WO2023230433A1 - Procédés et compositions pour la régulation des alcaloïdes dans le domaine du tabac - Google Patents

Procédés et compositions pour la régulation des alcaloïdes dans le domaine du tabac Download PDF

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WO2023230433A1
WO2023230433A1 PCT/US2023/067281 US2023067281W WO2023230433A1 WO 2023230433 A1 WO2023230433 A1 WO 2023230433A1 US 2023067281 W US2023067281 W US 2023067281W WO 2023230433 A1 WO2023230433 A1 WO 2023230433A1
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acid sequence
nucleic acid
tobacco
tobacco plant
seq
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PCT/US2023/067281
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English (en)
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Taejin Kim
Chengalrayan Kudithipudi
Yanxin Shen
Dongmei Xu
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Altria Client Services Llc
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Publication of WO2023230433A1 publication Critical patent/WO2023230433A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine

Definitions

  • this disclosure provides a method for producing a modified tobacco plant comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, where the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, where the recombinant DNA construct is not present in the endogenous nucleic acid sequence in a control tobacco plant of the same variety; and (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, where the at least one tobacco seed or plant germinated therefrom comprises the
  • Any tobacco plant, or part thereof, provided herein is specifically envisioned for use with any method provided herein.
  • any modified tobacco plant, or part thereof, is specifically envisioned for use with any method provided herein.
  • Any nucleic acid sequence, amino acid sequence, or other composition provided herein is specifically envisioned for use with any method provided herein.
  • Tobacco plants typically produce alkaloids at levels between 2% and 4% of their total dry weight. Nicotine is a major alkaloid compound in tobacco plants, and it often accounts for approximately 95% of the total alkaloid content of the plants. The remaining pool of alkaloids primarily comprises other structurally-related alkaloids such as anabasine, anatabine, and nornicotine.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in at least four different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-6. In another aspect, this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in at least five different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-6. In another aspect, this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in each of SEQ ID NOs: 1-6.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in each of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, where the first endogenous nucleic acid sequence is at least 90% identical to SEQ ID NO: 2, and where the second endogenous nucleic acid sequence is at least 90% identical to SEQ ID NO: 3.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in at least two different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in at least three different endogenous nucleic acid sequences selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in at least four endogenous nucleic acid sequences, where the at least four endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in at least five endogenous nucleic acid sequences, where the at least five endogenous nucleic acid sequences encode different polypeptides comprising an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence, where the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence at least 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence at least 99% identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence, wherein the endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, wherein the modified tobacco plant comprises at least one non-natural mutation in a first endogenous nucleic acid sequence, and at least one non-natural mutation in a second endogenous nucleic acid sequence, wherein the first endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, wherein the second endogenous nucleic acid sequence encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, and wherein the first and second end
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a first endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and a second endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, and where the first and second endogenous nucleic acid sequences are not identical to each other.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a first endogenous nucleic acid sequence encoding a polypeptide at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and a second endogenous nucleic acid sequence encoding a polypeptide at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, and where the first and second endogenous nucleic acid sequences are not identical to each other.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a first endogenous nucleic acid sequence encoding a polypeptide at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 and a second endogenous nucleic acid sequence encoding a polypeptide at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, and where the first and second endogenous nucleic acid sequences are not identical to each other.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first and second endogenous nucleic acid sequences are each at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 7-12, and where the first and second endogenous nucleic acid sequences are not identical to each other.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, wherein the first and second endogenous nucleic acid sequences are each at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-6, and where the first and second endogenous nucleic acid sequences are not identical to each other.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, where the first endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 2 and where the second endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 3.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, where the first endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 2 and where the second endogenous nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 3.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid encoding at least one small RNA molecule capable of binding to and reducing the expression 1 of a first endogenous nucleic acid sequence and a second endogenous nucleic acid sequence, where the first endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 7 and where the second endogenous nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 12.
  • a nucleic acid molecule encoding at least one small RNA molecule comprises a sequence at least 80% identical to one of SEQ ID NOs: 32 and 33. In an aspect, a nucleic acid molecule encoding at least one small RNA molecule comprises a sequence at least 85% identical to one of SEQ ID NOs: 32 and 33. In an aspect, a nucleic acid molecule encoding at least one small RNA molecule comprises a sequence at least 90% identical to one of SEQ ID NOs: 32 and 33. In an aspect, a nucleic acid molecule encoding at least one small RNA molecule comprises a sequence at least 95% identical to one of SEQ ID NOs: 32 and 33.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising a recombinant DNA construct comprising a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence that modulates the expression or activity of a gene, where the gene encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising at least one non-natural mutation in an endogenous nucleic acid sequence that modulates the expression or activity of a gene, where the gene encodes an RNA sequence at least 80% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising (a) a genetic modification in a gene; or (b) a genetic modification targeting the gene; where the genetic modification downregulates the expression or activity of the gene, where the gene encodes an amino acid sequence having at least 80% identity or similarity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • this disclosure provides a modified tobacco plant, or part thereof, comprising (a) a genetic modification in a gene; or (b) a genetic modification targeting the gene; where the genetic modification downregulates the expression or activity of the gene, where the gene encodes a nucleic acid sequence having at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • modified in the context of a plant, refers to a plant comprising a genetic alteration introduced for certain purposes and beyond natural polymorphisms.
  • a modified plant can comprise a non-natural mutation or a recombinant DNA construct.
  • a modified tobacco plant comprises a non-natural mutation.
  • a modified tobacco plant comprises a recombinant DNA construct.
  • a modified tobacco plant comprises a genetic modification.
  • a “mutation” refers to an inheritable genetic modification introduced into a gene to alter the expression or activity of a product encoded by a reference sequence of the gene.
  • a tobacco plant, or part thereof is homozygous for at least one non- natural mutation. In another aspect, a tobacco plant, or part thereof, is heterozygous for at least one non-natural mutation. In another aspect, a tobacco plant, or part thereof, is homozygous for an introduced recombinant DNA construct. In another aspect, a tobacco plant, or part thereof, is hemizygous for an introduced recombinant DNA construction. In a further aspect, a tobacco plant, or part thereof, is heterozygous for an introduced recombinant DNA construct.
  • a dominant negative allele abrogates or reduces the normal function of an allele in a heterozygous or homozygous state.
  • Dominant positive alleles can increase normal gene function (e.g., a hypermorph) or provide new functions for a gene (e.g., a neomorph).
  • a semi-dominant allele occurs when penetrance of a linked phenotype in individuals heterozygous for the allele is less than that which is observed in individuals homozygous for the allele.
  • a mutation provided herein creates a dominant negative allele of the mutated locus. In another aspect, a mutation provided herein creates a dominant positive allele of a mutated locus.
  • inducing a mutation comprises the use of a chemical mutagen.
  • a chemical mutagen comprises ethyl methanesulfonate (EMS).
  • inducing a mutation comprises the use of a transposon. In another aspect, inducing a mutation comprises the use of Agrobacterium. [0076] In a further aspect, inducing a mutation comprises the use of a nuclease.
  • inducing a mutation comprises the use of a CRISPR/CasX nuclease. In an aspect, inducing a mutation comprises the use of a CRISPR/CasY nuclease. In an aspect, inducing a mutation comprises the use of a Csml nuclease.
  • inducing a mutation comprises the use of a base editor.
  • a “base editor” refers to a catalytically impaired Cas nuclease fused to a nucleotide deaminase.
  • base editors further comprise DNA repair proteins.
  • a base editor is a cytosine base editor.
  • a cytosine base editor enables C-G to T-A transitions.
  • a base editor is an adenine base editor.
  • An adenine base editor enables A-T to G-C conversion.
  • a base editor is a C-to-G base editor.
  • a “substitution” refers to the replacement of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence.
  • a mutation comprises an inversion.
  • An “inversion” refers to when a segment of a polynucleotide or amino acid sequence is reversed end- to-end.
  • a “duplication” refers to when a segment of a polynucleotide or amino acid sequence is repeated. The repeated segment can immediately follow the original segment, or it can be separated from the original segment by one or more nucleotides or amino acids.
  • a mutation provided herein comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, a duplication, and an inversion.
  • a non-natural mutation comprises a mutation selected from the group consisting of a substitution, a deletion, an insertion, a duplication, and an inversion of one or more nucleotides relative to an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • a “frameshift mutation” refers to an insertion or deletion to a nucleic acid sequence that shifts the frame for translating the nucleic acid sequence to an amino acid sequence.
  • a “splice-site mutation” refers to a mutation in a nucleic acid sequence that causes an intron to be retained for protein translation, or, alternatively, for an exon to be excluded from protein translation. Splice-site mutations can cause nonsense, missense, or frameshift mutations. [0084] Mutations in coding regions of genes (e.g., exonic mutations) can result in a truncated protein or polypeptide when a mutated messenger RNA (mRNA) is translated into a protein or polypeptide.
  • mRNA messenger RNA
  • a premature stop codon refers to a nucleotide triplet within an mRNA transcript that signals a termination of protein translation.
  • a “premature stop codon” refers to a stop codon positioned earlier (e.g., on the 5 ’-side) than the normal stop codon position in an endogenous mRNA transcript.
  • several stop codons are known in the art, including “UAG,” “UAA,” “UGA,” “TAG,” “TAA,” and “TGA.”
  • a non-natural mutation comprises a mutation in a sequence region selected from the group consisting of a promoter, a 5'-UTR, a 3'- UTR, an exon, an intron, and a terminator.
