / g and 1000 ng / g. In some embodiments, at least one tobacco-specific nitrosamine or precursor thereof 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.

[0163] As defined herein, the term "comparable product" would be 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 (e.g., plant species, growth conditions, method of processing the plant, e.g., tobacco, etc.) are identical. A comparable product according to the present invention can refer, for example, to a tobacco plant cell or plant (e.g., a tobacco plant) or part thereof, such as a leaf (e.g., a tobacco leaf), harvested leaf (e.g., harvested tobacco leaf), cut and harvested leaf (e.g., cut and harvested tobacco leaf), processed leaf (e.g., processed tobacco leaf) or plant propagation material (e.g., a tobacco plant propagation material), obtainable or obtained from a plant that has not been modified according to the present invention to modulate the activity or expression of a Nic3 gene (or a Nic3 gene in combination with one or more Nic1 ERF genes, in combination with one or more Nic2 ERF genes), or a product comprising said plant or part thereof, such as a delivery system, or a combination thereof. A comparable product may also be known as a control or wild type.

[0164] As defined herein, the term "unmodified plant" refers to a plant (e.g., a tobacco plant) that has not been modified in accordance with the present invention to modulate the activity or expression of the Nic3 gene, and in which all other relevant characteristics (e.g., plant species, growing conditions, method of processing tobacco, etc.) are identical.

[0165] "Activity or expression" of the Nic3 gene (or the Nic1 ERF gene or the Nic2 ERF gene) can also refer to the level of transcription, translation, i.e., protein expression, of the Nic3 gene (or the Nic1 ERF or Nic2 ERF gene, respectively), or the activity of the protein encoded thereby. The activity of the Nic3 gene relates to its ability to function as a regulator of alkaloid biosynthesis, particularly nicotine biosynthesis. The activity of the Nic1 ERF gene (or the Nic2 ERF gene) relates to its ability to function as a transcription factor in alkaloid biosynthesis. The activity of the Nic3 gene (or the Nic1 ERF gene or the Nic2 ERF gene) can be determined by measuring the product of alkaloid synthesis, i.e., by measuring alkaloid content.

[0166] According to one aspect of the invention, gene expression can be reduced (or inhibited) by inhibiting transcription and / or translation. In one embodiment, gene activity or expression can be measured by the level of transcription, i.e., the amount of mRNA produced, or translation, i.e., the amount of mRNA produced. It may also refer to the level or amount of protein obtained.

[0167] In some embodiments, modulation of alkaloid content refers to an increase in alkaloid content, wherein the activity or expression of at least one Nic3 gene is modulated.

[0168] In some embodiments, modulation of alkaloid content refers to a decrease in alkaloid content, wherein the activity or expression of at least one Nic3 gene is modulated.

[0169] In some embodiments, modulation of alkaloid content refers to an increase in alkaloid content, in combination with modulation of the activity or expression of at least one Nic3 gene, and optionally, the activity or expression of at least one Nic1 ERF gene and / or Nic2 ERF.

[0170] In some embodiments, modulation of alkaloid content refers to a decrease in alkaloid content, in combination with modulation of the activity or expression of at least one Nic3 gene, and optionally, the activity or expression of at least one Nic1 ERF gene and / or Nic2 ERF.

[0171] In a further embodiment, the alkaloid content is measured from leaves. In one embodiment, the alkaloid content is measured from green leaves. In a further embodiment, the alkaloid content is measured from dried leaves, e.g., air-cured, flue-cured, fire-cured, etc. It is measured from fire-cured or sun-cured leaves. In a further embodiment, the alkaloid content is measured from flue-dried leaves. In a further embodiment, the alkaloid content is measured from air-dried leaves.

[0172] The term "alkaloid content" is used herein to mean the concentration and / or total amount of the entire group of compounds classified as alkaloids. Alkaloids typically present in tobacco include nicotine, anatabine, anabasine, myosmine, and nornicotine. In one embodiment, the content of one or more alkaloids selected from nicotine, anatabine, anabasine, myosmine, and nornicotine is adjusted. In one embodiment, the content of one or more alkaloids selected from nicotine, anatabine, anabasine, myosmine, and nornicotine is reduced. In one embodiment, the content of one or more alkaloids selected from nicotine, anatabine, anabasine, and nornicotine is increased. Suitably, the nicotine content is adjusted. In one embodiment, the nicotine content is reduced.

[0173] Any method known in the art for determining alkaloid concentration and / or total content can be used. One preferred method for analyzing alkaloid content includes analysis by gas chromatography-flame ionization detection (GC-FID).

[0174] In one embodiment, a method is provided for producing a plant (e.g., a tobacco plant) or part thereof, 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 and harvested leaf (e.g., cut and harvested tobacco leaf), processed leaf (e.g., processed tobacco leaf), cut and processed leaf (e.g., cut and processed tobacco leaf), product (e.g., delivery system) comprising said plant or part thereof, or combination thereof, obtainable by or obtained from a plant of the invention having a modulated alkaloid content, the method comprising modifying said tobacco to modulate the activity or expression of a Nic3 gene, or a combination of a Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene. Modulated alkaloid content can be determined by comparing the alkaloid content in a plant (e.g., a tobacco plant) or part thereof, 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 and harvested leaf (e.g., cut and harvested tobacco leaf), processed leaf (e.g., processed tobacco leaf), cut and processed leaf (e.g., cut and processed tobacco leaf), a product comprising a plant or part thereof of the invention, e.g., a delivery system, or combinations thereof, with comparable products.

[0175] Suitably, the alkaloid content can be adjusted in a plant, for example, a tobacco plant, for example, a modified tobacco plant. Suitably, the alkaloid content can be adjusted in leaves (for example, tobacco leaves, for example, tobacco leaves from a modified tobacco plant). Suitably, the alkaloid content can be adjusted in harvested leaves (for example, tobacco leaves harvested from a modified tobacco plant). Suitably, the alkaloid content can be adjusted in cut and harvested leaves (for example, cut and harvested tobacco leaves from a modified tobacco plant). Suitably, the alkaloid content can be adjusted in processed leaves (for example, processed tobacco leaves, for example, processed tobacco leaves from a modified tobacco plant). Suitably, the alkaloid content can be adjusted in cut and processed leaves (for example, cut and processed tobacco leaves, for example, cut and processed tobacco leaves from a modified tobacco plant). Suitably, the alkaloid content can be adjusted in cured leaves (for example, cured tobacco leaves from a modified tobacco plant). Suitably, the alkaloid content may be modulated in an extract of green leaves (e.g., green tobacco leaves from modified tobacco plants). Suitably, the alkaloid content may be modulated in a product comprising a plant of the invention or a part thereof (e.g., a delivery system, e.g., a delivery system produced from a modified tobacco plant or a part thereof). Suitably, the alkaloid content may be modulated in any of the above products or combinations thereof. Suitably, the modulated alkaloid content may be an increase in the alkaloid content. Suitably, the modulated alkaloid content may be a decrease in the alkaloid content.

[0176] In one embodiment, the content of one or more alkaloids selected from nicotine, anatabine, anabasine, myosmine, and nornicotine is reduced.

[0177] Suitably, the above-mentioned modulation of alkaloid content may be a reduction in nicotine content.

[0178] In one embodiment, the nicotine content of modified tobacco cells, or a modified plant or part thereof (e.g., tobacco plant), plant propagation material (e.g., tobacco plant propagation material), leaf (e.g., tobacco leaf), harvested leaf (e.g., harvested tobacco leaf), cut and harvested leaf (e.g., cut and harvested tobacco leaf), processed leaf (e.g., processed tobacco leaf), cut and processed leaf (e.g., cut and processed tobacco leaf), or a delivery system from a modified tobacco plant is reduced.

[0179] In one embodiment, the alkaloid content of a plant (e.g., a tobacco plant) or portion thereof may be modulated by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, respectively, when compared to the alkaloid content of a plant (e.g., a tobacco plant) or portion thereof that has not been modified to modulate the activity or expression of at least one Nic3 gene (or a combination of at least one Nic3 gene and at least one Nic1 ERF and / or at least one Nic2 ERF gene) grown under similar growth conditions. Suitably, the alkaloid content may be modulated (e.g., reduced) by about 2-fold to about 10-fold, preferably about 3-fold to about 10-fold, and suitably about 3-fold to about 5-fold. Suitably, the modification may be an increase or decrease in alkaloid content. Suitably, the modulation (e.g., reduction) may be an increase or decrease in nicotine, The modulation (e.g. reduction) of one or more alkaloids selected from anatabine, anabasine, myosmine and nornicotine may be achieved. Suitably, the nicotine content is reduced.

[0180] In one embodiment of the present invention, the alkaloid content of a tobacco plant cell or plant (e.g., a tobacco plant) or part thereof may be modulated by 1%, 2%, 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to a cell or plant (e.g., a tobacco plant) or part thereof that has not been modified according to the present invention. The modulation may be an increase or decrease in alkaloid content compared to an unmodified plant (e.g., a tobacco plant) or part thereof. Suitably, the modulation may be modulation of the total alkaloid content. Suitably, the modulation may be modulation of one or more alkaloids selected from nicotine, anatabine, anabasine, myosmine, and nornicotine. Suitably, the nicotine content is reduced.

[0181] In one embodiment, the method or use comprises cloning the Nic3 gene (or the Nic3 gene and Nic1 The modified alkaloid content of a plant (e.g., a tobacco plant) or part or cell thereof that has not been modified to modulate the activity or expression of a Nic2 ERF gene and / or a combination of Nic2 ERF genes, more particularly compared to or compared to expression by the plant (e.g., a tobacco plant) in the absence of the introduced modification.

[0182] In one embodiment, the method or use results in a modulated alkaloid content compared to a plant (e.g., a tobacco plant) or part or cell thereof that has not been modified to introduce a mutation in the Nic3 gene (or a combination of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene), more particularly compared to or compared to a plant (e.g., a tobacco plant) or part or cell thereof in the absence of the introduced modification.

[0183] In one embodiment, a plant (e.g., a tobacco plant) or part thereof or cell thereof has been modified to achieve modulation in alkaloid content relative to a plant (e.g., a tobacco plant) or part thereof that has not been modified to modulate the activity or expression of at least one Nic3 gene (or at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene), respectively. References herein to Nic3 and Nic1 and / or Nic2 (when referring to loci or genes) refer to the options i) Nic3 and Nic1, ii) Nic3 and Nic2, and iii) Nic3, Nic1 and Nic2.

[0184] The terms "modify" or "modified," as used herein, refer to cells (e.g., tobacco cells) or plants (e.g., tobacco plants) that have been altered or changed. The present invention includes the modification of plants using techniques for the genetic modification of plants or the non-genetic modification of plants. Such methods are well known in the art, and examples of genetic modification techniques include transformation, transgenic, cisgenic, and gene editing methods. Examples of non-genetic modification techniques include fast neutron mutagenesis, chemical mutagenesis, e.g., ethyl methanesulfonate (EMS) mutagenesis, and modern population analysis approaches.

[0185] In one embodiment, a natural variant having a modified Nic3 gene (or a combination of a modified Nic3 gene with at least one modified Nic1 ERF gene and / or at least one modified Nic2 ERF gene) is selected and that trait or gene is bred into a second plant having a commercially desirable trait.

[0186] In one embodiment, a cell or plant (e.g., a tobacco plant) according to the invention is a transgenic plant. It may be a nick cell plant.

[0187] In another embodiment, the cell plant (eg, tobacco plant) according to the present invention can be a non-transgenic cell or plant.

[0188] Suitably, a mutation in at least one Nic3 gene according to the present invention may be absent in K326.

[0189] Suitably, a mutation in at least one Nic3 gene according to the present invention may be absent in Green Briar.

[0190] Suitably, regulation of at least one Nic3 gene is absent in Burley 21.

[0191] In some embodiments, the modification that modulates the activity or expression of at least one Nic3 gene, thereby modulating alkaloid content, is reducing, preventing, or attenuating the transcription, translation, or expression of at least one Nic3 gene (or at least one Nic3 gene in combination with at least one Nic1 ERF gene and / or at least one Nic2 ERF gene); inhibiting the synthesis of, or the release from intracellular stores of, a polypeptide encoded by at least one Nic3 gene (or at least one Nic3 gene in combination with at least one Nic1 ERF gene and / or at least one Nic2 ERF gene); and Increasing the rate of degradation of a polypeptide encoded by at least one Nic3 gene (or at least one Nic3 gene in combination with at least one Nic1 ERF gene and / or at least one Nic2 ERF gene). is selected from the group consisting of:

[0192] In one embodiment, the modification that reduces the activity or expression of at least one Nic3 gene (or one or more modifications that reduce the activity of a combination of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene) comprises a mutation in one or more genes.

[0193] In one embodiment, the mutation results in the deletion of one or more entire Nic3 genes. Suitably, the mutation may result in the deletion of one or more Nic1 ERF genes. Suitably, the mutation may result in the deletion of one or more Nic2 ERF genes.

[0194] In one embodiment, one or more Nic3 genes may contain one or more mutations within the gene. Suitably, the one or more mutations result in reduced or eliminated gene activity in the mutated gene. In one embodiment, the one or more mutations result in an inactive gene. In one embodiment, the mutation results in an amino acid substitution. In one embodiment, the mutation is a nonsense mutation. Suitably, the mutation can disrupt the normal function of the protein encoded by the gene, e.g., the Nic3 gene, for example, disrupting DNA binding in the case of a transcription factor.

[0195] As an example, the method of the present invention comprises: providing mutations in a nucleic acid sequence encoding a protein listed in Table 3 or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; an amino acid sequence shown in Table 3 or at least 70% (preferably at least providing a mutation in the promoter of a nucleic acid sequence encoding a protein comprising an amino acid sequence having at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98% sequence identity with the nucleic acid sequence; providing mutations in the nucleic acid sequences of the Nic3 genes listed in Table 3 or in nucleotide sequences having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing a mutation in the promoter of a nucleic acid sequence of a Nic3 gene listed in Table 3 or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing an antisense RNA, siRNA or miRNA that reduces the level of a nucleic acid sequence encoding a Nic3 protein listed in Table 3 or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing an antisense RNA, siRNA or miRNA that reduces the level of a nucleic acid sequence listed in Table 3 or a sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; Suitably the protein listed in Table 3 or the amino acid sequence shown in Table 3 is selected from SEQ ID NOs: 75, 120, 127 and 129 (or their related sequences as described above). Suitably the gene or sequence listed in Table 3 is selected from SEQ ID NOs: 73, 118, 124 and 127 (or their related sequences as described above).

[0196] In one embodiment, the at least one mutation (or one or more mutations) in the Nic3 locus is in a Nic3 gene selected from SEQ ID NO: 73, 118, 124 or 127, or a sequence having at least 90% identity thereto, or a functional variant or functional fragment or ortholog of said gene, and said at least one mutation in the Nic3 gene is i) a mutation in SEQ ID NO: 73, or a sequence having at least 90% identity thereto, of the Nic3 gene, or a functional variant, functional fragment, or ortholog of said gene, resulting in a mutation in amino acid residues 74 to 258 or 483 to 538 of SEQ ID NO: 75, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or ortholog of said polypeptide; ii) a mutation in SEQ ID NO: 118, or a sequence having at least 90% identity thereto, which is the Nic3 gene, or a functional variant, functional fragment, or ortholog of said gene, resulting in a mutation in amino acid residues 120 to 584 of SEQ ID NO: 120, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or ortholog of said polypeptide; iii) SEQ ID NO: 126, or a sequence having at least 90% identity thereto or a mutation in SEQ ID NO: 124, or a sequence having at least 90% identity thereto, of the Nic3 gene, or a functional variant, functional fragment, or ortholog of said gene, resulting in a mutation in amino acid residues 166 to 406 or 483 to 970 of said functional variant, functional fragment, or ortholog of said polypeptide; and iv) SEQ ID NO: 127, which is the Nic3 gene, or a sequence having at least 90% identity thereto, resulting in a mutation in amino acid residues 171 to 406 or 509 to 967 of SEQ ID NO: 129, or a sequence having at least 90% identity thereto, or a functional variant, functional fragment, or ortholog of said polypeptide; or Mutations in functional variants or functional fragments or orthologues of said genes is selected from.

[0197] Amino acid residues 74-258 of SEQ ID NO:75 provide the N-terminal MYC domain of the MYC transcription factor. Amino acid residues 483-538 of SEQ ID NO:75 have several DNA binding sites (suitably providing sites for mutation), i.e., amino acid residues 488, 489, 492, 493, 500, 517, and 518 provide a bHLH domain. Amino acid residues 120-584 of SEQ ID NO:120 provide an LRR domain. Amino acid residues 166-406 of SEQ ID NO:124 provide an NB-ARC domain, and amino acid residues 483-970 provide an LRR domain. Amino acid residues 171-406 of SEQ ID NO:129 provide an NB-ARC domain, and amino acid residues 509-967 provide an LRR domain. Sites of mutation can also be described by reference to the encoding nucleotides, for example, amino acid residues 74-258 of SEQ ID NO:75 are encoded by nucleotides 631-1185 of SEQ ID NO:73.

[0198] As mentioned above, mutations are made in a particular gene or related sequence (as defined herein) to provide mutations in the listed amino acids or related sequences (as defined herein). Related sequences correspond to each other. For example, if mutations are made in a sequence with 90% sequence identity to SEQ ID NO: 75, the resulting variants are made to that related sequence, i.e., provide a sequence with the same sequence identity before taking into account the one or more mutations introduced. When a related sequence is mutated, the mutations are made in a sequence corresponding to the above-listed domains, i.e., take into account any changes in the number of amino acids that result in the generation of the related sequence.

[0199] In one embodiment, the mutation may be a deletion. For example, the domains described above may be partially or entirely deleted. In one embodiment, the mutation may be an insertion. In one embodiment, the mutation may introduce a premature stop codon. In one embodiment, the target site is unique to the target Nic3 gene and does not exist in other genes.

[0200] In one embodiment, the variant has reduced total alkaloids and / or reduced nicotine levels.

[0201] In one embodiment, the invention provides one or more mutations in the Nic1 ERF gene that encode a polypeptide comprising (or consisting of) an amino acid sequence set forth in Table 1, or a sequence having at least 90%, preferably at least 96%, identity thereto.

[0202] Suitably, the present invention may provide one or more mutations in the Nic1 ERF gene that encode a polypeptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 90%, preferably at least 96%, identity thereto.

[0203] Suitably, the present invention may provide one or more mutations in the Nic1 ERF gene comprising (or consisting of) the nucleotide sequence set out in SEQ ID NO: 5 or a sequence having at least 90%, preferably at least 96%, identity thereto.

[0204] As an example, the method of the present invention comprises: an amino acid sequence as set forth in SEQ ID NO:8; or SEQ ID NO:4; or SEQ ID NO:12; or SEQ ID NO:16; or SEQ ID NO:20; or SEQ ID NO:24; or SEQ ID NO:28; or SEQ ID NO:32; or SEQ ID NO:36, or at least 70% thereto (preferably at least 80%, preferably at least 90%, preferably at least providing a mutation in a nucleic acid sequence encoding a protein comprising an amino acid sequence having 96%, preferably at least 98%, sequence identity; providing a mutation in the promoter of a nucleic acid sequence encoding a protein comprising the amino acid sequence set forth as SEQ ID NO:8; or SEQ ID NO:4; or SEQ ID NO:12; or SEQ ID NO:16; or SEQ ID NO:20; or SEQ ID NO:24; or SEQ ID NO:28; or SEQ ID NO:32; or SEQ ID NO:36, or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing a mutation in the nucleic acid sequence of an ERF gene comprising SEQ ID NO:5; or SEQ ID NO:1; or SEQ ID NO:3; or SEQ ID NO:9; or SEQ ID NO:13; or SEQ ID NO:17; or SEQ ID NO:21; or SEQ ID NO:25; or SEQ ID NO:29; or SEQ ID NO:33 or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing a mutation in the promoter of a nucleic acid sequence of an ERF gene comprising SEQ ID NO:5; or SEQ ID NO:1; or SEQ ID NO:3; or SEQ ID NO:9; or SEQ ID NO:13; or SEQ ID NO:17; or SEQ ID NO:21; or SEQ ID NO:25; or SEQ ID NO:29; or SEQ ID NO:33 or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing an antisense RNA, siRNA or miRNA that reduces the level of a nucleic acid sequence encoding a protein comprising the amino acid sequence set forth as SEQ ID NO:8; or SEQ ID NO:4; or SEQ ID NO:12; or SEQ ID NO:16; or SEQ ID NO:20; or SEQ ID NO:24; or SEQ ID NO:28; or SEQ ID NO:32; or SEQ ID NO:36, or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing an antisense RNA, siRNA or miRNA that reduces the level of a nucleic acid sequence of SEQ ID NO:5; or SEQ ID NO:1; or SEQ ID NO:3; or SEQ ID NO:9; or SEQ ID NO:13; or SEQ ID NO:17; or SEQ ID NO:21; or SEQ ID NO:25; or SEQ ID NO:29; or SEQ ID NO:33, or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; may also include:

[0205] In one embodiment, one or more Nic1 ERF genes and / or one or more Nic2 ERF genes are modulated (eg, mutated).