  • an “endogenous” nucleic acid sequence refers to a nucleic acid sequence that occurs naturally in the genome of an organism. Endogenous nucleic acid sequences do not include heterologous sequences inserted into a genome via deliberate human intervention. Similarly, endogenous amino acid sequences are sequences that exist naturally via translation of an endogenous nucleic acid molecule. In an aspect, a nucleic acid sequence provided herein is an endogenous nucleic acid sequence.
  • heterologous refers to a sequence (nucleic acid or amino acid) that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • the term also is applicable to nucleic acid constructs, also referred to herein as “polynucleotide constructs” or “nucleotide constructs.”
  • a “heterologous” nucleic acid construct is intended to mean a construct that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • percent identity or “percent identical” as used herein in reference to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a window of comparison (the “alignable” region or regions), (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity.
  • two aliphatic (e.g., glycine, alanine, valine, leucine, isoleucine) amino acid residues can be substituted for each other in a conservative substitution;
  • two hydroxyl (e.g., serine, cysteine, threonine, methionine) amino acid residues can be substituted for each other in a conservative substitution;
  • two aromatic (e.g., phenylalanine, tyrosine, tryptophan) amino acid residues can be substituted for each other in a conservative substitution;
  • two basic (e.g., histidine, lysine, arginine) amino acid residues can be substituted for each other in a conservative substitution; and
  • two acid (e.g., aspartate, glutamate, asparagine, glutamine) amino acid residues can be substituted for each other in a conservative substitution.
  • the alignment and percent identity between two sequences can be as determined by the ClustalW algorithm, see, e.g., Chenna etal., “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acids Research 31: 3497-3500 (2003); Thompson et al., “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22: 4673-4680 (1994); Larkin MA et al., “Clustal W and Clustal X version 2.0,” Bioinformatics 23: 2947-48 (2007); and Altschul etal. "Basic local alignment search tool.” J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.
  • the “percent complementarity” can be calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to yield the percent complementarity of the two sequences.
  • Optimal base pairing of two sequences can be determined based on the known pairings of nucleotide bases, such as G- C, A-T, and A-U, through hydrogen binding.
  • polynucleotide or “nucleic acid molecule” is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • polynucleotides and nucleic acid molecules can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides.
  • deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues.
  • a “recombinant nucleic acid” refers to a nucleic acid molecule formed by laboratory methods of genetic recombination, such as, without being limiting, molecular cloning.
  • a “recombinant DNA construct” refers to a DNA molecule formed by laboratory methods of genetic recombination.
  • Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography.
  • a polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector.
  • a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a nucleic acid sequence provided herein is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 75% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 80% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 85% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30.
  • a nucleic acid sequence provided herein is at least 88% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 90% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 91% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 92% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30.
  • a nucleic acid sequence provided herein is at least 93% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 94% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 95% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 96% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30.
  • a nucleic acid sequence provided herein is at least 97% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 98% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is at least 99% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19- 30. In another aspect, a nucleic acid sequence provided herein is 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12 and 19-30.
  • an endogenous nucleic acid sequence provided herein is at least 70% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, an endogenous nucleic acid sequence provided herein is at least 75% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, an endogenous nucleic acid sequence provided herein is at least 80% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, an endogenous nucleic acid sequence provided herein is at least 85% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, an endogenous nucleic acid sequence provided herein is at least 88% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to
  • an endogenous nucleic acid sequence provided herein is at least 90% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, an endogenous nucleic acid sequence provided herein is at least 91% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, an endogenous nucleic acid sequence provided herein is at least 92% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, an endogenous nucleic acid sequence provided herein is at least 93% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • an endogenous nucleic acid sequence provided herein is at least 98% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, an endogenous nucleic acid sequence provided herein is at least 99% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, an endogenous nucleic acid sequence provided herein is 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • Detection can be accomplished using detectable labels.
  • label is intended to encompass the use of direct labels as well as indirect labels.
  • Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • an amino acid sequence provided herein is at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, an amino acid sequence provided herein is at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, an amino acid sequence provided herein is at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, an amino acid sequence provided herein is at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • an amino acid sequence provided herein is at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, an amino acid sequence provided herein is at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, an amino acid sequence provided herein is at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, an amino acid sequence provided herein is at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • an amino acid sequence provided herein is at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, an amino acid sequence provided herein is at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, an amino acid sequence provided herein is at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In another aspect, an amino acid sequence provided herein is 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 88% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 90% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence at least 99% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, an endogenous nucleic acid sequence provided herein encodes a polypeptide comprising an amino acid sequence 100% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • promoter refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site, and/or a TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene).
  • a promoter can be synthetically produced, varied, or derived from a known or naturally occurring promoter sequence or other promoter sequence.
  • a promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences.
  • a promoter of the present application can thus include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein.
  • a constitutive promoter is selected from the group consisting of a Cauliflower Mosaic Virus 35S promoter, a ubiquitin promoter, an actin promoter, an opine promoter, and an alcohol dehydrogenase promoter.
  • Promoters that drive expression during certain periods or stages of development are referred to as “developmental” promoters.
  • a root tissue-preferred promoter is a cassava vein mosaic virus (CsVMV) promoter.
  • CsVMV cassava vein mosaic virus
  • An “inducible” promoter is a promoter that initiates transcription in response to an environmental stimulus such as heat, cold, drought, light, or other stimuli, such as wounding or chemical application.
  • this disclosure provides a heterologous promoter. In another aspect, this disclosure provides a promoter that is operably linked to a heterologous polynucleotide. In another aspect, this disclosure provides a polynucleotide sequence that is operably linked to a heterologous promoter.
  • operably linked refers to a functional linkage between two or more elements.
  • an operable linkage between a polynucleotide of interest and a regulatory sequence is a functional link that allows for expression of the polynucleotide of interest.
  • Operably linked elements may be contiguous or non-contiguous.
  • a promoter provided herein is operably linked to a heterologous nucleic acid molecule.
  • a nucleic acid molecule provided herein is a small RNA molecule.
  • a nucleic acid molecule encodes a small RNA molecule.
  • a “small RNA molecule” refers to a non-coding RNA molecule of between 16 nucleotides and 50 nucleotides in length. In an aspect, a small RNA molecule comprises between 16 nucleotides and 40 nucleotides. In another aspect, a small RNA molecule comprises between 16 nucleotides and 30 nucleotides. In another aspect, a small RNA molecule comprises between 18 nucleotides and 50 nucleotides. In another aspect, a small RNA molecule comprises between 18 nucleotides and 40 nucleotides. In another aspect, a small RNA molecule comprises between 18 nucleotides and 30 nucleotides.
  • a small RNA molecule comprises between 18 nucleotides and 25 nucleotides. In another aspect, a small RNA molecule comprises between 20 nucleotides and 28 nucleotides. In another aspect, a small RNA molecule comprises between 20 nucleotides and 24 nucleotides. In another aspect, a small RNA molecule comprises between 21 nucleotides and 23 nucleotides. In another aspect, a small RNA molecule comprises 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
  • a small RNA molecule is selected from the group consisting of a double-stranded RNA, a small interfering RNA (siRNA), a /raw.s-acting siRNA, and a microRNA (miRNA).
  • this disclosure also provides guide RNAs (gRNAs) and prime editing gRNAs that bind to any one of SEQ ID NOs: 1 to 12.
  • miRNAs are generally of between about 19 to about 25 nucleotides (commonly about 20-24 nucleotides in plants), that guide cleavage in trans of target transcripts, negatively regulating the expression of genes involved in various regulation and development pathways. In some cases, miRNAs serve to guide in-phase processing of siRNA primary transcripts.
  • a small RNA molecule comprises 100% complementarity with a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In an aspect, a small RNA molecule comprises at least 95% complementarity over 21 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In an aspect, a small RNA molecule comprises at least 90% complementarity over 21 consecutive nucleotides of a nucleic acid molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • MIR genes have been reported to occur in intergenic regions, both isolated and in clusters in the genome, but can also be located entirely or partially within introns of other genes (both protein-coding and non-protein-coding). For a review of miRNA biogenesis, see Kim (2005) Nature Rev. Mol. Cell. Biol., 6:376-385. Transcription of MIR genes can be, at least in some cases, under promotional control of a MIR gene's own promoter.
  • the primary transcript termed a “pri- miRNA” can be quite large (several kilobases) and can be polycistronic, containing one or more pre-miRNAs (fold-back structures containing a stem-loop arrangement that is processed to the mature miRNA) as well as the usual 5' "cap” and polyadenylated tail of an mRNA.
  • Maturation of a mature miRNA from its corresponding precursors differs significantly between animals and plants.
  • pri-miRNAs and pre-miRNAs are believed to be largely processed to the mature miRNA entirely in the nucleus
  • the pri-miRNA transcript is processed in the nucleus by the animal-specific enzyme Drosha, followed by export of the pre-miRNA to the cytoplasm where it is further processed to the mature miRNA.
  • Mature miRNAs in plants are typically 21 nucleotides in length.
  • Transgenic expression of miRNAs can be employed to regulate expression of the miRNA's target gene or genes. Inclusion of a miRNA recognition site in a transgenically expressed transcript is also useful in regulating expression of the transcript. Recognition sites of miRNAs have been validated in all regions of an mRNA, including the 5' untranslated region, coding region, and 3' untranslated region, indicating that the position of the miRNA target site relative to the coding sequence may not necessarily affect suppression. Because miRNAs are important regulatory elements in eukaryotes, transgenic suppression of miRNAs is useful for manipulating biological pathways and responses.