[0206] Suitably, any one of the Nic3 gene and / or Nic1 ERF gene modifications (e.g., mutations) taught herein may be used in combination with one or more modifications of a Nic2 ERF gene, wherein the Nic2 ERF gene encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:72; or SEQ ID NO:40; or SEQ ID NO:44; or SEQ ID NO:48; or SEQ ID NO:52; or SEQ ID NO:56; or SEQ ID NO:60; or SEQ ID NO:64; or SEQ ID NO:68; or a functional variant, functional fragment, or ortholog thereof; or the Nic2 ERF gene comprises the nucleotide sequence set forth in SEQ ID NO:69; or SEQ ID NO:37; or SEQ ID NO:41; or SEQ ID NO:45; or SEQ ID NO:49; or SEQ ID NO:53; or SEQ ID NO:57; or SEQ ID NO:61; or SEQ ID NO:65; or a functional variant, functional fragment, or ortholog thereof.

[0207] As an example, the method of the present invention comprises: providing mutations in a nucleic acid sequence encoding a protein comprising the amino acid sequence set forth as SEQ ID NO:72; or SEQ ID NO:40; or SEQ ID NO:44; or SEQ ID NO:48; or SEQ ID NO:52; or SEQ ID NO:56; or SEQ ID NO:60; or SEQ ID NO:64; or SEQ ID NO:68; or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing a mutation in the promoter of a nucleic acid sequence encoding a protein comprising the amino acid sequence set forth as SEQ ID NO:72; or SEQ ID NO:40; or SEQ ID NO:44; or SEQ ID NO:48; or SEQ ID NO:52; or SEQ ID NO:56; or SEQ ID NO:60; or SEQ ID NO:64; or SEQ ID NO:68; or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing mutations in a nucleic acid sequence of an ERF gene comprising SEQ ID NO: 69; or SEQ ID NO: 37; or SEQ ID NO: 41; or SEQ ID NO: 45; or SEQ ID NO: 49; or SEQ ID NO: 53; or SEQ ID NO: 57; or SEQ ID NO: 61; or SEQ ID NO: 65; or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing a mutation in the promoter of a nucleic acid sequence of an ERF gene comprising SEQ ID NO: 69; or SEQ ID NO: 37; or SEQ ID NO: 41; or SEQ ID NO: 45; or SEQ ID NO: 49; or SEQ ID NO: 53; or SEQ ID NO: 57; or SEQ ID NO: 61; or SEQ ID NO: 65; or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing an antisense RNA, siRNA or miRNA that reduces the level of a nucleic acid sequence encoding a protein comprising the amino acid sequence set forth as SEQ ID NO:72; or SEQ ID NO:40; or SEQ ID NO:44; or SEQ ID NO:48; or SEQ ID NO:52; or SEQ ID NO:56; or SEQ ID NO:60; or SEQ ID NO:64; or SEQ ID NO:68; or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; providing an antisense RNA, siRNA or miRNA that reduces the level of a nucleic acid sequence that is SEQ ID NO: 69; or SEQ ID NO: 37; or SEQ ID NO: 41; or SEQ ID NO: 45; or SEQ ID NO: 49; or SEQ ID NO: 53; or SEQ ID NO: 57; or SEQ ID NO: 61; or SEQ ID NO: 65; or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; may also include:

[0208] In one embodiment, one or more mutations in a nucleic acid sequence encoding a protein comprising the amino acid sequence set forth as SEQ ID NO:4; or SEQ ID NO:8; or SEQ ID NO:12; or SEQ ID NO:16; or SEQ ID NO:20; or SEQ ID NO:24; or SEQ ID NO:28; or SEQ ID NO:32; or SEQ ID NO:36, or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto, and one or more mutations in a nucleic acid sequence encoding a protein comprising the amino acid sequence set forth as SEQ ID NO:1; or SEQ ID NO:3; or SEQ ID NO:5; or SEQ ID NO:9; or SEQ ID NO:13; or SEQ ID NO:17; or SEQ ID NO:21; or SEQ ID NO:25; or SEQ ID NO:29; or SEQ ID NO:33, or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%) sequence identity thereto. a mutation in at least one Nic1 ERF gene selected from the group consisting of one or more mutations in the nucleic acid sequence of an ERF gene comprising a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; and / or a mutation in at least one Nic2 ERF gene, in particular one or more mutations in a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52 or SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:68 or SEQ ID NO:72, or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto, or one or more mutations in a nucleic acid sequence comprising SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49 or SEQ ID NO:53, or SEQ ID NO:57, or SEQ ID NO:61, or SEQ ID NO:65 or SEQ ID NO:69, or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto, more particularly Nic2 ERF mutations are one or more mutations in a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 72 or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto, or in the nucleotide sequence of SEQ ID NO: 69 or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto.

[0209] In one embodiment, at least one mutation in a Nic1 ERF gene consists of one or more mutations in a nucleic acid sequence encoding a protein comprising the amino acid sequence set forth as SEQ ID NO: 8; or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto, or a nucleic acid sequence of an ERF gene comprising SEQ ID NO: 5; or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; and / or at least one Nic2 Mutations in the ERF gene may be used in combination, in particular one or more mutations in a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:40, SEQ ID NO:44, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:64, SEQ ID NO:68 or SEQ ID NO:72, or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto, or one or more mutations in a nucleotide sequence comprising SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:65 or SEQ ID NO:69, or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto.

[0210] In one embodiment, at least one mutation in the Nic1 ERF gene consists of one or more mutations in a nucleic acid sequence encoding a protein comprising the amino acid sequence set forth as SEQ ID NO: 8; or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto, or a nucleic acid sequence of an ERF gene comprising SEQ ID NO: 5; or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto; and / or an amino acid sequence set forth as SEQ ID NO: 72; or an amino acid sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto. % (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto, or one or more mutations in the nucleotide sequence set forth as SEQ ID NO: 69 or a nucleotide sequence having at least 70% (preferably at least 80%, preferably at least 90%, preferably at least 96%, preferably at least 98%) sequence identity thereto, may be used in combination.

[0211] The one or more Nic2 ERF genes may be one, two, three, four, five, six, seven, eight, or nine Nic2 ERF genes selected from Table 2. It may be an ERF gene.

[0212] In some embodiments, the modification that reduces the activity or expression of at least one Nic3 gene (or a combination of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene), thereby reducing alkaloid content, is one or more selected from the group consisting of point mutations, deletions, insertions, duplications, and inversions in one or more genes. Suitably, the modification is introduced by a method selected from random mutagenesis and targeted mutagenesis. Suitably, the modification may be introduced by a targeted mutagenesis method selected from, for example, meganucleases, zinc finger nucleases, TALEN, gene editing, and CRISPR.

[0213] The term "mutation" as used herein encompasses naturally occurring genetic variants or engineered variants.

[0214] Mutations refer to heritable genetic modifications introduced into a plant, part thereof, or cell that alter the activity or expression of a product encoded by a gene. These modifications may be in any sequence that controls the activity or expression of a gene, such as in the promoter, 5'UTR, exon, intron, 3'UTR, or terminator region. In one aspect, the mutation reduces, inhibits, or eliminates the expression or activity of the gene product. In another aspect, the mutation increases, elevates, or enhances the activity or expression of the gene product.

[0215] In particular, the term "mutation" refers to a variation in the amino acid sequence compared to the sequences shown in Table 1, Table 2 or Table 3 that reduces the expression or function of the protein.

[0216] In a preferred embodiment, each copy of a nucleic acid sequence set forth in Table 1, Table 2, or Table 3, or a sequence having at least 80% (at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) sequence identity thereto, present in the plant, is 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 N. tabacum can be mutated.

[0217] In a preferred embodiment, the plant or plant cell according to the invention is homozygous for the mutation.

[0218] In one embodiment, preferably, the plant or plant cell according to the invention expresses only the mutated nucleic acid. In other words, in some embodiments, there is no endogenous (or No endogenous (and functional) proteins are present. In other words, if any endogenous proteins are present, they are preferably in an inactive and / or truncated form.

[0219] The mutation may interrupt a nucleic acid sequence that encodes a protein as detailed herein.

[0220] The interruption may result in the nucleic acid sequence not being transcribed and / or translated.

[0221] The nucleic acid sequence may be disrupted, for example, by deleting or otherwise altering the ATG start codon of the nucleic acid sequence, such that translation of the protein is reduced or prevented.

[0222] The nucleic acid sequence may contain one or more nucleotide changes that reduce or prevent protein expression or affect protein trafficking. For example, protein expression can be reduced or prevented by introducing one or more premature stop codons, frameshifts, splice variants, or intolerant amino acid substitutions into the open reading frame.

[0223] A premature stop codon refers to a mutation that introduces a stop codon into an open reading frame, preventing translation of the entire amino acid sequence. A premature stop codon is a TAG ("amber") codon. "), TAA ("ochre"), or TGA ("opal" or "umber" ) codon.

[0224] Suitably, a premature stop codon may be introduced into Nitab4.5_0003090g0030.1 (ERF199) shown in any of SEQ ID NOs: 5 to 7.

[0225] Suitably, the premature stop codon in Nitab4.5_0003090g0030.1 (ERF199) may be a TGA ("opal" or "amber") premature stop codon, as set out in any of SEQ ID NOs: 5-7.

[0226] A frameshift mutation (also called a framing error or reading frameshift) is a mutation caused by the insertion or deletion (insertion or deletion) of several nucleotides in a nucleic acid sequence that is not divisible by three. Due to the triplet nature of gene expression by codons, the insertion or deletion can change the reading frame and result in a completely different translation from the original. Frameshift mutations often cause the mutated codon to code for a different amino acid. Frameshift mutations generally result in the introduction of a premature stop codon.

[0227] Splice variants insert, delete, or change several nucleotides at the designated site where splicing occurs during processing of precursor messenger RNA to mature messenger RNA. Deletion of a splice site can result in one or more introns remaining in the mature mRNA, leading to the production of an abnormal protein.

[0228] An impermissible amino acid substitution refers to a mutation that results in a non-synonymous amino acid substitution in a protein, which results in a reduced or eliminated function of the protein.

[0229] Any method known in the art for providing mutations in nucleic acid sequences can be used in the methods according to the invention. For example, homologous recombination can be used, in which the relevant nucleic acid sequence is mutated to generate a vector that is used to transform plants or plant cells. Recombinant plants or plant cells that express the mutated sequence can then be selected.

[0230] The nucleic acid sequence may be deleted in whole or in part. The deletion may be contiguous or may include multiple sections of the sequence. The deletion preferably removes a sufficient amount of the nucleotide sequence so that the nucleic acid sequence no longer encodes a functional protein. The deletion may, for example, remove at least 50, 60, 70, 80, or 90% of the coding portion of the nucleic acid sequence.

[0231] A deletion can be complete when 100% of the coding portion of the nucleic acid sequence is absent when compared to the corresponding genome of a comparable unmodified plant.

[0232] Methods for deleting nucleic acid sequences in plants are known in the art. For example, homologous recombination can be used, in which a vector is generated that lacks the relevant nucleic acid sequence(s) and is used to transform the plant or plant cell. Recombinant plants or plant cells that express the new portion of the sequence can then be selected.

[0233] Plant cells transformed with vectors such as those described above can be grown and maintained according to well-known tissue culture methods, for example, by culturing the cells in an appropriate culture medium supplemented with necessary growth factors, such as amino acids, plant hormones, vitamins, etc.

[0234] Modifications of nucleic acid sequences can be carried out using targeted mutagenesis (also called targeted nucleotide exchange (TNE) or oligo-directed mutagenesis (ODM)). Targeted mutagenesis methods include, but are not limited to, those employing zinc finger nucleases, TALENs (see WO 2011 / 072246 and WO 2010 / 079430), Cas9-like, Cas9 / crRNA / tracrRNA or Cas9 / gRNA CRISPR systems (see WO 2014 / 071006 and WO 2014 / 093622), meganucleases (see WO 2007 / 047859 and WO 2009 / 059195), or targeted mutagenesis methods (e.g., KeyBase® or TALENs) employing mutagenic oligonucleotides, possibly containing chemically modified nucleotides to enhance mutagenesis using sequence complementarity to genes in plant protoplasts.

[0235] Alternatively, 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) may be used. Mutagenesis systems such as the TILLING system (incorporated herein) can be used to generate plant lines containing genes encoding proteins with mutations. TILLING uses traditional chemical mutagenesis (e.g., ethyl methanesulfonate (EMS) mutagenesis), followed by high-throughput screening for mutations. In this manner, plants, seeds, and tissues containing genes with desired mutations can be obtained.

[0236] Methods can include mutagenizing plant seeds (e.g., EMS mutagenesis), pooling plant individuals or DNA, PCR amplifying the region of interest, heteroduplex formation and high-throughput detection, identifying mutant plants, and sequencing the mutant PCR products. It is understood that other mutagenesis and selection methods can equally be used to generate such modified plants. For example, seeds can be irradiated or chemically treated, and plants can be screened for modified phenotypes.

[0237] Modified plants can be distinguished from unmodified, i.e., wild-type, plants by molecular methods, for example, by the mutation(s) present in their DNA and by modified phenotypic characteristics. Modified plants can be homozygous or heterozygous for the mutation.

[0238] Suitably, the method may comprise transforming a cell of a plant (e.g., a tobacco plant) with a genetic construct capable of inhibiting the activity or expression of at least one Nic3 gene (or a construct capable of inhibiting the activity or expression of at least one Nic1 ERF gene and / or at least one Nic2 ERF gene in combination with at least one Nic3 gene).

[0239] In some embodiments, the modification that increases the activity or expression of at least one Nic3 gene (or a combination of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene), thereby increasing alkaloid content, is selected from the group consisting of: increasing, facilitating, or enhancing the transcription, translation, or expression of at least one Nic3 gene (or a combination of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene); increasing the synthesis of, or the release from intracellular stores of, a polypeptide encoded by at least one Nic3 gene (or a combination of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene); and decreasing the rate of degradation of a polypeptide encoded by at least one Nic3 gene (or a combination of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene).

[0240] Suitably, the method may include transforming cells of a plant (e.g., a tobacco plant) with a genetic construct comprising a nucleotide sequence encoding at least one exogenous Nic3 gene (or a combination of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene) or a protein capable of promoting or enhancing at least one endogenous Nic3 gene (or a combination of at least one endogenous Nic3 gene and at least one endogenous Nic1 ERF gene and / or at least one endogenous Nic2 ERF gene). It will be appreciated that each of these options will result in increased activity and expression of the polypeptide encoded by at least one Nic3 gene (or a combination of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene). The method may include regenerating a plant from the transformed cell.

[0241] Thus, there is provided the use of genetic constructs capable of increasing the activity and / or expression of a polypeptide encoded by at least one Nic3 (or a combination of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene) to increase the alkaloid content in plants transformed with the construct.

[0242] The genetic construct may encode a polypeptide comprising an amino acid sequence set forth in Table 1, Table 2 and / or Table 3, or a functional variant or functional fragment or ortholog thereof.

[0243] In some embodiments, the method or use according to the invention involves measuring the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene. This includes increasing the alkaloid content of a plant (e.g., a tobacco plant) or cell by increasing gene activity or expression.

[0244] The term "inhibiting" (e.g., inhibiting the activity or expression of the Nic3 gene) as used herein means that the activity or expression of a gene (e.g., the Nic3 gene) is lower or decreased compared to the activity or expression of the gene in a comparable product, or that the amount or activity of the protein produced by the gene is lower.

[0245] In one embodiment, the term "inhibiting" (e.g., inhibiting the activity or expression of the Nic3 gene) as used herein means that the activity or expression of the Nic3 gene is less than the gene activity or expression of the gene in a comparable product.

[0246] The activity of a particular Nic3, Nic1 ERF, or Nic2 ERF gene can be measured by measuring transcription of the gene. Methods for measuring transcription are well known in the art and include Northern blot, RNA-Seq, in situ hybridization, DNA microarray, and RT-PCR, among others. Alternatively, gene activity can be measured indirectly by measuring the level of a gene product, e.g., a protein encoded by the gene.

[0247] In some embodiments, the activity or expression of the Nic3 gene, the Nic1 ERF gene, or the Nic2 ERF gene may be modulated, i.e., increased or decreased by at least about 10%, 20%, 30%, or 40%, suitably at least about 50%, 60%, 70%, and more suitably at least about 80%, 90%, 95%, or 100%, compared to the activity or expression of said gene in a plant (e.g., a tobacco plant) that has not been modified according to the present invention.

[0248] Suitably, the expression or function of the Nic3 gene, the Nic1 ERF gene or the Nic2 ERF gene may be reduced, partially inactivated, inhibited, eliminated, knocked out or lost such that the protein expression or function of the gene is not detectable.

[0249] In one embodiment, at least one of the Nic3 gene, the Nic1 ERF gene, or the Nic2 The ERF gene is knocked out, in other words, the gene is completely disabled.

[0250] In a preferred embodiment, the Nic3 gene may have substantially no activity or expression, which preferably means that the plant may contain less than about 1% (suitably less than about 0.1%) of the activity or expression of the Nic3 gene compared to a plant that has not been modified to inhibit the activity or expression of the Nic3 gene.

[0251] In a preferred embodiment, the Nic1 ERF gene may have substantially no activity or expression, which preferably means that the plant may contain about 1% (suitably less than about 0.1%) of the activity or expression when compared to a plant that has not been modified to inhibit the activity or expression of the Nic1 ERF gene.

[0252] In a preferred embodiment, the Nic2 ERF gene may have substantially no activity or expression, which preferably means that the plant may contain less than about 1% (suitably less than about 0.1%) of the activity or expression of the Nic2 ERF gene compared to a plant that has not been modified to inhibit the activity or expression of the Nic2 ERF gene.

[0253] "ERF gene," as used herein, refers to a transcription factor gene that belongs to the ethylene response factor (ERF) subfamily.

[0254] "Nic1 ERF gene," as used herein, refers to the ERF gene identified by the inventors in WO 2018 / 237107 as mapping to the Nic1 region. As used herein, the Nic1 ERF genes are listed in Table 1, along with their corresponding nucleotide, cDNA, cds, and amino acid sequence identifiers.

[0255] Suitably, the at least one Nic1 ERF gene for use in the present invention is any one of those listed in Table 1.

[0256] The genomic sequence of each of the Nic1 and Nic2 ERFs listed in the table above is identical to their corresponding coding sequence, with the exception of the Nic1 ERF ERF17L3, whose genomic sequence (SEQ ID NO: 1) is not identical to its coding sequence (SEQ ID NO: 3).

[0257] "Nic2 ERF gene," as used herein, refers to an ERF gene that maps to the Nic2 region. The Nic2 ERF genes used herein are listed in Table 2 below, along with their corresponding nucleotide, cDNA, cds, and amino acid sequence identifiers.

[0258] Suitably, the Nic2 ERF gene for use in the present invention is any one of those listed in Table 2.

[0259] In one embodiment, at least one Nic3 gene referred to herein may be encoded by a polynucleotide sequence shown in Table 3.