  • promoters of MIR genes can have very specific expression patterns (e.g., cellspecific, tissue-specific, temporally specific, or inducible), and thus are useful in recombinant constructs to induce such specific transcription of a DNA sequence to which they are operably linked.
  • miRNAs their precursors, their recognition sites, and their promoters are described in detail in U.S. Patent Application Publication 2006/0200878 Al, incorporated by reference herein.
  • Non-limiting examples of these utilities include: (1) the expression of a native miRNA or miRNA precursor sequence to suppress a target gene; (2) the expression of an artificial miRNA or miRNA precursor sequence to suppress a target gene; (3) expression of a transgene with a miRNA recognition site, where the transgene is suppressed when the mature miRNA is expressed; (4) expression of a transgene driven by a miRNA promoter.
  • Designing an artificial miRNA sequence can be as simple as substituting sequence that is complementary to the intended target for nucleotides in the miRNA stem region of the miRNA precursor, as demonstrated by Zeng et al. (2002) Mol. Cell, 9:1327-1333.
  • One nonlimiting example of a general method for determining nucleotide changes in the native miRNA sequence to produce the engineered miRNA precursor includes the following steps: (a) Selecting a unique target sequence of at least 18 nucleotides specific to the target gene, e.g., by using sequence alignment tools such as BLAST (see, for example, Altschul et al. (1990) J. Mol. Biol., 215:403-410; Altschul et al.
  • 19-mers are selected that have all or most of the following characteristics: (1) a Reynolds score>4, (2) a GC content between about 40% to about 60%, (3) a negative AAG, (4) a terminal adenosine, (5) lack of a consecutive run of 4 or more of the same nucleotide; (6) a location near the 3' terminus of the target gene; (7) minimal differences from the miRNA precursor transcript.
  • the additional nucleotide at position 20 is preferably matched to the selected target sequence, and the nucleotide at position 21 is preferably chosen to either be unpaired to prevent spreading of silencing on the target transcript or paired to the target sequence to promote spreading of silencing on the target transcript; and (d) transforming the artificial miRNA into a plant.
  • RNAi knockdown of a candidate gene e.g., via the use of an artificial miRNA or an siRNA
  • a mutation e.g., missense or nonsense mutations
  • an RNAi knockdown of a candidate gene can both cause reduction of expression and/or decreased protein activity and can cause identical or similar phenotypes in plants. See, for example, Agrawal et al., Microbiology and Molecular Biology Reviews, 67:657-685 (2003).
  • a small RNA provided herein comprises at nucleic acid sequence at least 75% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 80% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 85% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • a small RNA provided herein comprises at nucleic acid sequence at least 90% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 95% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 96% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • a small RNA provided herein comprises at nucleic acid sequence at least 97% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 98% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 99% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence 100% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • a small RNA provided herein comprises at nucleic acid sequence at least 90% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 95% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 96% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • a small RNA provided herein comprises at nucleic acid sequence at least 97% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 98% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7- 12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence at least 99% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12. In an aspect, a small RNA provided herein comprises at nucleic acid sequence 100% identical or complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • a small RNA provided herein comprises a nucleic acid sequence at least 88.7% identical or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • a small RNA provided herein comprises a nucleic acid sequence at least 94.3% identical or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • a small RNA provided herein comprises a nucleic acid sequence 100% identical or complementary to at least 18 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, a small RNA provided herein comprises a nucleic acid sequence at least 85% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, a small RNA provided herein comprises a nucleic acid sequence at least 90% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • a small RNA provided herein comprises a nucleic acid sequence at least 95% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12. In another aspect, a small RNA provided herein comprises a nucleic acid sequence 100% identical or complementary to at least 20 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 70% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 75% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 85% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 91% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 92% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 93% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 94% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 95% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 96% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 97% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence encoding a polypeptide at least 98% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 70% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 75% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 88% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 90% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 91% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 92% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 93% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 94% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom.
  • a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 95% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom. In an aspect, a small RNA molecule provided herein is capable of binding to and reducing the expression of a nucleic acid sequence at least 96% identical to nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or an RNA transcribed therefrom.
  • a first nucleic acid molecule that is capable of binding to a second nucleic acid molecule binds to the second nucleic acid molecule.
  • a first nucleic acid molecule can “hybridize” a second nucleic acid molecule via non-covalent interactions (e.g., Watson-Crick base-pairing) in a sequence-specific, antiparallel manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength.
  • standard Watson-Crick basepairing includes: adenine pairing with thymine, adenine pairing with uracil, and guanine (G) pairing with cytosine (C) [DNA, RNA],
  • G guanine
  • C cytosine
  • G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA.
  • a guanine of a protein-binding segment (dsRNA duplex) of a subject DNA-targ eting RNA molecule is considered complementary to an uracil, and vice versa.
  • dsRNA duplex protein-binding segment of a subject DNA- targeting RNA molecule
  • the position is not considered to be non- complementary, but is instead considered to be complementary.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible.
  • the conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of complementation between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Lor hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or fewer nucleotides) the position of mismatches becomes important (see Sambrook el al.). Typically, the length for a hybridizable nucleic acid is at least about 10 nucleotides.
  • Illustrative minimum lengths for a hybridizable nucleic acid are: at least about 15 nucleotides; at least about 20 nucleotides; at least about 22 nucleotides; at least about 25 nucleotides; and at least about 30 nucleotides). Lurthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.
  • sequence of polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure).
  • an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity.
  • the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides.
  • Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined routinely using BLAST® programs (basic local alignment search tools) and PowerBLAST programs known in the art (see Altschul etal., J. Mol.
  • a small RNA molecule reduces the expression of any nucleic acid sequence to which it is capable of binding.
  • a non-natural mutation provided herein reduces the expression of the mutated nucleic acid sequence as compared to the non-mutated nucleic acid sequence in a control plant grown under comparable conditions.
  • Reduced expression of an endogenous nucleic acid sequence can be measured using any suitable method known in the art.
  • Non-limiting examples of measuring expression include quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), RNA blot (e.g., a Northern blot), RNA sequencing. Differences in expression can be described as an absolute quantification or a relative quantification. See, for example, Livak and Schmittgen, Methods, 25:402-408 (2001). If an endogenous nucleic acid sequence encodes a protein, changes in expression can be inferred by examining the accumulation of the encoded protein.
  • Non-limiting examples of measuring protein accumulation include Western blots and enzyme-linked immunosorbent assays (ELISAs).
  • a reduction in expression is measured using qRT-PCR. In another aspect, a reduction in expression is measured using an RNA blot. In another aspect, a reduction in expression is measured using RNA sequencing. In a further aspect, a reduction in expression is measured using a Western blot. In yet a further aspect, a reduction in expression is measured using an ELISA.
  • a non-natural mutation in a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12 results in a reduced level of expression of the nucleic acid sequence as compared to the nucleic acid sequence lacking the non-natural mutation in a control plant grown under comparable conditions.
  • a non-natural mutation in a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 results in a reduced level of expression of the nucleic acid sequence as compared to the nucleic acid sequence lacking the non-natural mutation in a control plant grown under comparable conditions.
  • a reduction in expression comprises a reduction of at least 1% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 5% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 10% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 25% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 50% as compared to expression in the same tissue of a control plant grown under comparable conditions.
  • a reduction in expression comprises a reduction of at least 75% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 90% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of at least 95% as compared to expression in the same tissue of a control plant grown under comparable conditions.
  • a reduction in expression comprises a reduction of between 1% and 25% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 25% and 90% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 50% and 90% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, a reduction in expression comprises a reduction of between 25% and 75% as compared to expression in the same tissue of a control plant grown under comparable conditions. [00144] In an aspect, a reduction in expression comprises a statistically significant reduction as compared to expression in the same tissue of a control plant grown under comparable conditions.
  • a non-natural mutation results in a reduced level of activity by a protein or polypeptide encoded by a nucleic acid sequence provided herein as compared to the activity of a control plant grown under comparable conditions.
  • a non-natural mutation in an endogenous nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12 reduces the level of activity by a protein or polypeptide encoded by the nucleic acid sequence as compared to activity of a protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco plant when grown under comparable conditions, where the nucleic acid sequence lacks the non-natural mutation in the control tobacco plant.
  • a non-natural mutation in an endogenous nucleic acid sequence where the endogenous nucleic acid sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18 reduces the level of activity by a protein or polypeptide encoded by the nucleic acid sequence as compared to activity of a protein or polypeptide encoded by the endogenous nucleic acid sequence in a control tobacco plant when grown under comparable conditions, where the nucleic acid sequence lacks the non-natural mutation in the control tobacco plant.
  • activity refers to the ability to carry out an enzymatic function.
  • activity of a decarboxylase protein or polypeptide refers to the ability of a decarboxylase to remove a carboxyl group from an organic molecule substrate.
  • the mutated decarboxylase may be able to (a) remove a carboxyl group from an organic molecule substrate at an increased rate as compared to a nonmutated decarboxylase protein; (b) be able to remove carboxyl groups from substrates that are not suitable substrates for a non-mutated decarboxylase; or (c) both (a) and (b).
  • the activity of decarboxylases can be measured using techniques standard in the art. For example, see Yu el al., Seely and Pegg, J. Biol. Chem., 258:2496-2500
  • a non-natural mutation results in increased expression of a nucleic acid sequence.
  • a non-natural mutation results in an increased level of expression of said nucleic acid sequence as compared to expression of said nucleic acid sequence in the same tissue of a control tobacco plant when grown under comparable conditions, wherein said nucleic acid sequence lacks the at least one non-natural mutation in said control tobacco plant.
  • an increased level of expression comprises an increase of at least 5% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 10% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 25% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 50% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 75% as compared to expression in the same tissue of a control plant grown under comparable conditions.
  • an increased level of expression comprises an increase of at least 100% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 200% as compared to expression in the same tissue of a control plant grown under comparable conditions. In another aspect, an increased level of expression comprises an increase of at least 500% as compared to expression in the same tissue of a control plant grown under comparable conditions.
  • a non-natural mutation results in an increased level of activity by a protein or polypeptide encoded by said nucleic acid sequence as compared to activity of a protein or polypeptide encoded by said nucleic acid sequence in a control tobacco plant when grown under comparable conditions, wherein said nucleic acid sequence lacks the at least one non-natural mutation in said control tobacco plant.
  • Nicotine is the primary natural alkaloid in commercialized cigarette tobacco, and it accounts for approximately 90% of the alkaloid content in Nicotiana tabacum.
  • other major alkaloids in tobacco include cotinine, nornicotine, myosmine, nicotyrine, anabasine and anatabine.
  • minor tobacco alkaloids include nicotine-n- oxide, N-methyl anatabine, N-methyl anabasine, pseudooxynicotine, 2,3 dipyridyl and others.
  • an alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine.
  • Alkaloid levels can be assayed by methods known in the art, for example by quantification based on gas-liquid chromatography, high performance liquid chromatography, radio-immunoassays, enzyme-linked immunosorbent assays, and mass spectrometry.
  • nicotinic alkaloid levels can be measured by a GC-FID method based on CORESTA Recommended Method No. 7, 1987 and ISO Standards (ISO TC 126N 394 E. See also Hibi et al., Plant Physiology 100: 826-35 (1992) for a method using gas-liquid chromatography equipped with a capillary column and an FID detector.
  • Alkaloid levels can be assayed from any plant tissue. Non-limiting examples include leaf and hairy root cultures. Additionally, alkaloid levels can be assayed in cured plant materials or in plant materials that have not been cured.
  • tobacco total alkaloids can be measured using a segmented-flow colorimetric method developed for analysis of tobacco samples as adapted by Skalar Instrument Co. (West Chester, PA) and described by Collins et al., Tobacco Science 13:79-81 (1969).
  • samples of tobacco are dried, ground, and extracted prior to analysis of total alkaloids and reducing sugars.
  • the method then employs an acetic acid/methanol/water extraction and charcoal for decolorization. Determination of total alkaloids was based on the reaction of cyanogen chloride with nicotine alkaloids in the presence of an aromatic amine to form a colored complex which is measured at 460 nm.
  • the level of an individual alkaloid is measured based on a freeze-dried cured leaf sample using liquid chromatography with tandem mass spectrometry (LC/MS/MS). In an aspect, the level of an individual alkaloid is measured based on a freeze-dried hairy root culture sample using liquid chromatography with tandem mass spectrometry (LC/MS/MS).
  • the nicotine or alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured after topping in a pooled leaf sample collected from leaf number 3, 4, and 5 after topping.
  • leaves from the same or comparable stalk position(s) and developmental stage(s) are intended so that the comparison can demonstrate effects due to genotype differences, not from other factors.
  • leaf 3 of a control plant is intended as a reference point for comparing with leaf 3 of a modified plant comprising a non-natural mutation or recombinant DNA construct.
  • “topping” refers to the removal of the stalk apex, including the shoot apical meristem, flowers, and up to several adjacent leaves, when a tobacco plant is near vegetative maturity and around the start of reproductive growth.
  • tobacco plants are topped in the button stage (soon after the flower begins to appear).
  • greenhouse or field-grown tobacco plants can be topped when 50% of the plants have at least one open flower. Topping a tobacco plant results in the loss of apical dominance and also induce increased alkaloid production.
  • the alkaloid level (or another leaf chemistry or property characterization; e.g., polyamines) of a tobacco plant is measured about 2 weeks after topping. Other time points can also be used.
  • alkaloid level (or another leaf chemistry or property characterization) of a tobacco plant is measured about 1 , 2, 3, 4, or 5 weeks after topping.
  • the nicotine, alkaloid, or polyamine level (or another leaf chemistry or property characterization; e.g., polyamines) of a tobacco plant is measured about 3, 5, 7, 10, 12, 14, 17, 19, or 21 days after topping.
  • a modified tobacco plant, or part thereof, comprising at least one nonnatural mutation in an endogenous nucleic acid produces at least one leaf comprising a reduced amount of at least one alkaloid as compared to the amount of the alkaloid in a control tobacco plant lacking the at least one non-natural mutation in the endogenous nucleic acid when grown under comparable conditions.
  • a modified tobacco plant, or part thereof, comprising a recombinant DNA construct provided herein produces at least one leaf comprising a reduced amount of at least one alkaloid as compared to the amount of the alkaloid in a control tobacco plant lacking the recombinant DNA construct when grown under comparable conditions.
  • a reduced level of at least one alkaloid comprises a reduction of at least 0.5% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 1% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 2% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 3% as compared to a control tobacco plant when grown under comparable conditions.
  • a reduced level of at least one alkaloid comprises a reduction of at least 4% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 5% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 10% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 15% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 20% as compared to a control tobacco plant when grown under comparable conditions.
  • a reduced level of at least one alkaloid comprises a reduction of at least 25% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 35% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 50% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 75% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at least 90% as compared to a control tobacco plant when grown under comparable conditions.
  • a reduced level of at least one alkaloid comprises a reduction of at between 1% and 20% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at between 1% and 10% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at between 1% and 5% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of at between 10% and 75% as compared to a control tobacco plant when grown under comparable conditions.
  • a reduced level of at least one alkaloid comprises a reduction of nicotine by at least 4% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nicotine by at least 5% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nicotine by at least 10% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nicotine by at least 15% as compared to a control tobacco plant when grown under comparable conditions.
  • a modified tobacco plant comprises a nicotine level of less than or equal to 11 mg/g of dry weight. In an aspect, a modified tobacco plant comprises a nicotine level of less than or equal to 10 mg/g of dry weight. In an aspect, a modified tobacco plant comprises a nicotine level of less than or equal to 9 mg/g of dry weight. In an aspect, a modified tobacco plant comprises a nicotine level of less than or equal to 8 mg/g of dry weight. In an aspect, a modified tobacco plant comprises a nicotine level of less than or equal to 7 mg/g of dry weight. In an aspect, a modified tobacco plant comprises a nicotine level of less than or equal to 6 mg/g of dry weight.
  • a reduced level of at least one alkaloid comprises a reduction of anatabine by between 1% and 20% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of anatabine by between 1% and 10% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of anatabine by between 1% and 5% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of anatabine by between 10% and 75% as compared to a control tobacco plant when grown under comparable conditions.
  • a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 4% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 5% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 10% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 15% as compared to a control tobacco plant when grown under comparable conditions.
  • a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 20% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 25% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 35% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 50% as compared to a control tobacco plant when grown under comparable conditions.
  • a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 75% as compared to a control tobacco plant when grown under comparable conditions. In an aspect, a reduced level of at least one alkaloid comprises a reduction of nornicotine by at least 90% as compared to a control tobacco plant when grown under comparable conditions.
  • Cells, tissues and organs can be from seed, fruit, leaf, cotyledon, hypocotyl, meristem, embryos, endosperm, root, shoot, stem, trichome, pod, flower, inflorescence, stalk, pedicel, style, stigma, receptacle, petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem, vascular tissue.
  • this disclosure provides a tobacco plant chloroplast.
  • this disclosure provides epidermal cells, stomata cell, leaf or root hairs, a storage root, or a tuber.
  • this disclosure provides a tobacco protoplast.
  • a “progeny tobacco plant” or “progeny tobacco seed” can be from any filial generation, e.g., Fi, F2, F3, F4, F5, Fe, F7, etc.
  • a tobacco plant, or part thereof is of a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Vietnamese variety.
  • a modified tobacco plant, or part thereof, provided herein is of a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Turkish variety.
  • a cured tobacco leaf or plant part is of a tobacco variety selected from the group consisting of a flue cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Vietnamese variety. Skilled artisans further understand that cured tobacco does not constitute a living organism and is not capable of growth or reproduction
  • Flue-cured tobaccos (also called “Virginia” or “bright” tobaccos) amount to approximately 40% of world tobacco production. Flue-cured tobaccos are often also referred to as “bright tobacco” because of the golden-yellow to deep-orange color it reaches during curing. Flue- cured tobaccos have a light, bright aroma and taste. Flue-cured tobaccos are generally high in sugar and low in oils. Major flue-cured tobacco growing countries are Argentina, Brazil, China, India, Africa and the United States of America.
  • tobacco plants or seeds or modified tobacco plants or seeds provided herein are of a flue-cured tobacco variety selected from the group consisting of the varieties listed in Table 2, and any variety essentially derived from any one of the foregoing varieties. See WO 2004/041006 Al.
  • modified tobacco plants or seeds provided herein are in a flue-cured variety selected from the group consisting of K326, K346, and NCI 96.
  • Air-cured tobaccos include “Burley,” “Maryland,” and “dark” tobaccos.
  • the common factor linking air-cured tobaccos is that curing occurs primarily without artificial sources of heat and humidity.
  • Burley tobaccos are light to dark brown in color, high in oil, and low in sugar. Burley tobaccos are typically air-cured in barns.
  • Major Burley growing countries include Argentina, Brazil, Italy, Malawi, and the United States of America.
  • tobacco plants or seeds or modified tobacco plants or seeds provided herein are of a Burley tobacco variety selected from the group consisting of the tobacco varieties listed in Table 3, and any variety essentially derived from any one of the foregoing varieties.