[0260] Suitably, at least one Nic3 gene referred to herein is i) a polynucleotide sequence set forth herein as SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:139, SEQ ID NO:142, SEQ ID NO:145, SEQ ID NO:148 or SEQ ID NO:151 (suitably SEQ ID NO:73, 118, 124 or 127); or a sequence having at least 80% identity thereto; or ii) a functional fragment of the polynucleotide sequence shown in i), wherein the functional fragment encodes the Nic3 gene; or iii) SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 87, a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth herein as SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:141, SEQ ID NO:144, SEQ ID NO:147, SEQ ID NO:150 or SEQ ID NO:153, suitably SEQ ID NO:75, 120, 126 or 129; or iv) High stringency of the polynucleotides taught in i), ii) or iii) above. a polynucleotide sequence capable of hybridizing under sequent conditions; or v) a polynucleotide having at least 80% (preferably 100%) of the polynucleotide set forth in i), ii) or iii) above; a polynucleotide sequence having preferably 85%, preferably 90%, preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%) identity to the vi) Due to the degeneracy of the genetic code, the polynucleotides shown in i), ii) or iii) Polynucleotide sequence different from It can be encoded by a polynucleotide sequence comprising:

[0261] In one embodiment, at least one Nic1 ERF gene referred to herein is i) a polynucleotide sequence set forth herein as SEQ ID NO:1, SEQ ID NO:3; SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29 or SEQ ID NO:33; or ii) a functional fragment of the polynucleotide sequence shown in i), which encodes a Nic1 ERF synthetic gene; or iii) SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth herein as SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32 or SEQ ID NO: 36; or iv) high stringency amplification of the polynucleotides taught in i), ii) or iii) above; a polynucleotide sequence capable of hybridizing under sensitivities, or v) a polynucleotide as set forth in i), ii) or iii) above, and a nucleic acid sequence encoding at least 70 % (preferably 80%, preferably 85%, preferably 90%, preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%) identity to the polynucleotide sequence of vi) Due to the degeneracy of the genetic code, the polynucleotides shown in i), ii) or iii) Polynucleotide sequence different from It can be encoded by a polynucleotide sequence comprising:

[0262] In one embodiment, at least one Nic2 ERF gene referred to herein is i) a polynucleotide sequence set forth herein as SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:65 or SEQ ID NO:69; or ii) a functional fragment of the polynucleotide sequence shown in i), which encodes the Nic1 ERF gene; or iii) SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth herein as SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68 or SEQ ID NO: 72; or iv) high stringency amplification of the polynucleotides taught in i), ii) or iii) above; a polynucleotide sequence capable of hybridizing under sensitivities, or v) a polynucleotide as set forth in i), ii) or iii) above, and a nucleic acid sequence encoding at least 70 % (preferably 80%, preferably 85%, preferably 90%, preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%) identity to the polynucleotide sequence of vi) Due to the degeneracy of the genetic code, the polynucleotides shown in i), ii) or iii) Polynucleotide sequences that differ from the ocid It can be encoded by a polynucleotide sequence comprising:

[0263] In one embodiment, at least one Nic3 gene for use in accordance with the present invention may be endogenous to the plant (eg, a tobacco plant).

[0264] In one embodiment, at least one Nic1 ERF gene for use in accordance with the present invention may be endogenous to the plant (eg, a tobacco plant).

[0265] In one embodiment, at least one Nic2 ERF gene for use in accordance with the present invention may be endogenous to the plant (eg, a tobacco plant).

[0266] Reference herein to an "endogenous" gene refers not only to the gene in question as found in the plant in its natural form (i.e., without any human intervention), but also to that same gene (or a substantially homologous nucleic acid / gene) in isolated form that is subsequently (re)introduced into a plant (transgene) or plant cell. For example, transgenic plants containing such transgenes may experience a substantial reduction in transgene expression and / or a substantial reduction in expression of the endogenous gene. An isolated gene may be isolated from an organism, or it may be artificial, for example, artificial by chemical synthesis.

[0267] In another embodiment, at least one Nic3 gene for use in accordance with the present invention may be exogenous to the plant (eg, a tobacco plant).

[0268] In another embodiment, at least one Nic1 ERF gene for use in accordance with the present invention may be exogenous to the plant (eg, a tobacco plant).

[0269] In another embodiment, at least one Nic2 ERF gene for use in accordance with the present invention may be exogenous to the plant (eg, a tobacco plant).

[0270] The term "exogenous gene" can mean that a gene transformed into an unmodified plant is derived from an external source, i.e., from a different species than that being transformed. An exogenous gene may comprise a nucleic acid sequence that is substantially the same as or different from that of an endogenous gene in the unmodified plant. An exogenous gene may be derived from the genomic or cDNA sequence corresponding to that gene from any species. An exogenous gene may form a chimeric gene. An exogenous gene may encode a polypeptide comprising an amino acid sequence listed in Table 1, or a functional variant, fragment, or ortholog thereof. An exogenous gene may comprise a nucleotide sequence listed in Table 2, or a functional variant, fragment, or ortholog thereof. An exogenous gene may comprise a nucleotide sequence listed in Table 3, or a functional variant, fragment, or ortholog thereof.

[0271] The present invention also provides the use of the Nic3 gene to regulate the alkaloid content of plants.

[0272] In one embodiment, the invention further provides the use of the Nic3 gene, and optionally the Nic1 ERF and / or the Nic2 ERF, to regulate the alkaloid content of a plant.

[0273] Methods for reducing gene expression or gene products are well documented in the art. Any of the methods described herein for modulating the activity or expression of the Nic3 gene can be used to alter the activity or expression of the Nic3 gene, and optionally the Nic1 ERF gene and / or the Nic2 ERF gene.

[0274] In one embodiment, the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and, optionally, the Nic1 ERF gene and / or the Nic2 ERF gene, may be inhibited by any method known in the art.

[0275] As a method for inhibiting the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene, These methods can include gene editing, targeted mutagenesis, RNA interference, antisense or sense co-suppression (see Wang and Wagner 2003, Planta Volume 216, Issue 4, pp 686-691, incorporated herein by reference). In one embodiment, inhibition of gene activity or expression can be achieved through the use of gene editing. Gene editing may be performed using any method known in the art. Some non-limiting examples are provided herein.

[0276] In one embodiment, inhibition of the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene, can be achieved using CRISPR-involving gene editing methods, including the use of the CRISPR / Cas9 system. CRISPR / Cas9 genome editing tools are commercially available, for example, "Guide-it" from Clontech (Avenue du President Kennedy 78100 Saint-Germain-en-Laye, France).

[0277] Suitably, to generate the gene editing vector pRGEB-M24, the rice snoRNA U3 promoter in vector pRGEB31 is transfected with a HindIII promoter as described in WO 2018 / 237107, which is incorporated herein by reference. and BsaI-assisted infusion cloning, the M24 promoter may be replaced with the M24 promoter amplified from pSiM24 (Sahoo, DK, Dey, N., and Maiti, IB (2014), incorporated herein by reference). pSiM24 is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants. PLoS One 9, e98988). For example, a pair of oligos can be used to identify each of the candidate genes. The polypeptides can be designed to be differentially targeted.

[0278] The oligo pair is first annealed to generate a double-stranded fragment with 4-nt 5' overhangs on both ends, and then ligated into BsaI-digested pRGEB-M24 vector.

[0279] Another method of gene editing is TALEN (transcriptional activation enzyme) using commercially available kits (e.g., from Addgene, 1 Kendall Sq. Ste. B7102, Cambridge, MA 02139, USA). In one embodiment, the activity or expression of at least one Nic3 gene or at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene is used. Inhibition of ERF gene activity or expression can be achieved using TALENs.

[0280] In another embodiment, the method may involve the use of zinc finger nucleases, such as the CompoZr® zinc finger nuclease technology available from Sigma-Aldrich. Another embodiment involves the use of meganucleases (e.g., as described in Silva et al. Curr Gene Ther. Feb 2011; 11(1): 11-27, the teachings of which are incorporated herein by reference). or further methods).

[0281] In one embodiment, the method for inhibiting the activity or expression of the Nic3 gene, or the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene, can be targeted mutagenesis. Any method of targeted mutagenesis can be used. In one embodiment, the method can be oligonucleotide-directed mutagenesis (ODM), such as KeyBase®, available from Keygene (Agro Business Park 90, 6708 PW Wageningen, The Netherlands). In another embodiment, inhibition of the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene, can be achieved through the use of a construct or vector (e.g., a plasmid).

[0282] The genetic constructs of the present invention may be in the form of an expression cassette suitable for inhibiting the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene in a host cell, or for increasing the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene in a host cell. The genetic construct may be introduced into a host cell without being incorporated into a vector. For example, the genetic construct may be a nucleic acid molecule or may be incorporated into a liposome or viral particle. Alternatively, purified nucleic acid molecules (e.g., histone-free DNA or naked DNA) may be inserted directly into a host cell by suitable means, such as direct endocytic uptake. The genetic construct may be directly introduced into cells of a host subject (e.g., a plant) by transfection, infection, microinjection, cell fusion, protoplast fusion, or ballistic bombardment. Alternatively, the genetic constructs of the present invention may be directly introduced into host cells using a particle gun.

[0283] Alternatively, the genetic construct may comprise or be contained in a recombinant vector for expression in a suitable host cell. The recombinant vector may be a plasmid, cosmid, or phage. Such recombinant vectors are highly useful for transforming host cells with the genetic construct of the present invention and replicating the expression cassette therein. A skilled artisan will recognize that the genetic construct of the present invention can be combined with many types of backbone vectors for expression purposes. The backbone vector may be a binary vector, which can be used in, for example, E. coli and Agrobacterium tumefaciens. capable of replicating in both a suitable base (Agrobacterium tumefaciens). The vector is a pBIN plasmid, e.g., pBIN19 (Bevan M., 1984, Nucleic Acids Research 12:8711-21).

[0284] In addition to sequences that inhibit the activity or expression of at least one Nic3 gene, or the activity or expression of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene, a recombinant vector can contain various other functional elements. For example, the vector may contain a promoter. In addition, the recombinant vector can be designed so that it replicates autonomously in the cytosol of the host cell. In this case, the recombinant vector may require elements that induce or regulate DNA replication. Alternatively, the recombinant vector can be designed so that it integrates into the genome of the host cell. In this case, DNA sequences that are more suitable for targeted integration (e.g., by homologous recombination) are contemplated.

[0285] Recombinant vectors may also contain DNA encoding genes that can be used as selectable markers in the cloning process, i.e., to allow for the selection of transfected or transformed cells and to allow for the selection of cells harboring vectors that have incorporated heterologous DNA. Vectors may also contain DNA involved in regulating the expression of coding sequences or for targeting the expressed polypeptide to a specific part of the host cell, for example, to protuberances or glandular trichomes. Thus, vectors may contain at least one additional element selected from the group consisting of selectable marker genes (e.g., antibiotic resistance genes); polypeptide termination signals; and protein target sequences (e.g., transit peptides).

[0286] In one embodiment, the method or use involves interfering with the activity or expression of the Nic3 gene, or the Nic3 gene and the Nic1 ERF gene and / or The method may involve inhibiting the activity or expression of the Nic3 gene or the Nic1 ERF gene. In one embodiment, the oligonucleotide is RNA-based. In one embodiment, the oligonucleotide is RNA interference (RNAi), e.g., dsRNAi. In one embodiment, the method may involve transforming cells of a plant (e.g., a tobacco plant) with an RNAi molecule, e.g., dsRNAi, that inhibits the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene. Suitably, the RNAi molecule may be provided from a vector that can be introduced into plant cells; for example, gene silencing may be used using a virus that contains a fragment of the relevant gene (e.g., a fragment of 100-300 nucleotides in length) and produces dsRNA to trigger RNA-mediated gene silencing.

[0287] In one embodiment, the activity or expression of at least one of the Nic3 gene, the Nic1 ERF gene and / or the Nic2 ERF gene is reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%, compared to the activity or expression of the polypeptide in a comparable plant or part or cell thereof.

[0288] In one embodiment, the activity or expression of at least one Nic3 gene, at least one Nic1 ERF gene, and at least one Nic2 ERF gene is reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%, compared to the activity or expression of the polypeptide in a wild-type plant or a comparable plant, or part or cell thereof.

[0289] The activity or expression of at least one Nic3 gene, or the activity or expression of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene, can be inhibited by any method known in the art. In any of the foregoing embodiments, the activity or expression of at least one Nic3 gene, or the activity or expression of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene can be inhibited by any method, including gene editing methods involving CRISPR, including the use of the CRISPR-Cas9 system, RNA interference (RNAi), antisense or sense co-suppression, gene editing, or targeted mutagenesis. In any of the foregoing embodiments, the activity or expression of at least one Nic3 gene, or the activity or expression of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene can be inhibited using RNAi methods, for example, using miRNA, siRNA, dsRNA, or shRNA.

[0290] In one embodiment, a construct that modulates the activity or expression of the Nic3 gene, or the activity or expression of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene, may be contained in a vector. Suitably, the vector may be a plasmid.

[0291] In one embodiment, a vector for use in the present invention is an Agrobacterium-based plasmid.

[0292] Thus, in one embodiment, the expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene, is modulated. Plants (e.g., tobacco plants) and plant propagation material (e.g., tobacco plant propagation material), leaves (e.g., tobacco leaves), cut and harvested leaves, processed leaves (e.g., processed tobacco leaves), or cut and processed leaves (e.g., cut and processed tobacco leaves) are provided.

[0293] In another embodiment, a cell (e.g., a tobacco cell), a plant (e.g., a tobacco plant) or portion thereof, and / or plant propagation material may comprise a construct that modulates the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene. In one embodiment, the construct reduces the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene. In another embodiment, the construct increases the activity or expression of the Nic3 gene, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene.

[0294] In a further embodiment, the cells (e.g., tobacco cells), plants (e.g., tobacco plants) or parts thereof and / or plant propagation material according to the present invention are i) a polynucleotide sequence shown in Table 3; or ii) a functional fragment of the polynucleotide sequence shown in i), wherein the functional fragment encodes the Nic3 gene; or iii) a polynucleotide encoding a polypeptide comprising an amino acid sequence set forth in Table 3 , or iv) high stringency amplification of the polynucleotides taught in i), ii) or iii) above; a polynucleotide sequence capable of hybridizing under sensitivities, or v) a polynucleotide as set forth in i), ii) or iii) above, and a nucleic acid sequence encoding at least 80 % (preferably 85%, preferably 90%, preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%) identity to the polynucleotide sequence of vi) Due to the degeneracy of the genetic code, the polynucleotides shown in i), ii) or iii) Polynucleotide sequences that differ from the ocid Suitably, the sequences of Table 3 are as described above.

[0295] In a further embodiment, the cells (e.g., tobacco cells), plants (e.g., tobacco plants) or parts thereof and / or plant propagation material according to the present invention are i) a polynucleotide sequence selected from Table 3, a polynucleotide sequence selected from Table 1, and a polynucleotide sequence selected from Table 2; or ii) a functional fragment of the polynucleotide sequence set forth in i), wherein the functional fragment encodes the Nic3 gene, the Nic1 ERF gene, or the Nic2 ERF gene; or iii) a polypeptide encoding a polypeptide comprising an amino acid sequence shown in Table 3, Table 1, or Table 2; nucleotides; or iv) high stringency amplification of the polynucleotides taught in i), ii) or iii) above; a polynucleotide sequence capable of hybridizing under sensitivities; or v) a polynucleotide as set forth in i), ii) or iii) above, and a nucleic acid sequence encoding at least 80 % (preferably 85%, preferably 90%, preferably 95%, more preferably 96%, more preferably 97%, more preferably 98%) identity to a polynucleotide sequence; or vi) Due to the degeneracy of the genetic code, the polynucleotides shown in i), ii) or iii) Polynucleotide sequences that differ from the ocid Suitably the sequences of Table 1, 2 or 3 are as described above.

[0296] In one embodiment, the cells (eg, tobacco cells) are grown in cell culture.

[0297] In one embodiment, at least one Nic3 gene (or at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene) is used to regulate alkaloid content (e.g., nicotine content) in a cell or cell culture (e.g., a tobacco cell culture).

[0298] In advantageous embodiments, inhibiting the activity or expression of at least one Nic3 gene, or the activity or expression of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene, can result in a decrease in alkaloid content. Suitably, inhibiting the activity or expression of at least one Nic3 gene, or the activity or expression of at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene can result in a decrease in nicotine content.

[0299] In another embodiment, increasing the activity or expression of the Nic3 gene (or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) can result in a decrease in alkaloid content. Suitably, increasing the activity or expression of the Nic3 ERF, or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene can result in a decrease in nicotine content.

[0300] In one embodiment, the plant or part thereof is a tobacco plant. In one embodiment, a tobacco plant or part thereof according to the invention is a burley or flue-cured plant modified according to the invention. In one embodiment, the invention relates to a burley or flue-cured plant modified according to the invention. In one embodiment, a tobacco plant according to the invention (e.g., a modified tobacco plant) is an Oriental or Turkish tobacco plant.

[0301] In one embodiment, the tobacco plant or part thereof is dried. In one embodiment, the tobacco plant or part thereof is dried, for example, air-dried, flue-dried, flame-dried, or sun-dried. In a further aspect, the tobacco plant or part thereof is flue-dried. In a further aspect, the tobacco plant or part thereof is air-dried.

[0302] Flue-curing is well known in the art and refers to the process of drying tobacco in a flue that is fed from a firebox or gas supply system. This process heat-cures the tobacco by slowly increasing the temperature over the drying period without exposing the tobacco to smoke. This method produces tobacco that is rich in sugars and has a medium to high nicotine level. The Smith Tobacco Barn is a variation of a traditional flue-cured tobacco curing barn. Here is an example.

[0303] Air-cured tobaccos include burley, Maryland, and dark tobacco. A common factor is that the curing is primarily done without artificial sources of heat and humidity. Burley tobacco is light to dark brown in color, high in oil, and low in sugar. Burley tobacco is air-cured in curing barns. Major burley-producing countries are Argentina, Brazil, Italy, Malawi, and the United States. Burley tobacco plants include, for example, Clay 402, Clay 403, Clay 502, Ky 14, Ky 907, Ky 910, Ky 8959, NC 2, NC 3, NC 4, NC 5, NC 2000, TN 86, TN 90, TN 97, R 610, R 630, R711, R 712, NCBH 129, Bu 21xKy 10, HB04P, Ky 14xL 8, Kt 200, Newton 98, Pedigo 561, Pf561, and Va 509 is an example.

[0304] Maryland tobacco has excellent burning characteristics, low nicotine, and a neutral aroma. Major Maryland-producing countries include the United States and Italy. Air-cured dark tobacco is distinguished from other types primarily by its fermentation process, which gives air-cured dark tobacco its medium to dark brown color and distinct aroma. Their leaves have a low sugar content but a high nicotine content. Air-cured dark tobacco is primarily used in the production of chewing tobacco and snuff. The primary growing regions for flue-cured dark tobacco are Tennessee, Kentucky, and Virginia in the United States.

[0305] The term "functional fragment" as used herein refers to a portion of a polynucleotide that can function in the same way as a polynucleotide. For example, if the polynucleotide is an ERF gene, the functional fragment must be able to function as an ERF gene, e.g., the functional fragment retains the activity of the ERF gene. The functional fragment may have an activity level equal to or greater than the activity level of the full-length polynucleotide.

[0306] In one embodiment, the functional fragment may be a portion of the Nic3 gene discussed herein comprising at least 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous nucleotides. Suitably, the functional fragment comprises a domain of the Nic3 gene having SEQ ID NO: 75, 120, 127, or 129, as described above. In some embodiments, the functional fragment may comprise at least 150 nucleotides of the Nic1 ERF discussed herein.

[0307] In one embodiment, a functional fragment can be a portion of the Nic1 ERF gene discussed herein that comprises at least 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous nucleotides. In some embodiments, a functional fragment can comprise at least 150 nucleotides of the Nic1 ERF gene discussed herein.

[0308] In one embodiment, a functional fragment of a Nic2 ERF gene can be a portion of a Nic2 ERF gene discussed herein that comprises at least 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 contiguous nucleotides. In some embodiments, a functional fragment can comprise at least 150 nucleotides of a Nic2 ERF gene discussed herein.

[0309] The term "functional variant," as used herein, refers to variations that may occur in a genomic sequence without significant loss of activity in either gene function and / or protein function. For example, some amino acids present in a polypeptide (or some nucleotides present in a polynucleotide) may be substituted without significant loss of activity. A functional variant may have a level of activity equal to or greater than that of the non-variant polynucleotide and / or polypeptide. A sequence that differs from a gene disclosed herein due to the degeneracy of the genetic code is a functional variant. A variant may differ from a sequence of interest by as few as 10, as many as 9, as many as 8, as many as 7, as many as 6, as many as 5, as many as 4, as many as 3, as many as 2, or as many as 1 amino acid.

[0310] The term "degeneracy of the genetic code," as used herein, refers to the multiplicity of codon combinations that specify an amino acid, and refers to the degeneracy of the codons that encode a polypeptide sequence. This refers to redundancy in the mRNA sequence. For example, in an mRNA molecule encoding a polypeptide with the amino acid isoleucine, isoleucine can be encoded by AUU, AUC, or AUA. This means that a DNA molecule encoding the RNA may have multiple sequences, but the resulting polypeptide will have the same sequence. In other words, polymorphic nucleotide sequences can encode the same polypeptide product. This means that one nucleic acid sequence can contain a sequence that encodes the same polypeptide sequence but has very little sequence identity with a second sequence.

[0311] Any nucleotide sequence described herein or a sequence that has a degree of sequence identity or sequence homology with the amino acid sequence of a polypeptide having the specific properties described herein may be a functional variant.

[0312] The term "ortholog," as used herein, refers to genes derived from a common ancestral gene and found in different species as a result of speciation. Orthologs can share at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity at the nucleotide and / or amino acid sequence level. Orthologous genes often share the same or similar functions, i.e., have conserved functions.

[0313] In some embodiments of the present invention, a promoter may be provided. The promoter for use in the present invention may be one or more selected from the group consisting of a constitutive promoter, a senescence-specific promoter, a tissue-specific promoter, a developmentally regulated promoter, and an inducible promoter. In one embodiment, the promoter may be a constitutive promoter.

[0314] A constitutive promoter directs gene expression throughout various parts of a plant continuously during plant development, although the gene may not be expressed at the same level in all cell types. An example of a known constitutive promoter is 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). , rice actin 1 gene (Zhang W, McElroy D, Wu R. (1991) Analysis of rice Act1 5' region activity in transgenic rice plants (Plant Cell 3 1155-65), and 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), which are incorporated herein by reference. Constitutive promoters include the carnation etched ring virus (CERV) promoter. Carnation etched ring virus DNA sequence: comparison with cauliflower mosaic virus and retroviruses (incorporated herein by reference (Hull R, Sadler J, Longstaff M 1986 EMBO Journal, 5(2):3083-3090) ).

[0315] The constitutive promoter may be selected from the carnation etched ring virus (CERV) promoter, the cauliflower mosaic virus (CaMV) 35S promoter, the promoter derived from the rice actin 1 gene or the maize ubiquitin 1 gene. Suitably, the promoter may be the CERV promoter.

[0316] Alternatively, in some embodiments, the promoter may not be the cauliflower mosaic virus (CaMV35S promoter). In one embodiment, the promoter may be a senescence-specific promoter. A "senescence-specific promoter" (SAG) is a promoter that encodes a senescence-associated gene. A senescence-specific promoter can be a promoter associated with controlling the expression of a gene. Thus, a promoter can restrict expression of a coding sequence (i.e., gene) to which it is operably linked substantially exclusively in senescent tissue. A senescence-specific promoter can therefore be a promoter capable of preferentially driving gene expression in plant tissue in a developmentally regulated manner such that expression of the 3' protein-coding region occurs substantially only when the plant tissue undergoes senescence. It will be appreciated that senescence tends to occur in older parts of the plant, e.g., older leaves, and not in younger parts of the plant, e.g., seeds.

[0317] An example of a plant known to express a large number of senescence-associated genes is Arabidopsis. Thus, promoters for senescence-associated genes in Arabidopsis are (The Plant Journal, 2003, 36, 629-642), incorporated herein by reference, conducted a detailed study of SAGs and their promoters using Arabidopsis thaliana as a model. The gene construct may comprise a promoter from any of the SAGs disclosed in this paper. For example, a suitable promoter may be selected from the group consisting of SAG12, SAG13, SAG101, SAG21, and SAG18, and functional variants and functional fragments thereof.

[0318] In one embodiment, the promoter may be the SAG12 or SAG13 promoter. In one embodiment, the promoter may be the SAG12 promoter, or a functional variant or functional fragment thereof, which would be known to a skilled artisan (Gan & Amasino, 1997, Plant Physiology, 113: 313-319, incorporated herein by reference). Suitable promoters and their sequences are described in WO 2010 / 097623. , incorporated herein by reference.

[0319] In another embodiment, the promoter may be a tissue-specific promoter. A tissue-specific promoter is one that directs the expression of a gene in a part (or a few parts) of a plant, usually throughout the life of those plant parts. The category of tissue-specific promoters generally also includes promoters whose specificity is not absolute, i.e., they may also direct expression at lower levels in tissues other than the preferred tissue. Several tissue-specific promoters are known in the art, examples of which include those associated with the patatin gene expressed in potato tubers and the high molecular weight glutenin gene expressed in wheat, barley, or maize endosperm. Any of these promoters can be used in the present invention.

[0320] Suitably, the tissue-specific promoter may be a leaf-specific promoter. Suitably, the leaf-specific promoter may include ASYMMETRIC LEAVES 1 (AS1).

[0321] In a particularly preferred embodiment, the tissue-specific promoter is a root-specific promoter.

[0322] In another embodiment, the promoter may be a developmentally-regulated promoter. A developmentally-regulated promoter directs a change in expression of a gene in one or more parts of a plant at a particular time 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.

[0323] In one embodiment, the promoter can be an inducible promoter. An inducible promoter can direct expression of a gene in response to an inducer. In the absence of the inducer, the gene The promoter sequence is not expressed. Inducers can act directly on the promoter sequence or by counteracting the effect of a repressor molecule. Inducers can be metabolites, proteins, growth regulators, or chemicals such as toxic elements, physiological stresses such as heat, wounding, or osmotic pressure, or the indirect result of the action of pathogens or pests. Developmentally regulated promoters can be described as endogenous inducers produced by the plant or as specific types of inducible promoters that respond to environmental stimuli at specific points in the plant's life cycle. Examples of known inducible promoters include those described by Warner SA, Scott R, Draper J. (1993) (Isolation of an asparagus intracellular PR gene (AoPR1) wound-responsive promoter by the inverse polymerase chain reaction and its characterization in transgenic tobacco. Plant J. 3 191-201.), which is incorporated herein by reference. those related to wound responses such as those described in, those related to temperature responses as disclosed by Benfey & Chua (1989) (Benfey, PN, and Chua, NH. (1989) Regulated genes in transgenic plants. Science 244 174-181), which is incorporated herein by reference, and those related to temperature responses as disclosed by Gatz (1995) (Gatz, C. (1995) Novel inducible / repressible gene expression systems. Methods in Cell Biol. 50 411-424), which is incorporated herein by reference. Examples of such steroids include chemically induced steroids, such as those described in

[0324] Thus, in one embodiment, the promoter may be selected from the group consisting of a CERV promoter, a cauliflower mosaic virus 35S promoter (full or truncated), a Rubisco promoter, a pea plastocyanin promoter, a nopaline synthase promoter, a chlorophyll r / b binding promoter, a high molecular weight glutenin promoter, an α,β-gliadin promoter, a hordein promoter, and a patatin promoter.

[0325] In one embodiment, the promoter may be the CaMV 35S promoter or a modified 35S promoter with a duplicated or doubled enhancer region (R. Kay et al. Science. 1987 Jun 5;236(4806):1299-302, incorporated herein by reference).

[0326] In one embodiment, the promoter may be the native promoter.

[0327] "Native promoter," as used herein, refers to a promoter that is endogenous to a gene, i.e., that is operably linked to the gene in nature.

[0328] Recombinant vectors may also contain DNA encoding genes that can be used as selectable markers in the cloning process, i.e., to allow for the selection of transfected or transformed cells and to allow for the selection of cells harboring vectors that have incorporated heterologous DNA. Vectors may also contain DNA involved in regulating the expression of coding sequences or for targeting the expressed polypeptide to a specific part of the host cell, such as the chloroplast. Thus, vectors may contain at least one additional element selected from the group consisting of a selectable marker gene (e.g., an antibiotic resistance gene); a polypeptide termination signal; and a protein target sequence (e.g., a chloroplast transit peptide).

[0329] Examples of suitable marker genes include antibiotic resistance genes, such as those conferring resistance to kanamycin, geneticin (G418), and hygromycin (npt-II, hyg-B); herbicide resistance genes, such as those conferring resistance to phosphinothricin and sulfonamide-based herbicides (bar and suI, respectively; European Patent Application Nos. 242246 and 0249637, which are incorporated herein by reference); and screenable markers, such as beta-glucuronidase (see, e.g., Marker genes include genes encoding ... The marker may be controlled by a second promoter that allows expression in cells that may or may not be in seeds, thereby allowing selection of cells or tissues containing the marker at any developmental stage of the plant. A suitable second promoter is the promoter for the nopaline synthase gene of Agrobacterium. The promoters used are those derived from genes encoding the 35S cauliflower mosaic virus (CaMV) transcript, but any other suitable second promoter can be used.

[0330] commercially desirable traits In one embodiment, a plant of the present invention has a reduced total alkaloid content, and / or a reduced content of one or more alkaloids selected from nicotine, nornicotine, anabasine, myosmine, and anatabine, and / or reduced nicotine, while at least maintaining its aroma characteristics and / or other commercially desirable traits. In one embodiment, a plant of the present invention produces leaves of a similar grade and / or quality as a plant that has not been modified according to the present invention.

[0331] In one embodiment, a plant of the present invention has a reduced nicotine content without a significant change in the plant's aroma profile (e.g., compared to the same plant not modified according to the present invention).

[0332] In one embodiment, a plant of the invention has a reduced nicotine content without a significant change (e.g., a decrease) in other commercially desirable traits of the plant (e.g., compared to the same plant not modified in accordance with the invention). In particular, the yield of the modified plant is preferably not reduced compared to the same plant not modified in accordance with the invention.

[0333] Therefore, in one embodiment, the methods and uses of the present invention relate to reducing the total alkaloid content and / or reducing one or more alkaloids selected from nicotine, nornicotine, anabasine and anatabine, and / or reducing nicotine, while maintaining aroma characteristics and / or other commercially desirable traits (e.g., yield).

[0334] The term "commercially desirable trait" includes traits such as yield, mature plant height, number of harvestable leaves, average node length, cutter leaf length, cutter leaf width, quality, abiotic (e.g., drought) stress tolerance, herbicide tolerance and / or biotic (e.g., insect, bacterial or fungal) stress tolerance.

[0335] The term "commercially desirable traits," as taught herein, includes traits such as drought tolerance, pest resistance, mature plant height, harvestable leaf number, average node length, cutter leaf length, cutter leaf width, and yield that are comparable to said traits in a flue-tube-dried progenitor of a comparable plant when grown under similar field conditions.

[0336] Unless otherwise specified, as used herein, tobacco yield refers to cured leaf yield calculated based on the weight of cured tobacco leaf per acre under standard field conditions according to standard agricultural and curing practices.

[0337] In one embodiment, a plant (e.g., tobacco plant) of the present invention has a yield of between 50% and 150%, between 55% and 145%, between 60% and 140%, between 65% and 135%, between 70% and 130%, between 75% and 125% of that of a comparable plant grown under similar field conditions. Between, 80% and 120%, Between 85% and 115%, Between 90% and 110%, Between 95% and 105%, Between 50% and 100%, Between 55% and 100%, Between 60% and 100%, Between 65% and 100%, Between 70% and 100%, Between 75% and 100%, Between 80% and 100%, Between 85% and 100%, Between 90% and 100 %, between 95% and 100%, between 100% and 150%, between 105% and 150%, between 110% and 150%, between 115% and 150%, between 120% and 150%, between 125% and 150%, between 130% and 150%, between 135% and 150%, between 140% and 150%, or between 145% and 150%.

[0338] In another aspect, the yield of a plant of the invention (e.g., a tobacco plant) is approximately 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 the yield of a comparable plant grown under similar field conditions.

[0339] In another aspect, the yield of tobacco plants of the present invention is comparable to the yield of comparable plants when grown under similar field conditions.

[0340] 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.

[0341] In one embodiment, a tobacco plant of the present invention provides a yield selected from the group consisting of about between 1200 and 3500, between 1300 and 3400, between 1400 and 3300, between 1500 and 3200, between 1600 and 3100, between 1700 and 3000, between 1800 and 2900, between 1900 and 2800, between 2000 and 2700, between 2100 and 2600, between 2200 and 2500, and between 2300 and 2400 pounds per acre.

[0342] In another aspect, the tobacco plants of the present invention provide a yield selected from the group consisting of about between 1200 and 3500, between 1300 and 3500, between 1400 and 3500, between 1500 and 3500, between 1600 and 3500, between 1700 and 3500, between 1800 and 3500, between 1900 and 3500, between 2000 and 3500, between 2100 and 3500, between 2200 and 3500, between 2300 and 3500, between 2400 and 3500, between 2500 and 3500, between 2600 and 3500, between 2700 and 3500, between 2800 and 3500, between 2900 and 3500, between 3000 and 3500, and between 3100 and 3500 pounds per acre.

[0343] In further embodiments, the tobacco plants of the present invention are between about 1200 and 3500, between 1200 and 3400, between 1200 and 3300, between 1200 and 3200, between 1200 and 3100, between 1200 and 3000, between 1200 and 2900, between 1200 and 2800, between 1200 and 2700, between 1200 and 2600, between 1200 and 2500, between 1200 and 2600, between 1200 and 2700, between 1200 and 2800, between 1200 and 2900, between 1200 and 2800, between 1200 and 2700, between 1200 and 2600, between 1200 and 2500, between 1200 and 2600, between 1200 and 2700, between 1200 and 26 ... and between 1200 and 1400 lbs / acre.

[0344] tobacco plant The present invention provides methods and uses directed to plants (eg, tobacco plants), as well as cells (eg, tobacco cells), plants (eg, tobacco plants) and plant propagation material.

[0345] The term "tobacco," as used herein, refers to plants of the genus Nicotiana used in the production of delivery systems. Non-limiting examples of suitable "tobacco" plants include N. tabacum and N. rustica (e.g., N. tabacum L., LA B21, LN KY171, T1 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico).

[0346] In one embodiment, a suitable tobacco plant can be any N. tabacum germplasm, strain or variety.

[0347] In another embodiment, suitable tobacco plants may be non-Tabacum species.

[0348] Tobacco materials can be derived from or obtained from various Nicotiana tabacum types, commonly known as burley, flue or bright, and dark. In some embodiments, the tobacco material is derived from a burley, Virginia, or dark tobacco plant. The tobacco plant can be selected from burley, rare, specialty, expanded, or the like.

[0349] The use of tobacco cultivars and elite tobacco cultivars is also contemplated herein. Tobacco plants for use herein may therefore be of Nicotiana species or elite tobacco cultivars. Particularly useful Nicotiana tabacum species include flue-cured Virginia types, Burley types, and Oriental types.

[0350] In some embodiments, the tobacco plant may be selected from, for example, one or more of the following species: L. cultivar TI1068, AA37-1, B 13P, Xanthi (Mitchell-Mor), KT D#3 Hybrid 107, Bel-W3, 79-615, Samsun Holmes NN, F4 derived from a cross of 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 86 SI, 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, NC 2000, PG 01, PG 04, P01, P02, P03, RG 11, RG 17, 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, Creole Missionary, PM092, Delcrest, Djebel 81, DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, PM102, Kutsage E1, KY 14×L8、KY 171、LA BU 21、McNair 944、NC 2326、NC 71、NC 297、NC 3、PVH 03、PVH 09、PVH 19、PVH 21 10、Red Russian JK-48, Yaka JB 125 / 3, ΤΙ-1068, KDH-960, TI-1070, TW136, PM204, PM205, Basma, TKF 4028, L8, TKF 2002, TN 90, GR141, Basma xanthi, GR149, GR153, and Petit Havana.

[0351] The range of different types of products includes: BD 64, CC 101, CC 200, CC 27, CC 301, CC 400, and CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF91 1, DT 538 LC, Galpao, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC HB3307PLC 403LC HB3307PLC 404LC 501 LC、K 149、K 326、K 346、K 358、K394、K 399、K 730、KDH 959、KT 200、KT204LC、KY10、KY14KY16KY 17、KY 171、KY 907、KY907LC、KTY14xL8 LC、Little Crittenden、McNair 373、McNair 944、msKY 14xL8、Narrow Leaf Madole、Narrow Leaf NBH 98、N-126、N-777LC、N-7371 LC、NC 100、NC 102、NC 2000、NC 291、NC 297、NC 299、NC 3、NC 4、NC 6、NC7、NC 606、NC 71、NC 72、NC 810、NC BH 129、NC 2002、Neal Smith Madole、OXFORD 207、PD 7302 LC、PD 7309 LC、PD 7312 LC「Periq'e」、バコ、PVH03、PVH09、PVH19、PVH50、PVH51、R 610、R 630、R 7-1 1、R 7-12、RG 17、RG 81、RG H51、RGH 4、RGH 51、RS 1410、Speight 168、Speight 172、Speight 179、Speight 1022 220、Speight 225、Speight 227、Speight 234、Speight G-28、Speight G-70、Speight H-6、Speight H20、Speight NF3、Tl 1406、Tl 129、NT 86、TN86LC、TN 90、TN 97、TN97LC、TN D94、TN D950、TR(Tom Rosson)Madole、VA 309、VA359、AA 37-1、B 13P、Xanthi(Mitchell-Mor)、Bel-W3、79-615、Samsun Holmes NN、KTRDC number 2. 609、PG 01、PG 04、P01、P02、P03、RG 1 1、RG 8、VA 509、AS44、Banquet A1, Basma Drama B84 / 31, Basma I Zichna ZP4 / B, Basma Xanthi 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, NC 2326, 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. Less converted variants of the above are also contemplated, even if not specifically identified herein.

[0352] The plant may be a hybrid produced by crossing any of the varieties disclosed herein.

[0353] The tobacco plant may be burley, flue-cured Virginia, or oriental.

[0354] In one embodiment, the plant propagation material can be obtained from a plant of the present invention (e.g., a tobacco plant). As used herein, "plant propagation material" refers to any plant material obtained from a plant from which additional plants can be produced. Suitably, the plant propagation material can be seeds. Suitably, the plant propagation material can be pollen.

[0355] In one embodiment, a cell (e.g., a tobacco cell), a plant (e.g., a tobacco plant) or part thereof and / or plant propagation material of the invention may comprise regulated activity or expression of the Nic3 gene (or the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene). In another embodiment, a cell (e.g., a tobacco cell), a plant (e.g., a tobacco plant) and / or plant propagation material may comprise a construct or vector according to the invention. In another embodiment, a cell (e.g., a tobacco cell), a plant (e.g., a tobacco plant) and / or plant propagation material is obtainable (e.g., obtained) by a method according to the invention.

[0356] Suitably, a plant (e.g., a tobacco plant) or part thereof according to the invention may comprise modulated activity or expression of the Nic3 ERF gene (or the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) when compared to a plant (e.g., a tobacco plant) or part thereof that has not been modified to modulate the activity or expression of the Nic3 gene (or the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene).

[0357] In one embodiment, a plant (e.g., a tobacco plant) or part thereof according to the invention comprises a cell (e.g., a tobacco cell) of the invention. In another embodiment, plant propagation material can be obtained (e.g., obtained) from a plant (e.g., a tobacco plant) of the invention.

[0358] In one embodiment, there is provided a use of a cell (e.g., a tobacco cell) as provided in the preceding embodiment for the production of a product (e.g., a delivery system). Additionally, there is provided a use of a plant (e.g., a tobacco plant) described herein for propagating a plant (e.g., a tobacco plant).

[0359] The present invention also provides, in another embodiment, the use of a plant (e.g., a tobacco plant) of the foregoing embodiments for the production of a product (e.g., a delivery system). In another embodiment, the present invention provides the use of a plant (e.g., a tobacco plant) of the present invention for growing a crop. In one embodiment, the use of a Nic3 gene (or a Nic3 gene and a Nic1 ERF gene and / or a Nic2 ERF gene) according to the present invention results in modulation of the alkaloid content of a plant (e.g., a tobacco plant).

[0360] In one embodiment, a method or use of the Nic3 gene (or the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) according to the present invention may result in modulation of alkaloid content. In another embodiment, use of the Nic3 gene (or the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) (e.g., reduced activity or expression thereof) may result in a decrease in the content of one or more alkaloids. Suitably, the content of one or more of anatabine, anabasine, myosmine, nornicotine, or nicotine may be reduced. Suitably, nicotine content is reduced. Suitably, this may be observed when the activity or expression of the Nic3 gene (or the activity of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) is reduced compared to a wild-type plant.

[0361] In another embodiment, the Nic3 gene (or the Nic3 gene and the Nic1 ERF gene) The method or use of the Nic2 and / or Nic3 ERF genes (e.g., their increased activity or expression) may result in an increase in the content of one or more alkaloids. Suitably, the content of one or more of anatabine, anabasine, nornicotine, or nicotine may be increased. Suitably, the nicotine content is reduced.

[0362] In one embodiment, a plant (e.g., a tobacco plant) or part thereof, such as a leaf, or harvested or harvested processed leaf, or a cell or product (e.g., a delivery system) comprises a modified (e.g., mutated or deleted) Nic3 gene of the invention (or a combination of a modified (e.g., mutated or deleted) Nic3 gene with a modified (e.g., mutated or deleted) Nic1 ERF gene and / or a modified (e.g., mutated or deleted) Nic2 ERF gene according to the invention).

[0363] In one embodiment, the present invention provides tobacco cell cultures (e.g., in the form of in vitro cultures). The tobacco cell cultures can be tobacco cell suspension cultures. These in vitro cultured tobacco cells can be incorporated into delivery systems, for example, as a replacement for traditional tobacco pieces, shreds, fine-cut or long-cut tobacco flakes, as an additive component, or as both a replacement and an additive.

[0364] In one embodiment, there is provided the use of a tobacco cell culture, such as a harvested and / or processed tobacco cell culture, or an extract therefrom, according to the present invention for the production of a delivery system.