  • modified tobacco plants or seeds provided herein are in a Burley variety selected from the group consisting of TN 90, KT 209, KT 206, KT212, and HB 4488.
  • tobacco plants or seeds or modified tobacco plants or seeds provided herein are of a Maryland tobacco variety selected from the group consisting of the tobacco varieties listed in Table 4, and any variety essentially derived from any one of the foregoing varieties.
  • tobacco plants or seeds or modified tobacco plants or seeds provided herein are of an cigar tobacco variety selected from the group consisting of the tobacco varieties listed in Table 7, and any variety essentially derived from any one of the foregoing varieties.
  • a tobacco plant, or part thereof is from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8.
  • a tobacco plant, or part thereof is from a variety listed in Table 2.
  • a tobacco plant, or part thereof is from a variety listed in Table 3.
  • a tobacco plant, or part thereof is from a variety listed in Table 4.
  • a tobacco plant, or part thereof is from a variety listed in Table 5.
  • a tobacco plant, or part thereof is from a variety listed in Table 6.
  • a tobacco plant, or part thereof is from a variety listed in Table 7.
  • a tobacco plant, or part thereof is from a variety listed in Table 8.
  • a modified tobacco plant, or part thereof is from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8.
  • a modified tobacco plant, or part thereof is from a variety listed in Table 2.
  • a modified tobacco plant, or part thereof is from a variety listed in Table 3.
  • a modified tobacco plant, or part thereof is from a variety listed in Table 4.
  • a modified tobacco plant, or part thereof is from a variety listed in Table 5.
  • a modified tobacco plant, or part thereof is from a variety listed in Table 6.
  • a modified tobacco plant, or part thereof is from a variety listed in Table 7.
  • a tobacco cell is from a variety selected from the group consisting of the tobacco varieties listed in Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8.
  • a tobacco cell is from a variety listed in Table 2.
  • a tobacco cell is from a variety listed in Table 3.
  • a tobacco cell is from a variety listed in Table 4.
  • a tobacco cell is from a variety listed in Table 5.
  • a tobacco cell is from a variety listed in Table 6.
  • a tobacco cell is from a variety listed in Table 7.
  • a tobacco cell is from a variety listed in Table 8.
  • a tobacco plant or variety provided herein is an inbred tobacco plant or variety.
  • an “inbred” tobacco variety is a variety that has been bred for genetic homogeneity.
  • a “hybrid” is created by crossing two plants from different varieties or species, such that the progeny comprises genetic material from each parent. Skilled artisans recognize that higher order hybrids can be generated as well. For example, a first hybrid can be made by crossing Variety C with Variety D to create a C x D hybrid, and a second hybrid can be made by crossing Variety E with Variety F to create an E x F hybrid. The first and second hybrids can be further crossed to create the higher order hybrid (C x D) x (E x F) comprising genetic information from all four parent varieties.
  • a modified tobacco plant provided herein is a hybrid tobacco plant. In another aspect, a modified tobacco seed provided herein is a hybrid tobacco seed.
  • this disclosure provides a method for producing a modified tobacco plant comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, where the at least one tobacco plant of the first tobacco variety comprises a non-natural mutation in an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, where the non-natural mutation is not present in the endogenous nucleic acid sequence in a control tobacco plant of the first tobacco variety; and (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, wherein the at least one tobacco seed or plant germinated therefrom comprises the non-natural mutation.
  • this disclosure provides a method for producing a modified tobacco plant comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, where the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, were the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, wherein the at least one progeny tobacco seed or plant germinated therefrom comprises the recombinant
  • this disclosure provides a method for producing a modified tobacco plant comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, where the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence at least 80% identical or similar to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, were the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, wherein the at least one progeny tobacco seed or plant germinated therefrom comprises the recombinant DNA construct.
  • this disclosure provides a method for producing a modified tobacco plant comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, where the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence at least 80% identical or similar to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12, were the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, where the at least one progeny tobacco seed or plant germinated therefrom comprises the recombinant DNA construct.
  • this disclosure provides a method for producing a modified tobacco plant comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, where the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18, where the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, where the at least one progeny tobacco seed or plant germinated therefrom comprises the recombinant DNA construct.
  • this disclosure provides a method for producing a modified tobacco plant comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, where the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6, where the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, where the at least one progeny tobacco seed or plant germinated therefrom comprises the recombinant DNA construct.
  • this disclosure provides a method for producing a modified tobacco plant comprising: (a) crossing at least one tobacco plant of a first tobacco variety with at least one tobacco plant of a second tobacco variety to produce at least one progeny tobacco seed, where the at least one tobacco plant of the first tobacco variety comprises a recombinant DNA construct, where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12, where the recombinant DNA construct is not present in a control tobacco plant of the first tobacco variety; and (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, where the at least one progeny tobacco seed or plant germinated therefrom comprises the recombinant DNA construct.
  • the first tobacco variety and second tobacco variety are the same tobacco variety.
  • the foregoing methods the first tobacco variety and second tobacco variety are the same tobacco variety.
  • crossing refers to the deliberate mating of two plants.
  • crossing comprises pollination and/or fertilization of a first tobacco plant by a second tobacco plant.
  • the two tobacco plants being crossed can be distantly related, closely related, or identical.
  • the two tobacco plants being crossed are both modified tobacco plants.
  • the two tobacco plants being crossed are of the same tobacco variety.
  • the two tobacco plants being crossed are of two different tobacco varieties.
  • one of the two tobacco plants being crossed is male sterile.
  • one of the two tobacco plants being crossed is female sterile.
  • at least one of the two tobacco plants being crossed is a hybrid tobacco plant.
  • at least one of the two tobacco plants being crossed is a modified tobacco plant.
  • a tobacco plant or variety provided herein is female sterile.
  • a modified tobacco plant or variety provided herein is female sterile.
  • female sterile plants can be made by mutating the STIG1 gene. See, for example, Goldman et al. 1994, EMBO Journal 13:2976-2984.
  • a modified tobacco plant provided herein is female sterile.
  • control plant refers to a plant of identical, or nearly identical, genetic makeup as the modified plant being compared, except for the non-natural mutation or recombinant DNA construct provided herein that was introduced to the modified plant.
  • a modified tobacco plant described herein is a low-alkaloid variety or low-alkaloid plant.
  • LA Burley 21 (LA BU21) is a low-alkaloid variety of tobacco.
  • LA BU21 is produced by incorporation of a low alkaloid gene(s) from a Cuban cigar variety into Burley 21 through several backcrosses. It has approximately 0.2% total alkaloids (dry weight) compared to the about 3.5% (dry weight) of its parent, Burley 21.
  • LA BU21 has a leaf grade well below commercially acceptable standards.
  • the present disclosure provides tobacco plants, or part thereof, comprising a low nicotine or low alkaloid-conferring mutation or transgene (e.g., a genetic modification in or targeting one or more ADC, AO, or ODC) and capable of producing a leaf comprising a comparable level of one or more polyamines relative to a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable level of one or more polyamines is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable level of one or more polyamines is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable level of one or more polyamines is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • the present disclosure provides ADC mutant or transgenic tobacco plants or parts thereof, AO mutant or transgenic tobacco plants or parts thereof, or ODC mutant or transgenic tobacco plants or parts thereof, capable of producing a leaf comprising a comparable chlorophyll level relative to a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable chlorophyll level is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • the present disclosure provides ADC mutant or transgenic tobacco plants or parts thereof, AO mutant or transgenic tobacco plants or parts thereof, or ODC mutant or transgenic tobacco plants or parts thereof, capable of producing a leaf comprising a comparable number of mesophyll cell per unit of leaf area relative to a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable number of mesophyll cell per unit of leaf area is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable number of mesophyll cell per unit of leaf area is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable number of mesophyll cell per unit of leaf area is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • the present disclosure provides ADC mutant or transgenic tobacco plants or parts thereof, AO mutant or transgenic tobacco plants or parts thereof, or ODC mutant or transgenic tobacco plants or parts thereof, capable of producing a leaf comprising a comparable epidermal cell size relative to a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable epidermal cell size is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable epidermal cell size is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable epidermal cell size is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • the present disclosure provides ADC mutant or transgenic tobacco plants or parts thereof, AO mutant or transgenic tobacco plants or parts thereof, or ODC mutant or transgenic tobacco plants or parts thereof, capable of producing a leaf comprising a comparable leaf yield relative to a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable leaf yield is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable leaf yield is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable leaf yield is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • the present disclosure provides ADC mutant or transgenic tobacco plants or parts thereof, AO mutant or transgenic tobacco plants or parts thereof, or ODC mutant or transgenic tobacco plants or parts thereof, exhibiting a comparable insect herbivory susceptibility relative to a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable insect herbivory susceptibility is within 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 2.5%, or 1% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable insect herbivory susceptibility is between 0.5% and 1%, between 1% and 2%, between 2% and 3%, between 3% and 4%, between 4% and 5%, between 5% and 6%, between 6% and 7%, between 7% and 8%, between 8% and 9%, between 9% and 10%, between 11% and 12%, between 12% and 13%, between 13% and 14%, between 14% and 15%, between 15% and 16%, between 16% and 17%, between 17% and 18%, between 18% and 19%, or between 19% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • a comparable insect herbivory susceptibility is between 0.5% and 5%, between 5% and 10%, or between 10% and 20% of the level in a comparable leaf of a control plant not comprising the same mutation or transgene.
  • Insect herbivory susceptibility level can be assayed by methods known in the art, for example, in an insect feeding assay.