[0365] Tobacco cells harvested from in vitro culture can be dried, eg, freeze-dried, eg, to produce a powder.

[0366] Those skilled in the art will recognize known methods for establishing in vitro cultures of tobacco cells. By way of example only, the following methods may be used: collecting seeds from desired tobacco plants and sterilizing the outside of these seeds to remove undesirable organisms, planting the seeds to grow desired tobacco plants, removing tissue from the tobacco plants (e.g., from tobacco stems) to use as explants, establishing callus cultures from the tobacco explants, establishing cell suspension cultures from the callus cultures, and harvesting culture material (e.g., including tobacco cells) to produce tobacco cell cultures.

[0367] Tobacco cells can be harvested by a variety of methods, including filtration, e.g., vacuum filtration. The sample can be washed in a filter by adding water, and the remaining liquid is removed by filtration, e.g., vacuum filtration.

[0368] The harvested tobacco cell culture can be further processed, for example, dried, e.g., air-dried and / or freeze-dried. The harvested tobacco cell culture or the dried harvested tobacco cell culture or an extract thereof can be incorporated into a delivery system according to the present invention.

[0369] In one embodiment, the present invention provides a tobacco plant or part thereof for use in molecular agriculture. Suitably, a plant or part thereof modified according to the present invention can be used in the production of proteins such as therapeutic substances, for example, antibiotics, virus-like particles, nutraceuticals, or small molecules.

[0370] In one embodiment, the invention provides a method for producing a protein (e.g., a therapeutic protein) comprising the step of: 1) translating at least one Nic3 ERF gene; or 2) translating at least one Nic3 gene and at least one Nic1 ERF gene; and / or at least one Nic2 ERF gene, and culturing the plant under conditions sufficient to allow production of the protein (e.g., a therapeutic protein).

[0371] In one aspect, the present invention provides a method for introgressing a low nicotine trait into a tobacco variety, comprising: a) crossing a first tobacco variety that includes a low-nicotine trait with a second tobacco variety that does not have the low-nicotine trait to produce one or more progeny tobacco plants; b) genotyping one or more progeny tobacco plants for a polymorphic marker linked to a low nicotine trait, wherein the polymorphic marker is within 20 cM, within 10 cM, within 5 cM, within 4 cM, within 3 cM, within 2 cM, within 1 cM, within 0.5 cM, or within less than 0.5 cM of the Nic3 locus; and c) selecting progeny tobacco plants containing the low-nicotine trait The present invention provides a method comprising:

[0372] In one aspect, the method may involve simultaneously or in parallel selecting for one or more molecular markers associated with or closely linked to the Nic1 locus and / or one or more molecular markers associated with or closely linked to the Nic2 locus.

[0373] product The present invention also provides products obtainable from or obtained from tobacco plants according to the present invention, including products obtainable from or obtained from tobacco plants in which the activity or expression of the Nic3 gene (or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) has been modulated, and which contain a modulated alkaloid content (e.g., reduced nicotine content).

[0374] In one aspect, the present invention provides a tobacco plant or part thereof or a plant cell according to the present invention; Tobacco plants or parts thereof propagated from tobacco plant propagation material according to the invention; harvested leaves of plants according to the invention; processed leaves according to the invention; or plants produced by the methods according to the invention. The delivery system includes:

[0375] As used herein, the term "delivery system" includes systems that deliver at least one substance to a user, including: combustible aerosol delivery systems (whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, tobacco substitutes, or other smokable materials), such as cigarettes, cigarillos, cigars, and tobacco for pipes or hand-rolled or homemade cigarettes; Non-combustible aerosol delivery systems that release compounds from aerosol-forming materials without burning the aerosol-forming materials, such as hybrid systems for generating aerosols using a combination of electronic cigarettes, tobacco heating products, and aerosol-forming materials; and Aerosol-free delivery systems that deliver at least one substance, which may or may not contain nicotine, 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 products, including snus or moist snuff. Includes:

[0376] According to this disclosure, a "combustible" aerosol delivery system is one in which the aerosol-generating material (or components thereof) that is a component of the aerosol delivery system burns or burns during use to facilitate delivery of at least one substance to a user.

[0377] In some embodiments, the delivery system is a combustible aerosol delivery system, such as a system selected from the group consisting of cigarettes, cigarillos, and cigars.

[0378] In some embodiments, the present disclosure relates to components for use in combustible aerosol delivery systems, such as filters, filter rods, filter segments, tobacco rods, splints, aerosol modifier-releasing components, e.g., capsules, threads, or beads, or papers, e.g., plug wrap, tipping paper, or cigarette paper.

[0379] According to this disclosure, a "non-flammable" aerosol delivery system is one in which the aerosol-generating materials (or components thereof) that are components of the aerosol delivery system do not burn or combust to facilitate delivery of at least one substance to a user.

[0380] In some embodiments, the delivery system is a non-flammable aerosol delivery system, such as a powered non-flammable aerosol delivery system.

[0381] In some embodiments, the non-flammable aerosol delivery system is also known as a vaping device or electronic nicotine delivery system (END). It should be noted that while electronic cigarettes are being marketed, the presence of nicotine in the aerosol-forming material is not required.

[0382] In some embodiments, the non-combustible aerosol delivery system is an aerosol-generating material heating system, also known as a heated system. An example of such a system is a tobacco heating system.

[0383] In some embodiments, the non-combustible aerosol delivery system is a hybrid system for generating aerosols using a combination of aerosol-forming materials, one or more of which can be heated. Each of the aerosol-forming materials can be, for example, in solid, liquid, or gel form and may or may not contain nicotine. In some embodiments, the hybrid system includes a liquid or gel aerosol-forming material and a solid aerosol-forming material. The solid aerosol-forming material can include, for example, a tobacco product or a non-tobacco product.

[0384] Typically, a non-flammable aerosol delivery system may include a non-flammable aerosol delivery device and a consumable item for use with the non-flammable aerosol delivery device.

[0385] In some embodiments, the present disclosure relates to consumables, sometimes referred to as supplies throughout this disclosure, that include aerosol-generating materials and are configured for use with non-flammable aerosol delivery devices.

[0386] In some embodiments, the non-flammable aerosol delivery system, e.g., the non-flammable aerosol delivery device, can include a power source and a controller. The power source can be, for example, an electrical power source or a heat-generating power source. In some embodiments, the heat-generating power source includes a carbon substrate that can receive energy and distribute power in the form of heat to an aerosol-generating material or a heat-transfer material in proximity to the heat-generating power source.

[0387] In some embodiments, the non-flammable aerosol delivery system includes an area for receiving consumables. , an aerosol generator, an aerosol-generating area, a housing, a mouthpiece, a filter, and / or an aerosol modifier.

[0388] In some embodiments, consumables for use with the non-flammable aerosol delivery device may include aerosol-generating materials, aerosol-generating material storage areas, aerosol-generating material transfer components, aerosol generators, aerosol-generating areas, storage boxes, overwraps, filters, mouthpieces, and / or aerosol modifiers.

[0389] Suitably, the delivery system may be prepared from (eg may comprise) a tobacco plant or part thereof according to the present invention.

[0390] Suitably, the delivery system may be prepared from tobacco cell cultures according to the present invention.

[0391] Suitably, the delivery system may be prepared from (eg, may comprise) a tobacco plant or part thereof grown from tobacco plant propagation material according to the present invention.

[0392] Suitably, the delivery system may be prepared from (eg may comprise) harvested leaves of a tobacco plant according to the present invention.

[0393] Suitably, the delivery system may be prepared from (eg may include) tobacco leaves that have been treated in accordance with the present invention.

[0394] Suitably, the delivery system may be prepared from (eg may include) dried tobacco material according to the present invention.

[0395] Suitably, the delivery system may be prepared from (eg may include) a tobacco blend according to the present invention.

[0396] In one embodiment, the delivery system is a combustible smoking article selected from the group consisting of cigarettes, cigarillos, and cigars.

[0397] 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 splint, an additive-releasing component, e.g., a capsule, a thread, a bead, or paper, e.g., plug wrap, tipping paper, or cigarette paper.

[0398] In one embodiment, the delivery system is a non-flammable aerosol delivery system.

[0399] In one embodiment, the delivery system includes one or more components of a non-flammable aerosol delivery system, such as a heater and an aerosolizable substrate.

[0400] In one embodiment, the aerosol delivery system is an electronic cigarette, also known as a vaping device.

[0401] In one embodiment, the electronic cigarette includes a heater, a power source capable of providing power to the heater, an aerosolizable substrate such as a liquid or gel, a housing, and optionally a mouthpiece.

[0402] In one embodiment, the aerosolizable substrate is contained in a substrate container. The substrate container is coupled to or includes a heater.

[0403] In one embodiment, the delivery system is a heating product that releases one or more compounds by heating, without burning, a substrate material. The substrate material is an aerosolizable material, which may be, for example, tobacco or other non-delivery systems that may or may not contain nicotine. In one embodiment, the heating product is a tobacco heating product.

[0404] In one embodiment, the heating product is an electronic device.

[0405] In one embodiment, the tobacco heating product includes a heater, a power source capable of supplying power to the heater, and an aerosolizable substrate, such as a solid or gel-like material.

[0406] In one embodiment, the heating product is a non-electronic product.

[0407] In one embodiment, the heating product includes an aerosolizable substrate, such as a solid or gel-like material, and a heat source that can provide thermal energy to the aerosolizable substrate without any electronic means, for example by burning a combustible material, such as charcoal.

[0408] In one embodiment, the heating product also includes a filter capable of filtering the aerosol generated by heating the aerosolizable substrate.

[0409] In some embodiments, the aerosolizable substrate material may include a vapor or aerosol generating agent or a humectant such as glycerol, propylene glycol, triacetin, or diethylene glycol.

[0410] In one embodiment, the delivery system is a hybrid system for generating an aerosol by heating, without combustion, a combination of substrate materials. The substrate materials may include, for example, solids, liquids, or gels, which may or may not contain nicotine. In one embodiment, the hybrid system includes a liquid or gel substrate and a solid substrate. The solid substrate may 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 substrate and tobacco.

[0411] In another embodiment, a product may comprise a construct of the invention that, when expressed in a plant (e.g., a tobacco plant), modulates the activity or expression of at least one Nic3 gene, thereby reducing alkaloid content (e.g., nicotine content).

[0412] In another embodiment, a product may comprise one or more constructs of the invention that modulate the activity or expression of the Nic3 gene (or the activity or expression of the Nic3 gene and the activity or expression of the Nic1 ERF gene and / or the activity or expression of the Nic2 ERF gene), and the product has a modulated alkaloid content (e.g., a reduced nicotine content).

[0413] In one embodiment, there is provided a use of a plant of the present invention (e.g., a tobacco plant) for producing a leaf (e.g., a tobacco leaf). Suitably, the leaf (e.g., a tobacco leaf) may be subjected to downstream uses such as processing. Thus, in one embodiment, the use of the aforementioned embodiment may provide a processed leaf (e.g., a processed tobacco leaf). Suitably, the tobacco leaf may be subjected to drying, fermentation, pasteurization, or a combination thereof.

[0414] In another embodiment, the leaf (e.g., tobacco leaf) may be cut. In some embodiments, the leaf (e.g., tobacco leaf) may be cut before or after being subjected to drying, fermentation, pasteurization, or a combination thereof.

[0415] In one embodiment, the present invention provides harvested leaves of a plant of the present invention (e.g., a tobacco plant). In one embodiment, the harvested leaves can be obtained from a plant (e.g., a tobacco plant) having regulated Nic3 gene activity or expression (or regulated Nic3 and Nic1 ERF and / or Nic2 ERF gene activity or expression). Suitably, the harvested leaves have a regulated alkaloid content. In a further embodiment, the harvested leaves can be obtained (e.g., obtained) from a plant (e.g., a tobacco plant) propagated from propagation material of the present invention. In another embodiment, harvested leaves obtainable from the methods or uses of the present invention are provided. Suitably, the harvested leaves can be cut and harvested leaves. In some embodiments, the harvested leaves can contain viable cells (e.g., viable tobacco cells). In some embodiments, the harvested leaves are free of viable cells (e.g., viable tobacco cells). In other embodiments, the harvested leaves can be subjected to further processing.

[0416] Some tobacco plants can be harvested by cutting the stem and harvesting all of the leaves at the same time (e.g., as with burley tobacco), while other tobacco plants (e.g., flue-cured tobacco) may be harvested at a stage in the process, such as priming, where individual leaves are removed from the stem as they mature.

[0417] "Priming," as used herein, refers to the removal of leaves from tobacco plants. It may also refer to the removal of mature or aged leaves from flue-dried plants.

[0418] Processed leaves (e.g., processed tobacco leaves) are also provided. Processed leaves (e.g., processed tobacco leaves) can be obtained from plants (e.g., tobacco plants) of the present invention. Suitably, processed leaves can be obtained from plants obtained according to any of the methods and / or uses of the present invention. In one embodiment, processed leaves (e.g., processed tobacco leaves) can be obtained from plants (e.g., tobacco plants) that have modulated Nic3 gene activity or expression (or Nic3 and Nic1 ERF gene and / or Nic2 ERF gene activity or expression) and modulated alkaloid content, preferably when compared to control leaves, i.e., leaves from plants (e.g., tobacco plants) that have not been modified according to the present invention. Processed leaves (e.g., processed tobacco leaves) may include modulation in Nic3 gene activity or expression (or Nic3 and Nic1 ERF gene and / or Nic2 ERF gene activity or expression) and modulated alkaloid content.

[0419] In another embodiment, processed leaves (e.g., processed tobacco leaves) can be obtained from plants (e.g., tobacco plants) propagated from plant (e.g., tobacco plant) propagation material according to the present invention. Processed leaves (e.g., processed tobacco leaves) of the present invention can be obtained by processing harvested leaves of the present invention.

[0420] The term "processed leaf," as used herein, refers to a leaf that has undergone one or more processing steps to which leaves are subjected in the art. A "processed leaf" is free or substantially free of viable cells.

[0421] As used herein, the term "treated tobacco leaf" refers to tobacco leaf that has undergone one or more processing steps that tobacco undergoes in the art. "Processed tobacco leaf" is free or substantially free of viable cells.

[0422] The term "viable cell" refers to a cell that is capable of proliferation and / or is metabolically active. Thus, when a cell is said to be not alive, also called "non-viable," the cell does not exhibit the characteristics of a viable cell.

[0423] The term "substantially no viable cells" means that less than about 5% of the total cells are viable, preferably less than about 3%, more preferably less than about 1%, and even more preferably less than about 0.1% of the total cells are viable.

[0424] In one embodiment, the processed tobacco leaf may be processed by one or more of drying, fermentation, and / or pasteurization. Suitably, the processed tobacco leaf may be processed by drying. The tobacco leaf may be dried by any method known in the art. In one embodiment, the tobacco leaf may be dried by one or more drying methods selected from the group consisting of air drying, flame drying, flue drying, and sun drying. Suitably, the tobacco leaf may be air dried. Suitably, the tobacco leaf may be flue dried.

[0425] Air-curing is typically done by hanging tobacco leaves to dry in well-ventilated barns, usually over a period of 4 to 8 weeks. Air-curing is particularly suited to burley tobacco.

[0426] Suitably, tobacco leaves may be flue-cured, which is typically done by hanging the leaves in large barns where hardwood is kept burning with continuous or intermittent low-smoke smoke, and usually takes between 3 days and 10 weeks, depending on the treatment and tobacco.

[0427] In another embodiment, tobacco leaves can be flue-cured. Flue-curing can involve wrapping tobacco leaves into tobacco sticks and hanging them from stepped poles in a drying barn. The barn usually has a flue leading from an externally fueled firebox. This typically results in heat-cured tobacco without exposure to smoke. Typically, the temperature is increased slowly during the drying process, and the entire process takes approximately one week.

[0428] Suitably, the tobacco leaves may be sun-cured, a process that typically involves exposing uncoated tobacco to sunlight.

[0429] Suitably, the treated tobacco leaves may be subjected to fermentation. Fermentation can be carried out in any manner known in the art. Typically, during fermentation, the tobacco leaves are stacked into a dry tobacco pile (bulk), which is covered, for example, with burlap to retain moisture. The residual moisture inside the leaves, combined with the weight of the tobacco, generates natural heat, which mellows the tobacco. The temperature at the center of the bulk is monitored once a day. In some methods, the entire bulk is opened weekly. The leaves are then removed, shaken, and moistened, and the bulk is rotated to replace the inner and outer leaves of the bulk, bringing the bottom leaves to the top. This ensures even fermentation throughout the bulk. The additional moisture on the leaves and the actual rotation of the leaves themselves generates heat, releasing the tobacco's natural ammonia and reducing nicotine, while also darkening the color and improving the tobacco's aroma. Typically, the fermentation process continues for up to six months, depending on the tobacco species, the position and thickness of the petiole on the leaf, and the intended use of the leaf.

[0430] Suitably, the processed tobacco leaf may be processed by pasteurization. Pasteurization may be particularly preferred when tobacco leaf is used to make a smokeless delivery system, most preferably snus. Pasteurization of tobacco leaf can be carried out by any method known in the art. For example, pasteurization ... This can be done as detailed in J Foulds, L Ramstrom, M Burke, K Fagerstrom. Effect of smokeless tobacco (snus) on smoking and public health in Sweden. Tobacco Control (2003) 12: 349-359.

[0431] In the production of snus, pasteurization is typically performed by heat treating tobacco with steam (reaching a temperature of approximately 100°C) for 24-36 hours. This results in a nearly sterile product, and without wishing to be bound by theory, one of the consequences of this is thought to be limiting further TSNA formation.

[0432] In one embodiment, the pasteurization may be steam pasteurization.

[0433] In some embodiments, the treated tobacco leaf may be shredded. The treated tobacco leaf may be shredded before or after treatment. Suitably, the treated tobacco leaf may be shredded after treatment.

[0434] In some embodiments, tobacco plants, harvested leaves of tobacco plants, and / or processed tobacco leaves can be used to extract nicotine. Nicotine extraction can be performed using any method known in the art. For example, a method for extracting nicotine from tobacco is taught in U.S. Patent No. 2,162,738, which is incorporated herein by reference.

[0435] In one aspect, the present invention provides a cured tobacco material made from a tobacco plant or part thereof according to the present invention.

[0436] In another aspect, the present invention provides a tobacco blend comprising a tobacco material produced from a tobacco plant or part thereof according to the present invention. In one aspect, the present invention provides a tobacco blend comprising a dried tobacco material according to the present invention.

[0437] Suitably, a tobacco blend according to the present invention may comprise approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% tobacco derived from a tobacco plant or part thereof according to the present invention, or a tobacco cell culture according to the present invention. Suitably, a tobacco blend may comprise approximately 10% tobacco derived from a tobacco plant or part thereof according to the present invention. Suitably, a tobacco blend may comprise approximately 20% tobacco derived from a tobacco plant or part thereof according to the present invention. Suitably, a tobacco blend may comprise approximately 30% tobacco derived from a tobacco plant or part thereof according to the present invention. Suitably, a tobacco blend may comprise approximately 40% tobacco derived from a tobacco plant or part thereof according to the present invention. Suitably, a tobacco blend may comprise approximately 50% tobacco derived from a tobacco plant or part thereof according to the present invention. Suitably, the tobacco blend may comprise approximately 60% tobacco derived from a tobacco plant or part thereof in accordance with the present invention. Suitably, the tobacco blend may comprise approximately 70% tobacco derived from a tobacco plant or part thereof in accordance with the present invention. Suitably, the tobacco blend may comprise approximately 80% tobacco derived from a tobacco plant or part thereof in accordance with the present invention. Suitably, the tobacco blend may comprise approximately 90% tobacco derived from a tobacco plant or part thereof in accordance with the present invention.

[0438] In one embodiment, a tobacco blend product of the present invention comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95 dry weight percent of tobacco cured from a tobacco plant or part thereof according to the present invention.

[0439] Suitably, the dried tobacco material may be air dried. Suitably, the dried tobacco material may be flue dried. Suitably, the dried tobacco material may be sun dried.

[0440] A delivery system or smoking article according to the present invention may comprise a tobacco material (eg, a dry tobacco material) according to the present invention.

[0441] In another aspect, the present invention provides a delivery system. Suitably, the delivery system may be a blended delivery system. In one embodiment, the delivery system may be prepared from a tobacco plant or part thereof of the present invention. In one embodiment, the delivery system may be prepared from a tobacco plant having regulated Nic3 gene activity or expression, or Nic3 gene and Nic1 ERF gene and / or Nic2 ERF gene activity or expression. The delivery system may comprise reduced Nic1 ERF gene activity or expression and reduced alkaloid content. Suitably, the tobacco plant or part thereof may be grown from tobacco plant propagation material according to the present invention.