  • a quarter inch layer of 0.7% agar in water is added to a 100 mm Petri dish and allowed to solidify.
  • Leaf discs are cut from the petri dish lid, placed in the plates and pushed gently into the agar.
  • Leaf discs are taken from plants at the 4-5 leaf stage. Discs were taken from lamina only to exclude major midribs.
  • a single disc is taken from each of the four largest leaves of the plant generating 4 replicates per plant. Four plants are sampled for a total of 16 biological replicates test line.
  • a single budworm (e.g., Heliothis sp., Helicoverpa sp.) at the second instar stage is added to the leaf and allowed to feed for 48 hours at ambient temperature. After 48 hours the budworm larvae are weighed and final larval weights are recorded.
  • budworm e.g., Heliothis sp., Helicoverpa sp.
  • a tobacco plant comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genome modification providing a comparable level of one or more traits selected from the group consisting of total leaf polyamine level, total root polyamine level, total leaf chlorophyll level, mesophyll cell number per leaf area unit, and leaf epidermal cell size; and where the control plant does not have both the first and the second genome modifications.
  • a first genome modification providing a lower level of nicotine or total alkaloid (e.g., in or targeting one or more ADC, AO, or ODC genes)
  • a second genome modification providing a comparable level of one or more traits selected from the group consisting of total leaf polyamine level, total root polyamine level, total leaf chlorophyll level, mesophyll cell number per leaf area unit, and leaf epidermal cell size; and where the control plant does not have both the first and the second genome
  • a tobacco plant, or part thereof comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genome modification providing a comparable level of total leaf polyamine level, where the control plant does not have both the first and the second genome modifications.
  • a tobacco plant, or part thereof comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genome modification providing a comparable level of total root polyamine level, where the control plant does not have both the first and the second genome modifications.
  • a tobacco plant comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genome modification providing a comparable level of mesophyll cell number per leaf area unit, where the control plant does not have both the first and the second genome modifications.
  • a first genome modification providing a lower level of nicotine or total alkaloid (e.g., in or targeting one or more ADC, AO, or ODC genes)
  • a second genome modification providing a comparable level of mesophyll cell number per leaf area unit, where the control plant does not have both the first and the second genome modifications.
  • a tobacco plant, or part thereof comprises relative to a control tobacco plant: a first genome modification providing a lower level of nicotine or total alkaloid (e.g., in or targeting one or more ADC, AO, or ODC genes), and a second genome modification providing a comparable level of leaf epidermal cell size, where the control plant does not have both the first and the second genome modifications.
  • a second genome modification is in or targeting an ADC, AO, or ODC gene.
  • a first genome modification, a second genome modification, or both comprise a transgene, a mutation, or both.
  • a genome modification, a second genome modification, or both comprise a transgene.
  • a first genome modification, a second genome modification, or both comprise a mutation.
  • a first genome modification, a second genome modification, or both are not transgene-based.
  • a first genome modification, a second genome modification, or both are not mutation-based.
  • tobacco plants provided herein comprise a reduced amount of total conjugated polyamines in leaves relative to the control tobacco plant. In one aspect, tobacco plants provided herein comprise a reduced amount of total conjugated polyamines in roots relative to the control tobacco plant.
  • conjugated polyamines include, but are not limited to, soluble conjugated polyamines such as phenolamides containing a backbone consisting of a free polyamine (e.g., putrescine, spermine, and/or spermidine) conjugated with one or more phenylpropanoids such as ferulic, caffeic and courmaric acids.
  • Conjugated polyamines also include, but are not limited to, insoluble conjugated polyamines incorporated into structural polymers such as lignin.
  • tobacco plants provided herein comprise a reduced amount of total free polyamines (e.g., putrescine, spermine, and spermidine) in leaves relative to the control tobacco plant.
  • tobacco plants provided herein comprise a reduced amount of total conjugated polyamines in roots relative to the control tobacco plant.
  • tobacco plants provided herein comprise a reduced amount of total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in leaves relative to the control tobacco plant.
  • tobacco plants provided herein comprise a reduced amount of total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in roots relative to the control tobacco plant. In an aspect, tobacco plants provided herein comprise a reduced amount of total free form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in leaves relative to the control tobacco plant. In one aspect, tobacco plants provided herein comprise a reduced amount of total conjugated form of one or more polyamines selected from the group consisting of putrescine, spermidine and spermine in roots relative to the control tobacco plant.
  • a characteristic or a trait of a tobacco plant described here are measured at a time selected from the group consisting of immediately before flowering, at topping, 1 week- post-topping (WPT), 2 WPT, 3 WPT, 4 WPT, 5 WPT, 6 WPT, 7 WPT, 8 WPT, and at harvest.
  • tobacco plants provided herein comprising a first and a second genome modification are capable of producing a leaf with a leaf grade comparable to that of a leaf from a control plant.
  • tobacco plants provided herein comprising a first and a second genome modification have a total leaf yield comparable to a control plant.
  • a tobacco plant of the present disclosure comprises a nicl mutation, a nic2 mutation, or both.
  • a modified tobacco plant provided herein further comprises a transgene or mutation directly suppressing the expression or activity of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, or seventeen or more genes or loci encoding a protein selected from the group consisting of agmatine deiminase (AIC), arginase, diamine oxidase, methylputrescine oxidase (MPO), NADH dehydrogenase, phosphoribosylanthranilate isomerase (PRAI), putrescine N- methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), S-adenosyl-methionine synthetase (SAMS), A622, NBB1, ber
  • a modified tobacco plant provided herein further comprises a mutation in an ERF gene of Nic2 locus (Nic2_ERF).
  • a modified tobacco plant provided herein further comprises one or more mutations in one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten genes selected from the group consisting of ERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.
  • a modified tobacco plant provided herein further comprises one or more mutations in ERF189, ERF115, or both.
  • a modified tobacco plant provided herein further comprises one or more transgenes targeting and suppressing a gene encoding one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten proteins selected from the group consisting oiERF32, ERF34, ERF39, ERF189, ERF115, ERF221, ERF104, ERF179, ERF17, and ERF168.
  • a modified tobacco plant provided herein further comprises a mutation in an ERF gene of Nicl locus (Nicl_ERF) (or Niclb locus as in WO/2019/140297). See also WO/2018/237107.
  • a modified tobacco plant provided herein further comprises one or more mutations in two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERF 101, ERF 110, ERFnew, ERF 199, ERF 19, ERF130, ERF16, ERF29, ERF210, and ERF91L2. See WO/2019/140297 and Kajikawa et al., Plant physiol. 2017, 174:999-1011.
  • a modified tobacco plant provided herein further comprises one or more mutations in one or more, two or more, three or more, four or more, five or more, or all six genes selected from the group consisting of ERFnew, ERF 199, ERF 19, ERF29, ERF210, and ERF91L2.
  • a modified tobacco plant provided herein further comprises one or more transgenes targeting and suppressing a gene encoding one or more, two or more, three or more, four or more, five or more, six or more, or seven or more genes selected from the group consisting of ERFlOl, ERF110, ERFnew, ERF199, ERF19, ERF130, ERF16, ERF29, ERF210, and ERF91L2.
  • a modified tobacco plant provided herein further comprise a first genetic modification comprising a mutation in a gene or locus encoding a protein selected from the group consisting of aspartate oxidase, agmatine deiminase (AIC), arginase, diamine oxidase, arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), and S-adenosyl- methionine synthetase (SAMS), A622, NBB1, BBL, MYC2, Nicl_ERF, Nic2_ERF, ethylene response factor (ERF) transcription factor, nicotine uptake permease
  • AIC aspartate
  • this disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, where the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • this disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, where the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 -6.
  • this disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, where the modified tobacco plant comprises a non-natural mutation in an endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, where the modified tobacco plant comprises a recombinant DNA construct, where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • this disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, where the modified tobacco plant comprises a recombinant DNA construct, and where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6.
  • this disclosure provides a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, where the modified tobacco plant comprises a recombinant DNA construct, and where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • this disclosure provides a method comprising preparing a tobacco plant using cured tobacco material from a modified tobacco plant, where the modified tobacco plant comprises a recombinant DNA construct, and where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • this disclosure provides a method comprising preparing a tobacco plant using cured tobacco material from a modified tobacco plant, where the modified tobacco plant comprises a recombinant DNA construct, and where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-6.
  • this disclosure provides a method comprising preparing a tobacco plant using cured tobacco material from a modified tobacco plant, where the modified tobacco plant comprises a recombinant DNA construct, and where the recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • “Curing” is the aging process that reduces moisture and brings about the destruction of chlorophyll giving tobacco leaves a golden color and by which starch is converted to sugar. Cured tobacco therefore has a higher reducing sugar content and a lower starch content compared to harvested green leaf.
  • tobacco plants or plant components provided herein can be cured using conventional means, e.g., flue-cured, barn-cured, fire-cured, air-cured or sun-cured. See, for example, Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford) for a description of different types of curing methods.
  • Cured tobacco is usually aged in a wooden drum (e.g., a hogshead) or cardboard cartons in compressed conditions for several years (e.g., two to five years), at a moisture content ranging from 10% to about 25%. See, U.S. Patent Nos. 4,516,590 and 5,372,149. Cured and aged tobacco then can be further processed. Further processing includes conditioning the tobacco under vacuum with or without the introduction of steam at various temperatures, pasteurization, and fermentation. [00233] Information regarding the harvesting of burley and dark tobacco varieties can be found in the 2019-2020 Burley and Dark Tobacco Production Guide (December 2018) published by the University of Kentucky, The University of Tennessee, Virginia Tech, and North Carolina State University, which is incorporated herein by reference in its entirety.
  • this disclosure provides cured tobacco material from any tobacco plant, or part thereof, provided herein. In an aspect, this disclosure provides cured tobacco material from any modified tobacco plant, or part thereof, provided herein.
  • cured tobacco material comprises tobacco material selected from the group selected from cured leaf material, cured stem material, cured bud material, cured flower material, and cured root material.
  • cured tobacco material comprises cured leaf material, cured stem material, or both.
  • cured tobacco material comprises cured leaf material.
  • cured tobacco material comprises cured stem material.
  • cured tobacco material comprises flue-cured tobacco material.
  • cured tobacco material comprises air-cured tobacco material.
  • cured tobacco material comprises fire-cured tobacco material.
  • cured tobacco material comprises sun-cured tobacco material.
  • cured tobacco material provided herein is selected from the group consisting of air-cured tobacco material, fire-cured tobacco material, sun-cured tobacco material, and flue-cured tobacco material.
  • cured tobacco material is from a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, an Oriental variety, and a Turkish variety.
  • cured tobacco leaf provided herein is selected from the group consisting of air-cured tobacco leaf, fire-cured tobacco leaf, sun-cured tobacco leaf, and flue-cured tobacco leaf.
  • cured tobacco leaf is from a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, an Oriental variety, and a Vietnamese variety.
  • Fermentation typically is characterized by high initial moisture content, heat generation, and a 10 to 20% loss of dry weight. See, for example, U.S. Patent Nos. 4,528,993, 4,660,577, 4,848,373, 5,372,149; U.S. Publication No. 2005/0178398; and Tso (1999, Chapter 1 in Tobacco, Production, Chemistry and Technology, Davis & Nielsen, eds., Blackwell Publishing, Oxford). Cured, aged, and fermented tobacco can be further processed (e.g., cut, shredded, expanded, or blended). See, for example, U.S. Patent Nos. 4,528,993; 4,660,577; and 4,987,907.
  • this disclosure provides fermented tobacco material from any tobacco plant, or part thereof, provided herein.
  • this disclosure provides fermented tobacco material from any modified tobacco plant, or part thereof, provided herein.
  • Tobacco products derived from plants of the present disclosure also include cigarettes and other smoking articles, particularly those smoking articles including filter elements, where the rod of smokable material includes cured tobacco within a tobacco blend.
  • a tobacco product of the present disclosure is selected from the group consisting of a cigarillo, a non-ventilated recess filter cigarette, a vented recess filter cigarette, a cigar, snuff, pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, hookah tobacco, shredded tobacco, and cut tobacco.
  • a tobacco product of the present disclosure is selected from the group consisting of a cigarette, a heated tobacco product, a kretek, a bidi cigarette, a cigar, a cigarillo, a non-ventilated cigarette, a vented recess filter cigarette, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco product, gum, a tablet, a lozenge, and a dissolving strip.
  • the modified tobacco plant, or part thereof, of embodiment 1 wherein said at least one non-natural mutation results in a reduced level of activity by a protein or polypeptide encoded by said nucleic acid sequence as compared to activity of a protein or polypeptide encoded by said nucleic acid sequence in a control tobacco plant when grown under comparable conditions, wherein said nucleic acid sequence lacks the at least one non- natural mutation in said control tobacco plant.
  • said promoter comprises a promoter selected from the group consisting of a constitutive promoter, a tissue-preferred promoter, a tissue-specific promoter, and an inducible promoter.
  • siRNA small interfering RNA
  • a /raw.s-acting siRNA a microRNA.
  • said at least one small RNA molecule comprises between 18 nucleotides and 30 nucleotides.
  • said at least one small RNA molecule comprises a nucleic acid sequence at least 90% complementary to a sequence selected from the group consisting of SEQ ID NOs: 7-12.
  • step (d) growing said modified tobacco plant regenerated in step (c).
  • step (c) growing said modified tobacco plant regenerated in step (c).
  • step (c) growing said modified tobacco plant regenerated in step (c).
  • step (c) growing said modified tobacco plant regenerated in step (c).
  • step (c) growing said modified tobacco plant regenerated in step (c).
  • step (c) growing said modified tobacco plant regenerated in step (c).
  • any one of embodiments 52 to 89 wherein said modified tobacco plant is of a variety selected from the group consisting of the varieties listed in Tables 2, 3, 4, 5, 6, 7, and 8.
  • the method of any one of embodiments 52 to 92, wherein said modified tobacco plant is female sterile.
  • said modified tobacco plant comprises a comparable or higher USDA leaf grade index as compared to a control tobacco plant lacking said non-natural mutation when grown under comparable conditions.
  • a method comprising preparing a tobacco product using cured tobacco material from a modified tobacco plant, wherein said modified tobacco plant comprises a recombinant DNA construct, and wherein said recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. .
  • said cured tobacco material comprises flue-cured tobacco material, air-cured tobacco material, fire-cured tobacco material, and sun-cured tobacco material.
  • said tobacco product is selected from the group consisting of a cigarette, a kretek, a bidi cigarette, a cigar, a cigarillo, a non-ventilated cigarette, a vented recess filter cigarette, pipe tobacco, snuff, snus, chewing tobacco, moist smokeless tobacco, fine cut chewing tobacco, long cut chewing tobacco, pouched chewing tobacco product, gum, a tablet, a lozenge, and a dissolving strip.
  • any one of embodiments 97 to 99, wherein said tobacco product is a smokeless tobacco product.
  • said smokeless tobacco product is selected from the group consisting of loose leaf chewing tobacco, plug chewing tobacco, moist snuff, nasal snuff, dry snuff, and snus.
  • said cured tobacco material is of a tobacco variety selected from the group consisting of a flue-cured variety, a bright variety, a Burley variety, a Virginia variety, a Maryland variety, a dark variety, a Galpao variety, an Oriental variety, and a Turkish variety.
  • a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein said recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding at least one small RNA molecule capable of binding to and reducing the expression of at least one endogenous nucleic acid sequence encoding a polypeptide at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. .
  • a method comprising transforming a tobacco cell with a recombinant DNA construct, wherein said recombinant DNA construct comprises a heterologous promoter operably linked to a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% identical or similar to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18. .
  • a method for producing a modified tobacco plant comprising:
  • a method for producing a modified tobacco plant comprising:
  • a method for producing a modified tobacco plant comprising:
  • step (b) selecting for at least one progeny tobacco seed, or a plant germinated therefrom, wherein said at least one tobacco seed or plant germinated therefrom comprises said recombinant DNA construct.
  • the method of embodiment 113, wherein said at least one alkaloid is selected from the group consisting of anabasine, anatabine, nicotine, and nornicotine. .
  • the method of embodiment 113 or 114, wherein said reduced amount of at least one alkaloid comprises a reduction of at least 1%. .
  • invention 110 or 111 wherein said endogenous nucleic acid sequence comprises a sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 12. .
  • the modified tobacco plant, or part thereof, of embodiment 118 wherein said endogenous nucleic acid sequence encodes a transcription factor that is capable of binding to a nucleic acid sequence encoding said gene. .
  • the modified tobacco plant, or part thereof, of embodiment 118, wherein said endogenous nucleic acid sequence encodes a protein that generates a precursor or substrate required for said polypeptide to function. .
  • the modified tobacco plant, or part thereof, of embodiment 118 wherein said expression or functional activity is increased expression or activity as compared to a control tobacco plant lacking said at least one non-natural mutation when grown under comparable conditions.
  • 124. The modified tobacco plant, or part thereof, of embodiment 118, wherein said tobacco plant produces a leaf comprising a reduced amount of at least one alkaloid as compared to the amount of said alkaloid in a control tobacco plant lacking said at least one non-natural mutation in said endogenous nucleic acid sequence when grown under comparable conditions.
  • a tobacco blend comprising the cured tobacco material of embodiment 125.
  • a tobacco product comprising the tobacco blend of embodiment 126.
  • a tobacco product comprising the cured tobacco material of embodiment 125.
  • a reconstituted tobacco comprising the cured tobacco material of embodiment 125.
  • a modified tobacco plant comprising: (a) a genetic modification in a gene; or (b) a genetic modification targeting said gene; wherein said genetic modification downregulates the expression or activity of said gene, wherein said gene encodes a nucleic acid sequence having at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 12.
  • decarboxylases Of the 189 decarboxylases identified, only two types of decarboxylases are known to act on a substrate comprising a six-membered ring: aromatic amino acid decarboxylases and tyrosine decarboxylases. Of the 189 identified decarboxylases, three decarboxylases are annotated as aromatic amino acid decarboxylases, and ten decarboxylases are annotated as tyrosine decarboxylases. After curating the thirteen decarboxylases, including removing enzyme isoforms, six decarboxylases are identified for further examination. See Table 9.
  • RNA is extracted from the root samples, and cDNA transcripts are generated from the RNA samples, using methods standard in the art. The resulting cDNA is used to measure the relative expression of each candidate gene with quantitative RT-PCR (qRT-PCR) using genespecific primers. See Table 10.
  • An expression vector is used as a backbone to generate multiple transformation vectors comprising recombinant DNA constructs (SEQ ID NO: 31).
  • the expression vector contains a Cassava Vein Mosaic Virus (CsVMV) promoter, a NOS terminator, and a cassette comprising a kanamycin selection marker (NPT II) operably linked to an Actin2 promoter and a NOS terminator.