[0442] The term "part thereof," as used herein, in the context of a plant (e.g., a tobacco plant), refers to a portion of the plant (e.g., a tobacco plant). Preferably, the "part thereof" is a leaf of the plant (e.g., a tobacco plant).

[0443] In another embodiment, the delivery system may be prepared from harvested leaves of the present invention. In a further embodiment, the delivery system may be prepared from processed tobacco leaves of the present invention. Suitably, the delivery system may be prepared from tobacco leaves that have been processed by one or more of curing, fermenting, and / or pasteurizing. Suitably, the delivery system may comprise cut tobacco leaves, which may be processed as in the previous embodiments.

[0444] In one embodiment, the delivery system may be a smoking article. As used herein, the term "smoking article" may include smokable products such as cigarettes, cigarettes, cigars, and cigarillos, whether based on tobacco, tobacco derivatives, expanded tobacco, reconstituted tobacco, or tobacco substitutes.

[0445] In another embodiment, the delivery system may be a smokeless delivery system. As used herein, the term "smokeless delivery system" refers to a delivery system that is smokeless and / or combustible. In one embodiment, the smokeless delivery system may include snus, snuff, chewing tobacco, or the like.

[0446] In further embodiments, the delivery system may be a tobacco heating device, a hybrid device, an electronic cigarette, or the like. Typically, in a heating device or hybrid device, the aerosol is generated by the conduction of heat from a heat source to a physically separate aerosol-forming substrate or material, which may be located in, around, or downstream from the heat source. During smoking, volatile compounds are released from the aerosol-forming substrate by heat conduction from the heat source and are carried along with the air drawn through the smoking article. As the released compounds cool, they condense to form an aerosol, which is inhaled by the user.

[0447] Aerosol-generating articles and devices for consuming or smoking tobacco heating devices are known in the art. These may include, for example, electrically heated aerosol-generating devices, in which the aerosol is generated by the conduction of heat from one or more electrical heating elements of the aerosol-generating device to an aerosol-forming substrate of the tobacco heating device.

[0448] Suitably, the tobacco heating device may be an aerosol generating device.

[0449] Preferably, the tobacco heating device can be a heated device. Heating devices are known in the art and release compounds by heating tobacco without burning it. Examples of suitable heated devices can be those taught in WO 2013 / 034459 or GB 2515502, which are incorporated herein by reference.

[0450] In one embodiment, the aerosol-forming substrate of the tobacco heating device may be a delivery system according to the present invention.

[0451] In one embodiment, the tobacco heating device may be a hybrid device.

[0452] molecular agriculture The present invention may be particularly useful in the field of plant molecular agriculture, where plants or parts thereof or plant cells (e.g., tobacco and other Nicotiana species) are used for the production of proteins, peptides, and metabolites, for example, for the production of therapeutic drugs and pharmaceuticals, such as antibiotics, virus-like particles, or nutraceuticals or small molecules.

[0453] "Molecular agriculture", as used herein, relates to the production of recombinant proteins and / or other secondary metabolites in plants, or parts thereof, or plant cells.

[0454] Suitably, molecular farming (or biopharming) is the process of growing recombinant proteins. and culturing said plant or part thereof or plant cell containing the nucleic acid sequence under conditions that allow expression of said recombinant protein. Suitably, the method may further comprise extracting the recombinant protein from the plant or part thereof or plant cell, and optionally purifying it. Suitably, molecular farming (or biofarming) may comprise modifying a plant or part thereof or plant cell by introducing a nucleic acid sequence that encodes a recombinant protein, culturing said plant or part thereof or plant cell containing the nucleic acid sequence under conditions that allow expression of said recombinant protein, and extracting and purifying the recombinant protein from the plant or part thereof or plant cell.

[0455] Suitably, molecular farming (or biofarming) may comprise culturing a plant or part thereof or plant cell under conditions that allow for expression of a secondary metabolite. Suitably, the method may further comprise extracting the secondary metabolite from the plant or part thereof or plant cell, and optionally purifying it. Suitably, molecular farming (or biofarming) may comprise culturing a plant or part thereof or plant cell under conditions that allow for expression of a secondary metabolite, and extracting and purifying the recombinant protein from the plant or part thereof or plant cell.

[0456] Methods for extracting and purifying recombinant proteins and / or secondary metabolites from plants or parts thereof or plant cells are known in the art, e.g., U.S. Pat. Nos. 9,220,295; 9,289,011; 9,175,052 and U.S. Patent Application No. 2016 / 0029663.

[0457] Thus, the plants or parts thereof or plant cells according to the present invention can be used for molecular agriculture. The plants or parts thereof or plant cells according to the present invention can be used to reduce or eliminate the presence of nicotine and / or other nicotinic alkaloids in plants or parts thereof or plant cells. The plants or parts thereof or plant cells according to the present invention can be used to reduce or eliminate the presence of nicotine and / or other nicotinic alkaloids in products extracted and / or purified from the plants or parts thereof or plant cells.

[0458] Advantageously, the use of low-nicotine plants or rootsocks in molecular agriculture will reduce downstream processing costs associated with purifying products from plants or parts thereof or plant cells. Tobacco plants are attractive bioreactors for the production of recombinant proteins due to their potential for large-scale, low-cost production.

[0459] Suitable plants or parts thereof or plant cells for use in molecular agriculture include, but are not limited to, Nicotiana species. Suitably, the plant or parts thereof or plant cells for use in molecular agriculture may be Nicotiana benthamiana. Suitably, the plant or parts thereof or plant cells for use in molecular agriculture may be The plant cell may be Nicotiana tabacum.

[0460] In one aspect, a tobacco plant is provided for use in molecular agriculture. For example, the tobacco plant according to the present invention can be used for the production of recombinant proteins. Recombinant proteins that can be produced in tobacco plants include, for example, antigens, antibodies, enzymes, vaccines, and growth factors for vaccine production.

[0461] Monoclonal antibodies and fragments thereof, such as immunoglobulin G (IgG) and immunoglobulin A (IgA), IgA and IgG shimmer molecules, molecules secreted by IgG and IgA, single chain variable fragments, fragment antigen binding, and heavy and light chain variable fragments, can be produced by molecular farming using plants or parts thereof or plant cells according to the invention.

[0462] Pharmaceutical proteins can be produced by molecular farming using plants or parts thereof or plant cells according to the present invention, for example, pharmaceutical proteins expressed in plants include erythropoietin, interferon, hirudin, aprotinin, Leu-enkephalin, human growth hormone somatotropin.

[0463] Non-pharmaceutical proteins can be produced by molecular farming using the plants or parts thereof or plant cells according to the present invention, for example, plant-derived non-pharmaceutical proteins include avidin, trypsin, aprotinin, β-glucocerebrosidase, peroxidase and cellulose.

[0464] Suitably, plants or parts thereof or plant cells for use in molecular agriculture according to the present invention may comprise an average alkaloid level or an average nicotine level of about 0.01%, 0.02%, 0.05%, 0.0.75%, 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 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%, 3%, 4% or 5% based on dry weight. Suitably, plants or parts thereof or plant cells for use in molecular agriculture according to the present invention may comprise an average alkaloid level or an average nicotine level of less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.075%, less than 0.05%, less than 0.02% or less than 0.01%.

[0465] Suitably, the molecular agriculture according to the present invention may be carried out at a concentration of about 0.01%, 0.02%, 0.05%, 0.0.75%, 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 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%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.1% soluble fiber, based on dry weight. 0.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 4% or 5%. Suitably, molecular farming according to the present invention can produce products, extracts or purified products (e.g. recombinant proteins) comprising average alkaloid or nicotine levels of less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.075%, less than 0.05%, less than 0.02% or less than 0.01%.

[0466] Polynucleotides / Polypeptides / Constructs In certain embodiments of the invention, a construct that modulates the activity or expression of at least one Nic3 gene (or at least one Nic3 gene and at least one Nic1 ERF gene and / or at least one Nic2 ERF gene) may be transformed into a plant cell, suitably under the direction of a promoter.

[0467] In certain embodiments of the present invention, a construct that reduces (i.e., inhibits) the activity or expression of the Nic3 gene (or the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) may be transformed into a plant cell under the direction of a promoter. The genetic construct may be a gene editing construct, or may be a small interfering RNA (siRNA) molecule or a short hairpin loop ( The nucleic acid molecule may include an RNAi molecule, which may include an shRNA (shRNA) molecule.

[0468] In certain embodiments of the invention, constructs that increase the activity or expression of the Nic3 gene (or the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene), e.g., constructs encoding equivalent endogenous genes, may be transformed into plant cells under the direction of a promoter.

[0469] The construct may be introduced into a plant according to the present invention using a suitable vector, e.g., a plant transformation vector. The plant transformation vector may contain an expression cassette comprising, in the 5' to 3' transcriptional direction, a promoter sequence, a construct sequence targeting the Nic3 gene (or targeting the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene), and optionally a 3' untranslated terminator sequence containing a termination signal for RNA polymerase and a polyadenylation signal for polyadenylases. The promoter sequence may be present in one or more copies, and such copies may be identical to or variants of the promoter sequences described above. Terminator sequences may be obtained from plant, bacterial, or viral genes. Suitable terminator sequences include, for example, the pea rbcS E9 terminator sequence, the nopaline synthase of Agrobacterium tumefaciens, and the E9 terminator sequence of the pea rbcS gene. The nos terminator sequence derived from the ribosomal gene and the 35S terminator sequence derived from the cauliflower mosaic virus. Other suitable terminator sequences will be readily apparent to those skilled in the art.

[0470] Constructs of the present invention may also include gene expression enhancing mechanisms to increase promoter strength. An example of such an enhancer element is that derived from a portion of the promoter of the pea plastocyanin gene, the subject of International Patent Application WO 97 / 20056, incorporated herein by reference. Suitable enhancer elements may be, for example, the nos enhancer element derived from the nopaline synthase gene of Agrobacterium tumefaciens and the 35S enhancer element derived from the cauliflower mosaic virus.

[0471] These regulatory regions may be derived from the same gene as the promoter DNA sequence, or may be derived from different genes, either from Nicotiana tabacum or other organisms, such as Solanaceae or Cestroideae. All regulatory regions must be from the same gene as the promoter DNA sequence of the tissue being transformed. It should be operable in cells.

[0472] The promoter DNA sequence can be derived from the desired gene, e.g., the gene that the promoter is intended to direct, e.g., the gene encoding the Nic3 gene of the present invention, the same gene as the coding sequence used in the present invention, or it can be derived from a different gene, e.g., from Nicotiana tabacum or another organism, e.g., from a plant of the Solanaceae family or the subfamily Noctuidae.

[0473] 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. The plant transformation vector will contain such sequences in addition to the expression cassette as they are necessary for the transformation process. These sequences may include Agrobacterium vir genes, one or more T-DNA border sequences, and a selectable marker or other means of identifying transgenic plant cells.

[0474] The term "plant transformation vector" refers to a construct capable of in vivo or in vitro expression. Preferably, the expression vector is integrated into the genome of the organism. The term "integrated" preferably covers stable integration into the genome.

[0475] 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 to obtain stable maintenance of the inserted genetic material. An overview of the general technique can be found in Potrykus (Annu Rev Plant Physiol Plant Mol Biol

[1991] 42:205-225) and Friedrich (1991) 42:205-225, both of which are incorporated herein by reference. and Christon (AgroFood-Industry Hi-Tech March / April 1994 17-27) You can see it.

[0476] Typically, in Agrobacterium-mediated transformation, a binary vector carrying the desired foreign DNA, i.e., a Nic3 construct, is transferred from an appropriate Agrobacterium strain to a target plant by co-cultivating Agrobacterium with explants derived from the target plant. Transformed plant tissue is then regenerated on selective medium containing a selectable marker and a plant growth hormone. An alternative method is the floral dip method (Clough & Bent, 1998 Plant J. 1998 Dec;16(6):735-43, incorporated herein by reference), in which flower buds of intact plants are contacted with a suspension of an Agrobacterium strain containing a chimeric gene, and after seed production, transformed individuals are germinated and identified by growth on selective medium. Direct infection of plant tissue with Agrobacterium is a widely used and simple technique, which is described in Butcher DN et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: DS Ingrams and JP Helgeson, 203-208, incorporated herein by reference.

[0477] Further suitable transformation methods include direct gene transfer into protoplasts, for example, using polyethylene glycol or electroporation techniques, particle guns, microinjection, and the use of silicon carbide fibers. Plant transformation using ballistic transformation, such as silicon carbide whisker technology, is taught in Frame BR, Drayton PR, Bagnaall SV, Lewnau CJ, Bullock WP, Wilson HM, Dunwell JM, Thompson JA & Wang K (1994), which is incorporated herein by reference. Fertile transgenic plants by silicon carbide whisker-mediated transformation The generation of Cassava plants is taught in The Plant Journal 6: 941-948, which is incorporated herein by reference, and viral transformation techniques are taught, for example, in Meyer P, Heidmann I & Niedenhof I (1992), which is incorporated herein by reference. The use of cassava mosaic virus as a vector system for plants is taught in Gene 110: 213-217, which is incorporated herein by reference. Further teachings on plant transformation can be found in An example can be found in EP 0 449 375, which is incorporated herein by reference.

[0478] 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 contain one vector, but may also contain two vectors. In the case of two vectors, the vector system is usually called a binary vector system. Binary vector systems are described in more detail in Gynheung Anetal, (1980) Binary Vectors, Plant Molecular Biology Manual A3, 1-19, which is incorporated herein by reference. It is listed.

[0479] One widely used plant cell transformation system uses the Ti plasmid from Agrobacterium tumefaciens or the Ri plasmid from Agrobacterium rhizogenes, as described by An et al., (1986), Plant Physiol. 81, 301-305, and Butcher DN et al., (1980), Tissue Culture Methods for Plant Pathologists, eds.: DS Ingrams and JP Helgeson, 203-208, which are incorporated herein by reference. After each method of introduction of a desired exogenous gene in plants according to the present invention, the presence and / or insertion of additional DNA sequences may be necessary. The use of T-DNA for the transformation of plant cells has been intensively studied and is described in EP 120516, Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters BB, Amsterdam, 1985, Chapter 1, which is incorporated herein by reference. V, Fraley et al., Crit. Rev. Plant Sci., 4:1-46, and Anatol. EMBO J (1985) 4: 277-284.

[0480] Plant cells transformed with constructs that modulate the activity or expression of the Nic3 gene (or the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) can be grown and maintained according to well-known tissue culture methods, for example, by culturing the cells in an appropriate culture medium supplemented with essential growth factors, such as amino acids, plant hormones, vitamins, and the like.

[0481] The term "transgenic plant" in the context of the present invention includes any plant containing a construct that regulates the activity or expression of the Nic3 gene (or the combination of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) according to the present invention. Thus, a transgenic plant is a plant that has been transformed with a construct according to the present invention. Preferably, the transgenic plant exhibits regulated Nic3 gene activity or expression (or the activity or expression of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene) and regulated alkaloid content according to the present invention. The term "transgenic plant" does not refer to a native nucleotide coding sequence in its natural environment, i.e., under the control of its native promoter (which is also in its natural environment).

[0482] In one embodiment, the Nic3 gene, the Nic1 ERF gene, the Nic2 gene, The ERF gene, construct, plant transformation vector, or plant cell is in isolated form. The term "isolated" means that the sequence is free from at least one other component with which the sequence is naturally associated and found in nature in its natural state. It means not having it qualitatively.

[0483] In one embodiment, the Nic3 gene, the Nic1 ERF gene, the Nic2 gene, The ERF gene, construct, plant transformation vector, or plant cell is in a purified form. The term "purified" means in a relatively pure state, e.g., at least about 90% pure, or at least about 95% pure, or at least about 98% pure.

[0484] As used herein, the term "nucleotide sequence" refers to an oligonucleotide or polynucleotide sequence, as well as variants, homologs, fragments, and derivatives thereof (such as portions thereof). Nucleotide sequences may be of genomic, synthetic, or recombinant origin, and they may be double-stranded or single-stranded, representing either the sense or antisense strand.

[0485] The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably, the nucleotide sequence refers to a DNA sequence, more preferably a cDNA sequence, encoding the present invention.

[0486] In a preferred embodiment, the nucleotide sequence, i.e., the Nic3 gene, the Nic1 ERF gene, or the Nic2 ERF gene, as it relates to and is within the scope of the present invention includes the native nucleotide sequence in its natural environment and linked to its naturally associated sequences (also in their natural environment). For ease of reference, we refer to this preferred embodiment as a "native nucleotide sequence." In this regard, the term "native nucleotide sequence" refers to the entire nucleotide sequence in its native environment and operably linked to the entire promoter with which it is naturally associated (which promoter is also in its native environment).

[0487] Nucleotide sequences encoding proteins having the specific properties of the Nic3 gene, Nic1 ERF, or Nic2 ERF as defined herein, or proteins suitable for modification, can be identified and / or isolated and / or purified from any cell or organism that produces the protein. Various methods for identifying, isolating, and / or purifying nucleotide sequences are well known in the art. For example, once a suitable sequence has been identified and / or isolated and / or purified, PCR amplification techniques can be used to prepare two or more sequences.

[0488] In yet a further alternative, the nucleotide sequence encoding the Nic3 gene or the Nic1 ERF or the Nic2 ERF can be purified using established standard methods, such as the phosphoramidite method described by Beucage SL et al., (1981) Tetrahedron Letters 22, pp. 1859-1869, which is incorporated herein by reference, or the method described by Matthes et al., (1984) EMBO J. 3, pp. 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 appropriate vectors.

[0489] 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."

[0490] The present invention also encompasses the use of amino acid sequences or any nucleotide sequences of polypeptides having the specific properties defined herein, i.e., sequences having a degree of sequence identity or homology with the Nic3 gene, Nic1 ERF gene, or Nic2 ERF gene encoding such polypeptides (hereinafter referred to as "homologous sequences"). Here, the term "homolog" refers to an entity having a degree of homology with the subject amino acid sequence and subject nucleotide sequence. Here, the term "homology" can be considered equivalent to "identity."

[0491] Homologous amino acid sequences and / or nucleotide sequences and / or fragments should provide and / or encode polypeptides that retain and / or enhance the functional activity of the Nic3 or Nic1 ERF or Nic2 ERF gene. Typically, a homologous sequence will contain or encode, for example, the same active site as the reference amino acid sequence. Although homology can 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 preferred to express homology in terms of sequence identity. Homologous sequences typically retain functional domains or motifs.

[0492] In one embodiment, a homologous sequence comprises an amino acid or nucleotide sequence that has one, two, or more additions, deletions, and / or substitutions compared to the reference sequence.

[0493] Comparison of homology or identity can be performed by eye, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology between two or more sequences. The percent homology or percent identity can be calculated over contiguous sequences, i.e., one sequence is aligned with the other, and each amino acid in one sequence is directly compared, one residue at a time, to the corresponding amino acid in the other sequence. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

[0494] While this is a very simple and consistent method, it cannot take into account, for example, that in an otherwise identical pair of sequences, a single insertion or deletion at a subsequent amino acid residue would misalign the alignment and thus potentially significantly reduce the percent homology when the entire alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology.

[0495] However, these more sophisticated methods assign a "gap penalty" to each gap that occurs in the alignment, such that, given the same number of identical amino acids, sequence alignments with as few gaps as possible (which reflects a greater relatedness between the two compared sequences) will achieve a higher score than those with many gaps. "Affine gap costs" are typically used, which impose a relatively high cost on the existence of a gap and a smaller penalty on each subsequent residue within the gap. This is the most commonly used gap scoring system. High gap penalties naturally result in optimized alignments with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, it is preferred to use the default values ​​when using such software for sequence comparisons.

[0496] The calculation of maximum % homology is therefore based on the optimal alignment taking into account gap penalties. A suitable computer program for performing such alignments is Vector NTI (Invitrogen Corp.). Examples of software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) Short Protocols in Molecular Biology, 4th Ed - Chapter 18), BLAST2 (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, BLAST2, and FASTA are available for offline and online searching (see Ausubel et al. 1999, pages 7-58 to 7-60).

[0497] Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​generally used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLAST program. The BLOSUM62 matrix is ​​the default matrix for the Vector NTI suite of programs. Vector NTI programs typically use either the published default values ​​or a custom symbol comparison table if provided (see user manual for further details). For some applications, it is preferred to use the default values ​​of the Vector NTI package.

[0498] Alternatively, the percentage 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 produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0499] If gap penalties are used in determining sequence identity, the following parameters are preferably used for pairwise alignments:

[0500] [Table 4]

[0501] [Table 5]

[0502] In one embodiment, CLUSTAL uses the gap penalties and gap extensions defined above. In some embodiments, the gap penalties used for BLAST or CLUSTAL alignments may differ from those detailed above. It will be understood that the standard parameters for performing BLAST and CLUSTAL alignments may change periodically, and that appropriate parameters may be selected based on the detailed standard parameters for the BLAST or CLUSTAL alignment algorithm at any given time.