  • Nucleic acid vectors comprising transgenes of interest e.g., SEQ ID NOs: 1 to 12
  • TN90 tobacco plants are grown in MagentaTM GA- 7 boxes and leaf discs are cut and placed into Petri plates.
  • Agrobacterium tumefaciens cells comprising a transformation vector are collected by centrifuging a 20 mL cell suspension in a 50 mL centrifuge tube at 3500 RPM for 10 minutes. The supernatant is removed and the Agrobacterium tumefaciens cell pellet is resuspended in 40 mL liquid re-suspension medium.
  • Tobacco leaves, avoiding the midrib are cut into eight 0.6 cm discs with a #15 razor blade and placed upside down in a Petri plate.
  • Leaf discs are transferred to co-cultivation Petri plates (1/2 MS medium) and discs are placed upside down in contact with filter paper overlaid on the co-cultivation TOM medium (MS medium with 20 g/L sucrose; 1 mg/L indole-3 -acetic acid; and 2.5 mg/L 6-benzyl aminopurine (BAP)).
  • the Petri plate is sealed with parafilm prior to incubation in dim light (60- 80 mE/ms) with 18 hours on, 6 hours off photoperiods at 24 degrees Celsius for three days. After incubation, leaf discs are transferred to regeneration/selection TOM K medium Petri plates (TOM medium plus 300 mg/L kanamycin).
  • Modified tobacco plants from Example 3, along with controls, are grown in 25 centimeter pots in a greenhouse with 75 parts per million fertilizer. At flowering stage, plants are topped. Two weeks after topping, lamina samples are collected from the third, fourth, and fifth leaves from the top of the plant, and alkaloid levels are measured using a method in accordance with CORESTA Method No 62, Determination of Nicotine in Tobacco and Tobacco Products by Gas Chromatographic Analysis, February 2005, and those defined in the Centers for Disease Control and Prevention’s Protocol for Analysis of Nicotine, Total Moisture and pH in Smokeless Tobacco Products, as published in the Federal Register Vol. 64, No. 55 March 23, 1999 (and as amended in Vol. 74, No. 4, January 7, 2009).
  • Plants are also planted in the field, harvested, and tested for alkaloids and TSNA levels in cured tobacco. Both leaf yield and leaf grade are also assessed for modified plants.
  • CsVMV Cassava Vein Mosaic Virus
  • Hairy root cultures can serve as an alternative to plant cell suspensions and serve as useful proxies for whole plants when studying secondary metabolites. See, for example, Zhou et al., Appl. Microbiol Biotechnol., 90:1229-1239 (2011) and Hidalgo et al., Scientific Reports, 7:45331 (2017) for additional information regarding hairy root cultures.
  • Transformation and hairy root induction is performed as described in Hidalgo et al., Scientific Reports, 7 7976 (2017). Briefly, leaf discs from tobacco plantlets are grown in vitro on MS medium. The leaf discs are co-infected with Agrobacterium rhizogenes line ATCC15834 to introduce the transformation vectors described above and to induce hairy root formation. Approximately two to four weeks after transformation, hairy roots appear in leaf discs grown on solid MS medium comprising 30 grams per liter (g/L) sucrose and 500 milligrams per liter (mg/L) cefotaxime.
  • Hairy roots are then excised and individually cultured on solid MS medium comprising 30 g/L sucrose, 500 mg/L cefotaxime, and 50 mg/L kanamycin to select for hairy root cultures that are successfully transformed.
  • Transformed hairy root cultures are grown in the dark at 25°C. Approximately two to three weeks later, liquid cultures are inoculated with transgenic roots and are grown at 25°C until they are used in further analyses.
  • Table 11 provides the relative expression of each overexpressed candidate gene as compared to the expression of tobacco eukaryotic translation elongation factor 1 alpha (EFla; SEQ ID NO: 34) in dark tobacco hairy root cultures.
  • EFla tobacco eukaryotic translation elongation factor 1 alpha
  • Table 11 Relative expression of overexpressed genes as compared to tobacco EFla (Data for EFla is not shown, but is set to 1. Values over 1 indicate higher expression relative to EFla, and values below 1 indicate lower expression relative to EFla). Expression as compared to control is determined by dividing the relative expression of the overexpressed gene in the experimental lines by the relative expression of the relevant control line.
  • Alkaloid levels are also measured in the hairy root cultures that are overexpressing a candidate gene discussed above in Example 5 and control hairy root cultures comprising an empty vector.
  • Table 17 Anabasine levels measured in hairy root cultures that are overexpressing a candidate gene. All anabasine levels are in milligrams per gram of dry weight.
  • Separate transformation vectors comprising an artificial miRNA designed to reduce the transcription or translation of one each of SEQ ID NOs: 7-12 driven by CsVMV are constructed using the vector described in Example 3.
  • An additional transformation vector is generated to simultaneously target g66856 (SEQ ID NO: 7) and gl l429 (SEQ ID NO: 12) using a miRNA construct (SEQ ID NO: 32) driven by a CsVMV promoter as described in Example 3.
  • Another transformation vector is generated to simultaneously target g44542 (SEQ ID NO: 9) and g8073 (SEQ ID NO: 8) using a miRNA construct (SEQ ID NO: 33) driven by a CsVMV promoter as described in Example 3.
  • Tobacco lines plants or hairy root cultures) expressing these RNAi constructs are collectively referred to as “knockdown lines” below.
  • the vectors are used to (a) transform dark tobacco hairy root cultures using the Agrobacterium rhizogenes line ATCC15834 as described in Example 5 and/or (b) generate modified tobacco plants as described in Example 3. Control hairy root cultures and control modified tobacco plants are transformed with an “empty” vector that lacks the miRNA constructs.
  • Table 18 provides the relative expression of each candidate gene as compared to the expression of tobacco eukaryotic translation elongation factor 1 alpha (EFla; SEQ ID NO: 34) in dark tobacco hairy root cultures that express either SEQ ID NO: 32 or SEQ ID NO: 33 under the control of a CsVMV promoter. For each of Tables 18, 19, and 20, expression is measured using qRT-PCR, and the values shown are averages from three biological replicates for each line.
  • Table 18 Relative expression of candidate genes as compared to tobacco EFla in hairy root culture knockdown lines. (Data for EFl a is not shown, but is set to 1. Values over 1 indicate higher expression relative to EFla, and values below 1 indicate lower expression relative to EFla). Expression as compared to control is determined by dividing the relative expression of the candidate gene in the knockdown lines by the relative expression of the relevant control line. SEQ ID NO: 32 targets g66856 and gl 1429, while SEQ ID NO: 33 targets g44542 and g8073.
  • Table 19 Relative expression of QPT in hairy root cultures knockdown lines as compared to tobacco EFla (Data for EF 1 a is not shown, but is set to 1. Values over 1 indicate higher expression relative to EFla, and values below 1 indicate lower expression relative to EFla). Expression as compared to control is determined by dividing the relative expression of QPT in the overexpression lines by the relative expression of the relevant control line. SEQ ID NO: 32 targets g66856 and gl 1429, while SEQ ID NO: 33 targets g44542 and g8073. Table 20. Relative expression of PMT in hairy root cultures knockdown lines as compared to tobacco EFla (Data for EF 1 a is not shown, but is set to 1.
  • Expression as compared to control is determined by dividing the relative expression of PMT in the overexpression lines by the relative expression of the relevant control line.
  • SEQ ID NO: 32 targets g66856 and gl 1429, while SEQ ID NO: 33 targets g44542 and g8073.
  • Example 8 Measuring alkaloid content in transformed tobacco hairy root culture knockdown lines
  • Alkaloid levels are measured in hairy root culture knockdown lines using the method described above in Example 6. Results are shown in Tables 21-24, and the values are averages measured in three biological replicates for each line. Percent change compared to control in Tables 21-24 is calculated by using the formula: ((Control Level - Overexpression Line Level) / (Control Level)) x 100 for each measured alkaloid. Negative values derived from this formula are depicted as increases, and positive values as decreases.
  • Table 23 Anatabine levels measured in hairy root culture knockdown lines. All anatabine levels are in milligrams per gram of dry weight.
  • Table 24 Anabasine levels measured in hairy root culture knockdown lines. All anabasine levels are in milligrams per gram of dry weight.
  • Modified tobacco plants (TO generation) comprising the aforementioned knockdown constructs are generated and grown as described in Example 3. Then, the resulting modified plants are topped, and alkaloid levels are measured as described in Example 4.
  • Mutations are produced in each of the genes identified in Table 9 by specifically editing SEQ ID NOs: 1-6, separately, in the tobacco genome. Higher order mutants are also produced (e.g., a double mutant comprising a mutation in each of SEQ ID NOs: 1 and 6; a double mutant comprising a mutation in each of SEQ ID NOs: 2 and 3; a quadruple mutant comprising a mutation in each of SEQ ID NOs: 1, 2, 3, and 6; a sextuple mutant comprising a mutation in each of SEQ ID NOs: 1-6).
  • Tobacco protoplasts are transfected using polyethylene glycol (PEG) with plasmids encoding a CRISPR protein or a CRISPR protein and specific guide RNA (gRNA) targeting individual genes at desired positions.
  • PEG polyethylene glycol
  • gRNA specific guide RNA

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Abstract

La présente divulgation concerne des plantes de tabac, des parties de plantes, des graines, des compositions et des procédés liés à la modulation de l'expression des gènes de la décarboxylase dans le tabac pour réguler les niveaux d'alcaloïdes.
PCT/US2023/067281 2022-05-23 2023-05-22 Procédés et compositions pour la régulation des alcaloïdes dans le domaine du tabac WO2023230433A1 (fr)

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