[0503] Suitably, the degree of identity for nucleotide sequences may be 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.

[0504] Suitably, the degree of identity for nucleotide, cDNA, cds or amino acid sequences may be determined over the entire sequence.

[0505] The sequences may also have deletions, insertions, or substitutions of amino acid residues that result in silent changes and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, so long as the secondary binding activity of the substance is maintained. 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 with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0506] Conservative substitutions can be made, for example, according to Table 4 below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

[0507] [Table 6]

[0508] The present invention also encompasses possible homologous substitutions (both substitution and replacement are used herein to mean the replacement of an existing amino acid residue with an alternative residue), i.e., basic to basic, acidic to acidic, polar to polar, etc. Non-homologous substitutions, i.e., the substitution of one class of residue for another class of residue, or, instead, ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienyl Substitutions may also be made that involve the inclusion of unnatural amino acids such as guanylalanine, naphthylalanine, and phenylglycine.

[0509] The replacement also * and alpha-disubstituted * Amino acids, N-alkyl amino acids * , lactic acid * , halide derivatives of natural amino acids, e.g., trifluorotyrosine * , p-Cl-phenylalanine * , p-Br-phenylalanine * , pI-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 * , methyl derivatives of phenylalanine (Phe), e.g., 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) * This can be achieved with unnatural amino acids, including * is used for the purposes of the above discussion (for homologous or non-homologous substitutions) to indicate the hydrophobicity of the derivative, while # is used to indicate the hydrophilicity of the derivative, and # * indicates amphiphilic characteristics.

[0510] Variant amino acid sequences may contain, in addition to amino acid spacers such as glycine or β-alanine residues, suitable spacer groups, including alkyl groups such as methyl, ethyl, or propyl groups, that may be inserted between any two amino acid residues in the sequence. A further variation involves the presence of one or more amino acid residues in peptoid form, as will be well understood by those skilled in the art. For the avoidance of doubt, the term "peptoid form" is used to refer to variant amino acid residues in which the nitrogen atom of the residue has an α-carbon substituent rather than an α-carbon. Methods for preparing peptides in peptoid form are known in the art, e.g., Simon RJ et al., PNAS (1992) 89(20), 9367-9371, and Horwell DC Trends Biotechnol. (1995) 13(4), 132-134.

[0511] Nucleotide sequences for use in the present invention may contain synthetic or modified nucleotides. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and / or the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For purposes of the present invention, it is understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be made to enhance the in vivo activity or life span of the nucleotide sequences of the present invention.

[0512] The present invention also encompasses sequences that are complementary to the nucleic acid sequences of the present invention, or sequences that can hybridize to either the sequences of the present invention or to sequences complementary thereto. As used herein, the term "hybridization" refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing, and to the polymerase chain reaction (PCR). This includes the amplification process carried out in the technology.

[0513] The present invention also relates to nucleotide sequences that can hybridize to the nucleotide sequences of the present invention (including complementary sequences of the sequences presented herein). Preferably, hybridization is determined under stringent conditions (e.g., 50°C and 0.2xSSC {1xSSC = 0.15M NaCl, 0.015M NaCitrate, pH 7.0}). More preferably, hybridization is determined under high stringency conditions (e.g., 65°C and 0.1xSSC {1xSSC = 0.15M NaCl, 0.015M NaCitrate, pH 7.0}).

[0514] In one aspect, the sequences for use in the present invention are synthetic sequences, i.e., sequences prepared by in vitro chemical or enzymatic synthesis, including, but not limited to, sequences made with optimal codon usage for the host organism.

[0515] The term "expression vector" refers to a construct capable of in vivo or in vitro expression. In one embodiment, a vector of the invention expresses the Nic3 gene described herein. In one embodiment, a vector of the invention further expresses the Nic1 ERF and / or Nic2 ERF genes described herein. Preferably, the expression vector is integrated into the genome of a suitable host organism. The term "integrated" preferably covers stable integration into the genome.

[0516] The nucleotide sequence for use in the present invention can be present in a vector in which the nucleotide sequence is operably linked to a regulatory sequence capable of providing expression of the nucleotide sequence by a suitable host organism. The construct for use in the present invention can be transformed into a suitable host cell as described herein to provide expression of the polypeptide of the present invention. The selection of a vector, such as a plasmid, cosmid, or phage vector, often depends on the host cell into which it will be introduced. The vector can be used in vitro, for example, to produce RNA, or can be used to transfect, transform, transduce, or infect host cells.

[0517] In some applications, the nucleotide sequences for use 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. By way of example, the present invention encompasses vectors comprising the nucleotide sequence of the Nic3 gene described herein operably linked to such regulatory sequences, i.e., the vector is an expression vector. Suitably, the vector may additionally comprise the nucleotide sequence of the Nic1 ERF gene and / or the Nic2 ERF gene described herein, operably linked to regulatory sequences.

[0518] The term "operably linked" refers to a juxtaposition wherein the components described 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 way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0519] The term "regulatory sequence" includes promoters and enhancers and other expression regulation signals. The term "promoter" is used in its normal sense in the art, e.g., an RNA polymerase binding site. The nucleotide sequence within the construct encoding the Nic3 gene or a combination of the Nic3 gene and the Nic1 ERF gene and / or the Nic2 ERF gene may be operably linked to at least a promoter.

[0520] The term "construct" is synonymous with terms such as "cassette" or "vector," but includes any vector for use in accordance with the present invention that is directly or indirectly linked to a promoter. The present invention includes a nucleotide sequence for

[0521] An example of an indirect linkage is the provision of a suitable spacer group, such as an intron sequence, such as the Sh1-intron or ADH intron, between the promoter and the nucleotide sequence of the invention. The same applies to the term "fused" in the context of the present invention, which includes direct or indirect linkage. In some cases, the term does not refer to the natural combination of a wild-type gene promoter and a nucleotide sequence encoding a protein normally associated therewith, when both are in their natural environment. The construct may further contain or express a marker, which allows for the selection of the genetic construct.

[0522] A review of the general techniques used in plant transformation can be found in the articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol

[1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March / April 1994 17-27), which are incorporated herein by reference. Further teachings on plant transformation can be found in European Patent Application Publication No. 0 449 375, which is incorporated herein by reference.

[0523] In one embodiment, provided herein are SNPs for use in genotyping plants (e.g., tobacco plants) having a low-alkaloid (e.g., low-nicotine) trait. Suitably, the SNPs may be selected from Tables 5 to 9 below. Suitably, at least two SNPs may be selected, where a first SNP may be selected from any of Tables 5 to 7 and a second SNP may be selected from any of Tables 5 to 7. Suitably, at least two SNPs may be selected, where a first SNP may be selected from Table 5 and a second SNP may be selected from Table 5. Suitably, at least two SNPs may be selected, where a first SNP may be selected from Table 6 and a second SNP may be selected from Table 6. Suitably, at least two SNPs may be selected, where a first SNP may be selected from Table 7 and a second SNP may be selected from Table 7.

[0524] In one embodiment, provided herein are markers for use in genotyping plants (eg, tobacco plants) having low alkaloids (eg, low nicotine traits).

[0525] In one embodiment, provided herein are SNPs for use in genotyping the Nic3 locus in plants (e.g., tobacco plants). Suitably, the SNPs can be selected from Tables 5-7 below.

[0526] In one embodiment, provided herein are markers for use in genotyping the Nic3 locus in plants (eg, tobacco plants).

[0527] [Table 7] JPEG2025169355000011.jpg185134JPEG2025169355000012.jpg189136JPEG202 5169355000013.jpg190136JPEG2025169355000014.jpg189134JPEG2025169355 000015.jpg187136JPEG2025169355000016.jpg187135JPEG2025169355000017. jpg188138JPEG2025169355000018.jpg187132JPEG2025169355000019.jpg28133

[0528] [Table 8] JPEG2025169355000021.jpg186134JPEG2025169355000022.jpg189134JPEG2025169355000023.jpg97133

[0529] [Table 9] JPEG2025169355000025.jpg188137JPEG2025169355000026.jpg43135

[0530] In one embodiment, provided herein are markers for use in identifying plants having low nicotine levels.

[0531] SNPs or markers for use in genotyping the Nic1 and / or Nic2 loci are available in WO2018237107, which is incorporated herein by reference.

[0532] "SNP" or "single nucleotide polymorphism," as used herein, refers to a sequence variation that occurs when a single nucleotide (A, T, C, or G) in a genomic sequence is altered or variable compared to a reference sequence. When a SNP is mapped to a site on the genome, a "SNP marker" exists.

[0533] "Marker" or "SNP marker," as used herein, refers to a nucleic acid or amino acid sequence that is sufficiently unique to characterize a specific locus on the genome. A polymorphic trait can be used as a marker if it is differentially inherited and exhibits linkage disequilibrium with the phenotypic trait of interest. When a trait is said to be linked to a given marker, it will be understood that the actual DNA segment whose sequence influences the trait usually segregates with the marker.

[0534] Unless otherwise defined, 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 definition of many of the terms used in this disclosure. This provides a general dictionary of the term to those skilled in the art.

[0535] 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 embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, all nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0536] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure that may be incorporated by reference in their entirety. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0537] Amino acids are described herein using the amino acid name, three-letter abbreviation, or one-letter abbreviation. The term "protein" as used herein 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."

[0538] In the present disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that due to degeneracy, a polypeptide can be encoded by more than one nucleotide sequence.

[0539] Other definitions of terms can be found throughout the specification. Before describing exemplary embodiments in more detail, it is to be understood that the disclosure is not limited to the particular embodiments described, and therefore, It is to be understood, of course, that variations may occur. The scope of the present disclosure will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0540] The various embodiments described herein are presented solely to aid in the understanding and teaching of the claimed features. These embodiments are provided merely as a representative sample of embodiments and are not all-inclusive and / or limiting. The advantages, embodiments, examples, functions, features, structures, and / or other aspects described herein are not intended to be limitations on the scope of the invention as defined by the appended claims or equivalents thereof, and it will be understood that other embodiments may be utilized and modifications may be made without departing from the scope of the claimed invention. Various embodiments of the invention may suitably comprise, consist of, or consist essentially of any suitable combination of the disclosed elements, components, features, parts, steps, means, etc., other than those specifically described herein. Additionally, this disclosure may include other inventions not claimed herein but which may be claimed in the future.

[0541] Where a range of values ​​is provided, unless the context clearly indicates otherwise, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range is also specifically disclosed. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value within that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and each range including either limit, neither limit, or both limits within that smaller range, subject to any specifically excluded limit in the stated range, is also encompassed within the disclosure. When a stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included within the disclosure.

[0542] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to an "enzyme" or "nitrate reductase" includes a plurality of such candidate substances and equivalents thereof known to those skilled in the art, and so forth.

[0543] advantage Surprisingly, it has been discovered that by modulating the activity or expression of the Nic3 gene as taught herein, for example, by providing a mutation in the Nic3 locus, the alkaloid content (e.g., nicotine content) and / or TSNA precursor content of tobacco cells and tobacco plants, or portions thereof, can be modulated. This allows for the production of delivery systems with modulated alkaloid (e.g., reduced nicotine) and / or reduced TSNA precursor content and commercially desirable traits desired by consumers of the delivery systems. In particular, tobacco cells and tobacco plants, or portions thereof, with reduced nicotine content can be produced by providing at least one mutation in the Nic3 locus and, optionally, at least one mutation in the Nic1 locus and / or at least one mutation in the Nic2 locus.

[0544] The present inventors have identified, for the first time, a new genetic locus capable of conferring an ultra-low nicotine phenotype. Prior to the present invention, it was not known that modulation of the activity or expression of the Nic3 gene described herein could be used to modulate alkaloid and / or TSNA content.

[0545] The inventors have determined that modulation of a new genetic locus, referred to herein as the Nic3 locus, can reduce the alkaloid content (e.g., nicotine content) of the modified plant to surprisingly low levels. In particular, the inventors have determined that by providing at least one mutation in the Nic3 locus, and optionally at least one mutation in the Nic1 locus and / or at least one mutation in the Nic2 locus, the alkaloid content (e.g., nicotine content) can be reduced to surprisingly low levels.

[0546] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto. [Example] [Example]

[0547] Development of populations segregating for Nic3 A flue-cured tobacco variety (FC101) containing nic1 and nic2 was found to have lower nicotine levels than predicted based on these two loci alone.

[0548] We hypothesize that a third locus, Nic3, controls reduced nicotine levels in this variety, and here we present studies to identify the underlying gene controlling this locus.

[0549] plant material To develop a population segregating for Nic3, an F2 population of 262 individuals was generated from a cross between FC101 (nic1 nic2 nic3) and LAFC53 (nic1 nic2 Nic3).

[0550] All individuals, along with five replicates of the two parents, were sown in a greenhouse and then planted in a field in Kernersville, North Carolina during the normal US growing season.

[0551] Plants were grown for up to 140 days after planting before harvest. Lower, middle, and upper stem leaf positions were harvested when they were mature. The upper leaves (top 5-7 leaves) were then dried in a six-rack flue-tube drying barn as per standard flue-tube drying practices. [Example]

[0552] Phenotypic analysis of parents and F2s obtained from a cross between FC101 (nic1 nic2 nic3) and LAFC53 (nic1 nic2 Nic3) Nicotine and nornicotine were measured in three technical replicates for both parents as well as 218 individuals from the F2 population.

[0553] We measured the alkaloids via standard gas chromatographic methods.

[0554] result Analysis of the nicotine and nornicotine content of the two parents showed that FC101 contained significantly lower levels of both alkaloids compared to LAFC53 (Figure 1).

[0555] Nicotine levels in the F2 plants obtained from FC101 × LAFC53 were found to be continuously distributed (Fig. 2A), and therefore the Nic3 genotype was determined based on the phenotypic values. It was not possible to draw a clear inference.

[0556] Nornicotine content in F2s was found to be largely uniform, with some individuals exhibiting naturally high levels of nornicotine (Fig. 2B). [Example]

[0557] Marker development and linkage analysis DNA was extracted from leaf samples of all F2 lines and their respective parents using the CTAB method. Custom Tobacco 50K Infinium iSelect HD BeadChips (Illumina Inc., San Diego, CA) were used. All F2 lines and parental DNA samples were selected for SNP genotyping using GenomeStudio version 2.0 (Illumina Inc., San Diego, CA). SNP clusters were generated using GenomeStudio version 2.0 (Illumina Inc., San Diego, CA), and all identified polymorphic markers were used for further analysis. The software Joinmap version 2.0 was used with the regression mapping function at default settings. A genetic linkage map for each population was constructed using the software program 4.0 (Stam, 1993).

[0558] To roughly map the nic3 locus, we used the Haley-Knott regression method using the stepwiseqtl function of R / QTL (Broman & Sen, 2009; Manichaikul et al. 2009). Multiple QTL mapping was performed on two populations using genotypic probabilities calculated at a maximum distance of 1 cM and 1000 permutations to determine the logarithm of the odds (LOD) significance threshold for incorporating both additive QTL and epistatic interactions at α = 0.01 per experiment.

[0559] result iSelect HD BeadChip genotyping of F2 individuals derived from FC101 x LAFC53 To identify the Nic3 locus through QTL analysis, we then genotyped the F2 individuals using custom 50K Infinium iSelect HD BeadChips. Using BeadChip, the present inventors identified the polymorphisms between FC101 and LAFC53. Approximately 4,400 SNP markers were identified.

[0560] These markers could be mapped to 2992 unique loci in the F2 population.

[0561] QTL analysis of the F2 population identified a linkage group containing a large number of markers significantly associated with total nicotine content (maximum LOD score of 22.17), explaining 37.4% of the variance in this trait (Figure 3).

[0562] These markers are from the 30k Infinium HD Consensus Map 2015 of N. tabacum It was placed with markers on linkage group 5 (Edwards, K.D., Fernandez-Pozo, N., Drake-Stowe, K., Humphry M., Evans, A.D., Bombarely, A., Allen, F., Hurst, R., White, B., Kernodle, SP, Bromley, JR, Sanchez-Tamburrino, JP, Lewis, RS, and Mueller, LA (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18, 448 (pseudo-chromosome 5 of the genome).

[0563] result Using markers identified as closely linked to the peak QTL for nicotine content, we identified a genomic region encompassing the Nic3 locus bounded by markers Nt1AG1750 (SEQ ID NO: 311) and Nt1AC2307 (SEQ ID NO: 312) (206 cM to 398 cM in Figure 3).

[0564] Using BioNano hybrid assemblies of (Edwards, KD, Fernandez-Pozo, N., Drake-Stowe, K., Humphry M., Evans, AD, Bombarely, A., Allen, F., Hurst, R., White, B., Kernodle, SP, Bromley, JR, Sanchez-Tamburrino, JP, Lewis, RS, and Mueller, LA (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18, 448), we were able to identify scaffolds that mapped to pseudosex chromosomes covering most of this region. Although they could not be located within the pseudosex chromosomes, (Edwards, KD, Fernandez-Pozo, N., Drake-Stowe, K., Humphry M., Evans, AD, Bombarely, A., Allen, F., Hurst, R., White, B., Kernodle, SP, Bromley, JR, Sanchez-Tamburrino, JP, Lewis, RS, and Mueller, LA (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18, 448) Markers that could be uniquely mapped to scaffolds in the genome were integrated based on their location on the genetic map. We then used a genetic map to refine the genetic models of genes within this identified region. (Edwards, KD, Fernandez-Pozo, N., Drake-Stowe, K., Humphry M., Evans, AD, Bombarely, A., Allen, F., Hurst, R., White, B., Kernodle, SP, Bromley, JR, Sanchez-Tamburrino, JP, Lewis, RS, and Mueller, LA (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC We used RNA-seq information from Genomics 18, 448).

[0565] Candidate genes were then selected based on their predicted functions.

[0566] We identified a MYC transcription factor (Nitab4.5_0002539g0040.1) that contains a SNP in its coding region (marker ID Nt2AG2015) resulting in amino acid changes (K87E, where K is wild type) and G84V. F2 individuals classified as containing the FC101 or LAFC53 allele at this marker exhibited clear segregation with respect to nicotine and nornicotine content (Figure 4), indicating that this alteration may be responsible for the low-nicotine phenotype. [Example]

[0567] Physical mapping and candidate gene identification SNP markers found to be strictly genetically linked to the Nic3 locus were identified using the R / QTL lodint function ( Broman & Sen, 2009 ) with an LOD reduction of 1.5. The regions were identified and defined using the improved tobacco genome assembly (Edwards, KD, Fernandez-Pozo, N., Drake-Stowe, K., Humphry M., Evans, AD, Bombarely, A., Allen, F., Hurst, R., White, B., Kernodle, SP, Bromley, JR, Sanchez-Tamburrino, JP, Lewis, RS, and Mueller, LA (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18, 448 Markers within the region of interest surrounding the Nic3 locus were used to identify BioNano hybrid scaffolds that delimit the region (i.e., pseudochromosomal regions) and therefore identify Nic 3 was identified. Gaps in the pseudochromosome sequence were filled with markers that could be uniquely mapped to the genome scaffold but were absent from the BioNano hybrid scaffold, based on their relative placement in the genetic map.

[0568] The candidate gene models in the updated regions were then compared with the RNA-seq data (Edwards, K.D., Fernandez-Pozo, N., Drake-Stowe, K., Humphry, M., Evans, A.D., Bombarely, A., Allen, F., Hurst, R., White, B., Kernodle, SP, Bromley, JR, Sanchez-Tamburrino, JP, Lewis, RS, and Mueller, LA (2017) A reference Genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18, 448) , and corrected as necessary. [Example]

[0569] Identification of candidate genes To identify genes within the Nic3 locus involved in the nicotine regulation observed in FC101, each gene will be silenced individually in a low-nicotine background (i.e., a nic1nic2 background), e.g., by virus-induced gene silencing (VIGS) as described in WO 2020 / 025963, and alkaloid content will be measured. [Example]

[0570] Regulation of candidate gene activity Two approaches are used to confirm that a target amino acid is required for protein function: 1. Gene editing to mutate residues of interest (e.g., G84V and / or K87E in the case of MYC2) in a low-nicotine background (i.e., nic1nic2 background) 2. Overexpression of the non-mutated gene plus the gene-edited variant (e.g., full-length MYC2, MYC2 G84V, MYC2 K87E, and MYC2 G84V K87E).

[0571] Functional domains: To correlate the very low nicotine phenotype with the function of our genes of interest, two approaches are used: 1. Gene editing to delete a functional domain (e.g., the DNA binding site in the case of MYC2) 2. Overexpression of the full-length protein as well as versions containing deletions in functional domains (e.g., full-length MYC2 and the MYC2 delta DNA-binding domain).

[0572] The alkaloid content is measured. [Example]

[0573] Virus-induced gene silencing of genes at the Nic3 locus TRV vectors containing both (TRV RNA1, SEQ ID NO: 570) and (TRV RNA2, SEQ ID NO: 571-574) containing targeted nucleotide sequences (from SEQ ID NO: 73 (Nitab4.5_0002539g0040.2), 118 (Nitab4.5_0002683g0080.2), 124 (Nitab4.5_0005412g0010.2), and 127 (Nitab4.5_0005412g0020.2)) were propagated separately in A. tumefaciens. These cultures were mixed (1:1) and syringe-infiltrated into two-week-old LaBY21 plants with a nic1nic2 background (carrying the ERF199 and ERF189 mutations in the Nic1 and Nic2 genes, respectively, as disclosed in WO 2018 / 237107). The effect of silencing was evaluated by assessing the expression levels of the target genes 5 weeks after virus infection (data not shown).

[0574] The TRV RNA2 sequence is shown in Figures 5 to 8, with gene-specific sequences shown in bold and underlined.

[0575] result Figure 9 shows the nicotine content of 6-week-old LaBY21(nic1nic2) leaves expressing silenced constructs for the indicated genes. Contents are expressed relative to the control and include three biological replicates analyzed by one-way ANOVA. Values ​​are shown as mean ± SEM. Asterisks indicate statistical significance with a P value of ≤ 0.001.

[0576] Silencing of genes at the Nic3 locus leads to a reduction in nicotine content compared to nic1 nic2.

[0577] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry and bioengineering or related fields are intended to be within the scope of the following claims.

[0578] (References) Adams, AC, De Godoy Lusso, MS, Pramod, S., and Xu, D. (2016). Compositions and Methods for Producing Tobacco Plants and Products Having Altered Alkaloid Levels. US patent application 20160374387A1. Bindler, G., Plieske, J., Bakaher, N., Gunduz, I., Ivanov, N., Hoeven, R., Ganal, M., and Donini, P. (2011). A high density genetic map of tobacco (Nicotiana tabacum L.) obtained from large scale microsatellite marker development. Theoretical and Applied Genetics 123, 219-230. Collins, G. B., Legg, P. D., and Kasperba.Mj (1974). Use of Anther-Derived Haploids in Nicotiana .1. Isolation of breeding lines differing in total alkaloid content. Crop Science 14, 77-80. Chakrabarty, R., Banerjee, R., Chung, S. M., Farman, M., Citovsky, V., Hogenhout, S. A., Tzfira, T., and Goodin, M. (2007). PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions. Molecular Plant-Microbe Interactions 20, 740-50. Edwards, K. D., Fernandez-Pozo, N., Drake-Stowe, K., Humphry M., Evans, A. D., Bombarely, A., Allen, F., Hurst, R., White, B., Kernodle, S. P., Bromley, J. R., Sanchez-Tamburrino, J. P., Lewis, R. S., and Mueller, L.A. (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genomics 18, 448. Hibi, N., Higashiguchi, S., Hashimoto, T., and Yamada, Y. (1994). Gene-Expression in Tobacco Low-Nicotine Mutants. Plant Cell 6, 723-735. Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G., and Fraley, R. T. (1985). A simple and general method for transferring genes into plants. Science 227, 1229-1231. Kajikawa, M., Sierro, N., Kawaguchi, H., Bakaher, N., Ivanov, N. I., Hashimoto, T., and Shoji, T. (2017). Genomic insights into the evolution of the nicotine biosynthesis pathway in tobacco. Plant Physiology 174, 999-1011. Kidd, S. K., Melillo, A. A., Lu, R. H., Reed, D. G., Kuno, N., Uchida, K., Furuya, M., and Jelesko, J. G. (2006). The A and B loci in tobacco regulate a network of stress response genes, few of which are associated with nicotine biosynthesi s. Plant Mol Biol 60, 699-716. Langmead, B., and Salzberg, S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-9. Legg P, Chaplin J, Collins G. (1969). Inheritance of percent total alkaloids in Nicotiana tabacum L.: populations derived from crosses of low alkaloid lines with burley and flue-cured varieties. Journal of Heredity 60: 213-217. Legg P., and G., C. (1971). Inheritance of percent total alkaloids in Nicotiana tabacum L. II. genetic effects of two loci in Burley21 X LA Burley 21 populations. Canadian Journal of Genetics and Cytology 13, 287-291. Legg PD, Collins GB, Litton CC. 1970. Registration of La Burley-21 Tobacco Germplasm. Crop Science 10(2): 212. McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M., and DePristo, M. A. (2010). The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Research 20, 1297-303. Neff, M. M., Turk, E., and Kalishman, M. (2002). Web-based primer design for single nucleotide polymorphism analysis. Trends in Genetics 18, 613-5. Nielsen, M. T., Legg, P. D., and Collins, G. B. (1988). Registration of HI and LI burley 21 tobacco germplasms. Crop Science 28, 206-207. Qin, Q., Li, D., Dai, X., Zhao, P., Miller, R., Jack, A., and Yang, S. (2015) Development of user-friendly marker for Nic2 in tobacco. SRC, Tobacco Science Research Conference, 69, abstract 79. Reed, D. G., and Jelesko, J. G. (2004). The A and B loci of Nicotiana tabacum have non-equivalent effects on the mRNA levels of four alkaloid biosynthetic genes. Plant Science 167 1123 - 1130. Rigola, D., van Oeveren, J., Janssen, A., Bonne, A., Schneiders, H., van der Poel, H. J. A., van Orsouw, N. J., Hogers, R. C. J., de Both, M. T. J., and van Eijk, M. J. T. (2009) High-Throughput Detection of Induced Mutations and Natural Variation Using KeyPoint TM Technology. PLOS ONE 4, e4761. https: / / doi.org / 10.1371 / journal.pone.0004761. Rushton PJ, Bokowiec MT, Han SC, Zhang HB, Brannock JF, Chen XF, Laudeman TW, Timko MP. 2008. Tobacco transcription factors: Novel insights into transcriptional regulation in the Solanaceae. Plant Physiology 147: 280-295. Sahoo, D. K., Dey, N., and Maiti, I. B. (2014). pSiM24 is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants. PLoS One 9, e98988. Shoji, T., Kajikawa, M., and Hashimoto, T. (2010). Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. The Plant Cell 22, 3390-3409.Sierro, N., Battey, J. N., Ouadi, S., Bakaher, N., Bovet, L., Willig, A., Goepfert, S., Peitsch, M. C., and Ivanov, N. V. (2014). The tobacco genome sequence and its comparison with those of tomato and potato. Nature Communications 5, 3833.Stam, P. (1993). Construction of integrated genetic linkage maps by means of a new computer package - Joinmap. Plant Journal 3, 739-744. Valleau W. 1949. Breeding low-nicotine tobacco. Journal of Agricultural Research 78: 171-181. Van Ooijen J. W. (2009). MapQTL 6: Software for the mapping of quantitative trait loci in experimental populations of diploid species. Wageningen (The Netherlands): Kyazma BV. Voelckel, C., Krugel, T., Gase, K., Heidrich, N., van Dam, N. M., Winz, R., and Baldwin, I. T. (2001). Anti-sense expression of putrescine N-methyltransferase c onfirms defensive role of nicotine in Nicotiana sylvestris against Manduca sexta. Chemoecology 11, 121-126. Zhu, B., Cai, G., Hall, E. O., and Freeman, G. J. (2007). In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43, 354-9.

Claims

1. 1. A method for modulating (e.g., decreasing) the alkaloid content (e.g., nicotine content) of a tobacco plant or part thereof, or a tobacco plant cell, comprising: a) a functional fragment of the Nic3 gene set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a part thereof, comprising at least 500 consecutive nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or an orthologue of said gene; and optionally, b) at least one Nic1 ERF gene; and / or c) at least one Nic2 ERF gene; The method further comprises a step of modifying the plant or a part or cell thereof by regulating the activity or expression of

2. 1. A method for modulating (e.g., decreasing) the alkaloid content (e.g., nicotine content) of a tobacco plant or part thereof, or a tobacco plant cell, comprising modifying the plant or part thereof or cell by introducing at least one mutation into the Nic3 gene set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a functional fragment of the gene, or an ortholog of the gene, comprising said portion comprising at least 500 contiguous nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, and optionally introducing at least one mutation into the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation into the Nic2 locus (e.g., the Nic2 ERF gene).

3. 1. A method for modulating (e.g., reducing) the content of tobacco-specific nitrosamine (TSNA) precursors in a tobacco plant or part thereof, or in a tobacco plant cell, comprising: i) a) a functional fragment of the Nic3 gene as set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a portion thereof, comprising at least 500 consecutive nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or an orthologue of said gene; and optionally, b) at least one Nic1 ERF gene; and / or c) at least one Nic2 ERF gene or modulating the activity or expression of ii) introducing at least one mutation into the Nic3 gene set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or into a functional fragment of said gene, or an ortholog of said gene, comprising a portion thereof comprising at least 500 contiguous nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, and optionally introducing at least one mutation into the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation into the Nic2 locus (e.g., the Nic2 ERF gene); The method further comprises a step of modifying the plant or a part thereof, or a cell by

4. a) a functional fragment of the Nic3 gene set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a portion thereof, comprising at least 500 consecutive nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or an orthologue of said gene, and optionally at least one Nic1 ERF gene and / or at least one Nic2 ERF gene; or b) at least one mutation in the Nic3 gene as set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a functional fragment of said gene comprising a portion thereof, said portion comprising at least 500 contiguous nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or an orthologue of said gene, and optionally at least one mutation in the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation in the Nic2 locus (e.g., the Nic2 ERF gene); 10. The use of a compound of claim 1, wherein the compound is selected from the group consisting of: a) a tobacco plant or a part thereof; b) a tobacco plant cell; c) a tobacco plant cell; d) a tobacco plant cell;

5. 1. A method for producing a plant or part thereof, tobacco plant cell, tobacco plant propagation material, tobacco leaf, cut and harvested tobacco leaf, processed tobacco leaf, or cut and processed tobacco leaf having a modified (e.g., reduced) alkaloid content (e.g., nicotine content), comprising: i) a) a functional fragment of the Nic3 gene set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a portion thereof, comprising at least 500 consecutive nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or an orthologue of said gene; and optionally, b) at least one Nic1 ERF gene; and / or c) to modulate the activity or expression of at least one Nic2 ERF gene; or ii) to introduce at least one mutation in the Nic3 gene set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a functional fragment of said gene or an orthologue of said gene comprising a portion thereof comprising at least 500 consecutive nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, and optionally at least one mutation in the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation in the Nic2 locus (e.g., the Nic2 ERF gene); The method further comprising the step of modifying the tobacco plant or part thereof or tobacco cell.

6. 6. The method or use of any one of claims 1 to 5, wherein the nicotine content is reduced compared to a tobacco plant or part thereof or tobacco cell that has not been modified to introduce at least one mutation into the Nic3 gene as set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a functional fragment of said gene, or an ortholog of said gene, comprising at least 500 contiguous nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, and optionally at least one mutation in the Nic1 locus and / or at least one mutation in the Nic2 locus.

7. 1. A tobacco plant or part thereof, or tobacco cell, modified to achieve a reduced alkaloid content (e.g., nicotine content) compared to an unmodified tobacco plant or part thereof, or tobacco cell, wherein the modification comprises: i) a) a functional fragment of the Nic3 gene as set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a portion thereof, comprising at least 500 consecutive nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or an orthologue of said gene; and optionally, b) at least one Nic1 ERF gene; and / or c) at least one Nic2 ERF gene or modulated activity or expression of ii) at least one mutation in the Nic3 gene as set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a functional fragment of said gene comprising a portion thereof comprising at least 500 contiguous nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or an ortholog of said gene, and optionally at least one mutation in the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation in the Nic2 locus (e.g., the Nic2 ERF gene); The tobacco plant or part thereof, or tobacco cell, comprising:

8. 8. Tobacco plant propagation material obtainable from a tobacco plant or part thereof or tobacco cell according to claim 7, or from a tobacco plant or part thereof or tobacco cell produced by the method of any of claims 5 or 6.

9. a) the activity or expression of a Nic1 ERF gene selected from those listed in Table 1 is modulated; or the at least one mutation in the Nic1 locus is in a Nic1 ERF gene selected from SEQ ID NO:5; or SEQ ID NO:9; or SEQ ID NO:13; or SEQ ID NO:17; or SEQ ID NO:21; or SEQ ID NO:25; or SEQ ID NO:29; or a sequence having at least 90% sequence identity thereto; or a functional fragment of said gene comprising a portion thereof comprising at least 500 contiguous nucleotides of SEQ ID NO:5, 9, 13, 17, 21, 25, or 29, or a sequence having at least 90% sequence identity thereto, or an ortholog of said gene; and / or b) the activity or expression of a Nic2 ERF gene selected from those listed in Table 2 is modulated; or said at least one mutation in the Nic2 locus is in a Nic2 ERF gene selected from SEQ ID NO:69; SEQ ID NO:37; or SEQ ID NO:41; or SEQ ID NO:45; or SEQ ID NO:49; or SEQ ID NO:53; or SEQ ID NO:57; or SEQ ID NO:61; or SEQ ID NO:65; or a sequence having at least 90% sequence identity thereto; or a functional fragment of said gene, or an ortholog of said gene, comprising said portion thereof comprising at least 500 contiguous nucleotides of SEQ ID NO:69, 37, 41, 45, 49, 53, 57, 61 or 65, or a sequence having at least 90% sequence identity thereto; The method or use according to any one of claims 1 to 6, the plant or part or cell thereof according to claim 7, or the plant propagation material according to claim 8.

10. i) the activity or expression of SEQ ID NO: 5 is modulated; or said at least one mutation in the Nic1 locus is in SEQ ID NO: 5; and / or ii) the activity or expression of SEQ ID NO: 69 is modulated; or the at least one mutation in the Nic2 locus is in SEQ ID NO: 69; A method or use according to any one of claims 1 to 6 and 9, a plant or part thereof according to claim 7 or 9, or a plant propagation material according to claim 8 or 9.

11. 11. The method or use according to any one of claims 1 to 6, 9 and 10, the plant or part thereof according to claim 7 or 9, or the plant propagation material according to claims 8 to 10, wherein at least one mutation is in the Nic3 gene as set forth in SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or in a functional fragment of the gene, or an ortholog of the gene, comprising at least 500 contiguous nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, and results in a mutation in amino acid residues 120 to 584 of SEQ ID NO: 120, or a sequence having at least 90% sequence identity thereto, or of a functional fragment or ortholog of the polypeptide.

12. 12. Use of a plant or part thereof according to any one of claims 7 and 9 to 11, or a plant produced by the method according to any one of claims 5 or 6, or 9 to 11, for plant breeding.

13. 12. Use of a plant or part thereof according to any one of claims 7 and 9 to 11, or a plant produced by the method according to any one of claims 5 or 6, or 9 to 11, for the production of a product.

14. 12. Use of a plant or part thereof according to any one of claims 7 and 9 to 11, or a plant produced by the method according to any one of claims 5 or 6, or 9 to 11, for growing crops.

15. Use of a plant or part thereof according to any one of claims 7 and 9 to 11, or a plant produced by the method according to any one of claims 5 and 6 and 9 to 11, for producing leaves.

16. 12. Harvested leaves from a plant according to any one of claims 7 and 9 to 11, or harvested leaves obtainable from a plant propagated from the propagation material according to any one of claims 7 to 11, or obtainable from a plant obtained by the use according to any one of claims 4, 6 and 9 to 15, or obtainable from a plant produced by the method according to any one of claims 5 and 6 and 9 to 15.

17. 17. Harvested leaves of the plant of claim 16, which are cut and harvested leaves.

18. obtainable (e.g. obtained) from a plant obtainable from the use according to any one of claims 4, 6 and 9 to 15; It can be obtained (e.g., obtained) by processing a plant according to any one of claims 7 and 9 to 11. obtainable (e.g. obtained) from a plant propagated from the plant propagation material of any one of claims 8 to 11; or obtainable (e.g. obtained) by processing harvested leaves of a plant according to claim 16 or 17; or obtainable (e.g. obtained) from a plant produced by the method of any one of claims 5, 6 and 9 to 11; Processed leaf, preferably processed tobacco leaf, preferably non-viable processed tobacco leaf.

19. 19. The processed leaves of claim 18, wherein the leaves have been processed by drying, fermenting, pasteurizing or a combination thereof, preferably to reduce the content of one or more TSNAs selected from N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N'-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB), preferably to modulate (e.g. reduce) the content of NNN and / or NNK, more preferably to reduce the content of NNN.

20. 20. The processed leaf of claim 18 or 19, which is a cut and processed leaf.

21. obtainable (e.g. obtained) from a plant obtainable from the use according to any one of claims 4, 6 and 9 to 15; It can be obtained (e.g., obtained) by processing a plant according to any one of claims 7 and 9 to 11. obtainable (e.g. obtained) from a plant propagated from the plant propagation material of any one of claims 8 to 11; or obtainable (e.g. obtained) by processing harvested leaves of a plant according to claim 16 or 17; or It can be obtained from a plant produced by the method according to any one of claims 5, 6 and 9 to 11. Cured tobacco material made from plants or parts thereof.

22. 22. A tobacco blend comprising the dry tobacco material of claim 21.

23. A tobacco plant according to any one of claims 7 and 9 to 11 or a part thereof according to claims 7 and 9 to 11, A tobacco plant or part thereof propagated from the tobacco plant propagation material according to any one of claims 8 to 11. Harvested leaves of the plant according to claim 16 or 17.

20. The processed leaves according to claim 18 or 19. or A plant produced by the method of any one of claims 5, 6 and 9 to 11. A delivery system prepared from

24. 24. The delivery system of claim 23, which is a combustible smoking article.

25. 24. The delivery system of claim 23, which is a smokeless delivery system.

26. 26. The delivery system of claim 25, which is a non-combustible aerosol delivery system, such as a tobacco heating device or an aerosol generating device.

27. A combustible smoking article, a non-combustible aerosol delivery system, a smokeless delivery system, or a tobacco heating device, comprising the plant or part thereof or an extract thereof (e.g., a tobacco extract) described in any one of claims 7 to 11, or the dried tobacco material described in claim 21, or the tobacco blend described in claim 22.

28. Use of the nucleotide sequence of the Nic3 gene as set forth in SEQ ID NO: 118, or a sequence having at least 90% sequence identity thereto, or a functional fragment of said gene comprising at least 500 contiguous nucleotides of SEQ ID NO: 118 or a sequence having at least 90% sequence identity thereto, or a portion thereof, or an ortholog of said gene, and optionally the nucleotide sequence of the Nic1 locus (e.g., the Nic1 ERF gene) and / or the Nic2 locus (e.g., the Nic2 ERF gene), to select plants having reduced alkaloid content (e.g., nicotine content) and / or reduced content of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs.

29. 1. A plant mutant having at least one genetic variation in the Nic3 gene set forth in SEQ ID NO:118, or a sequence having at least 90% sequence identity thereto, or a functional fragment of said gene, or an ortholog of said gene, comprising at least 500 contiguous nucleotides of SEQ ID NO:118 or a sequence having at least 90% sequence identity thereto, and optionally at least one genetic variation in the Nic1 locus (e.g., in the Nic1 ERF gene) and / or at least one genetic variation in the Nic2 locus (e.g., in the Nic2 ERF gene), wherein the genetic variation reduces alkaloid content (e.g., nicotine content) and / or reduces the content of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs in the mutant tobacco plant compared to a comparable plant not having the genetic variation.

30. 30. Progeny or seeds of a mutant plant having the genetic mutation of claim 29.

31. 1. Harvested leaves, processed leaves, or cured tobacco material produced from a plant comprising at least one mutation in the Nic3 gene as set forth in SEQ ID NO:118, or a sequence having at least 90% sequence identity thereto, or a functional fragment of said gene, or an ortholog of said gene, comprising at least 500 consecutive nucleotides of SEQ ID NO:118 or a sequence having at least 90% sequence identity thereto, wherein said portion thereof comprises at least 500 consecutive nucleotides of SEQ ID NO:118 or a sequence having at least 90% sequence identity thereto, and optionally at least one mutation in the Nic1 locus (e.g., the Nic1 ERF gene) and / or at least one mutation in the Nic2 locus (e.g., the Nic2 ERF gene), wherein said plant has a reduced nicotine content and / or a reduced content of tobacco-specific nitrosamines (TSNAs) or precursors of TSNAs compared to a comparable plant that does not have said mutations in the Nic3 locus and, optionally, the Nic1 locus and / or the Nic2 locus.

Images