WO2024079137A1 - Increasing leaf biomass and nitrogen use efficiency by regulating ntp2 - Google Patents

Increasing leaf biomass and nitrogen use efficiency by regulating ntp2 Download PDF

Info

Publication number
WO2024079137A1
WO2024079137A1 PCT/EP2023/078084 EP2023078084W WO2024079137A1 WO 2024079137 A1 WO2024079137 A1 WO 2024079137A1 EP 2023078084 W EP2023078084 W EP 2023078084W WO 2024079137 A1 WO2024079137 A1 WO 2024079137A1
Authority
WO
WIPO (PCT)
Prior art keywords
ntntp2
plant
seq
sequence
polypeptide
Prior art date
Application number
PCT/EP2023/078084
Other languages
French (fr)
Inventor
Lucien Bovet
Prisca Campanoni
Simon GOEPFERT
Joanne Deborah SCHWAAR
Nicolas Sierro
Original Assignee
Philip Morris Products S.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Philip Morris Products S.A. filed Critical Philip Morris Products S.A.
Publication of WO2024079137A1 publication Critical patent/WO2024079137A1/en

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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]

Definitions

  • the present invention generally relates to a mutant, non-naturally occurring or transgenic plant leaf or part of the plant leaf having modulated expression or activity of NTP2.
  • the present invention relates to a mutant, non-naturally occurring or transgenic plant or part of the plant (such as leaf) having reduced or inhibited expression or activity of NTP2 which confers an increase in biomass and Nitrogen Use Efficiency (NUE) response, without decreasing nitrate levels in the plant or part of the plant.
  • NUE Nitrogen Use Efficiency
  • NUE nitrogen utilization efficiency
  • TSNAs tobacco-specific nitrosamine
  • NNN N- nitrosonornicotine
  • NNK 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone
  • TSNAs have been shown to be strong carcinogens in numerous animal studies (Hecht (1998) Chem Res Toxicol, Jun;11 (6):559- 603; Ma etal. (2016) Carcinogenesis, Feb 9;39(2):232-241 ; Kovi et al. (2016) Toxicol Pathol., Feb;46(2):184-192; Carlson et al. (2016) Chem Res Toxicol., May 21 ;31 (5):358-370).
  • NtNTP2-S and NtNTP2-T Two genes (NtNTP2-S and NtNTP2-T) from Nicotiana tabacum are disclosed herein which belong to the nitrate transporter family 1 NRT1.4 (PTR2 family of peptide transporters) according to homology searches against Arabidopsis thaliana AtNTP2 At2g26690. Reducing or inhibiting (for example, switching off) the expression or activity of the endogenous NtNTP2- T or both NtNTP2-S and NtNTP2-T in Nicotiana tabacum using various different methods is found to confer an advantageous phenotype that improves an agronomic characteristic as compared to a control plant grown in the same conditions.
  • nitrate levels are not decreased;
  • biomass for example, leaf biomass
  • NUE response intended as biomass per unit of nitrogen fertilization applied, is increased.
  • ‘unit’ in the context of a unit of nitrogen fertilization applied means ‘kg per hectare’, as discussed herein
  • NtNTP2 reducing or inhibiting the function of NtNTP2 using, for example, mutation or RNAi, surprisingly results in a change (for example, an increase) in root development by generating more and thinner roots compared to wild type plants grown in the same conditions. Without being bound by any particular theory, this may increase the ability of the plants to uptake nutrients from the soil.
  • the advantageous phenotype of the present invention is also surprising in view of the results reported by Chiu et al. (2004) Plant Cell Physiol., 45(9), 1139-1148, which discloses results for Arabidopsis thaliana homozygous AtNTP2 insertional mutants.
  • the Arabidopsis thaliana homozygous AtNTP2 insertional mutants developed (i) nitrate levels that were 50-64% lower; (ii) increased plant leaf width due to cell expansion; and (iii) no mention of NUE improvement.
  • NtNtp2 is an excellent target for developing plants which have increased yield and higher NUE response (ratio biomass per unit of nitrogen applied) compared to wild type plants grown in the same conditions, whereas this is not demonstrated for AtNTP2 in the mentioned publication.
  • NtNTP2-T a mutant, non-naturally occurring or transgenic plant or part of the plant having reduced or inhibited expression or activity of NtNTP2-T, or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S
  • said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7;
  • the plant or the part of the plant (i) does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response, indicated as biomass per units of nitrogen applied, as compared to the control plant grown in the same conditions.
  • the plant or the part of the plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • the plant or the part of the plant (for example, leaf): (i) does not have decreased nitrate levels as compared to the control plant grown in the same conditions; and (ii) has at least a 5% biomass increase as compared to the control plant grown in the same conditions; and (iii) has at least a 5% increase in NUE response as compared to the control plant grown in the same conditions.
  • the plant or the part of the plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • the plant or the part of the plant (for example, leaf): (i) does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and (ii) has at least a 5% biomass increase as compared to the control plant grown in the same fertilization conditions; and (iii) has at least a 5% increase in NUE response as compared to the control plant grown in the same fertilization conditions.
  • the plant or the part of the plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • mutant, non-naturally occurring or transgenic plant or part of the plant can be modified in various ways with the aim of reducing or inhibiting the expression or activity of NtNTP2-T, or reducing or inhibiting the expression or activity of NtNTP2-T and NtNTP2-S.
  • the mutant, non-naturally occurring or transgenic plant or part of the plant in which the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited comprises: (i) one or more sequence-specific polynucleotides that can interfere with the transcription of NtNTP2-T or NtNTP2-T and NtNTP2-S ⁇ , (ii) one or more sequence-specific polypeptides that can interfere with the stability of NtNTP2-T or NtNTP2-T and NtNTP2-S; (iii) one or more sequence-specific polynucleotides that can interfere with the enzymatic activity of NtNTP2-T or NtNTP2-T and NtNTP2-S or the binding activity of NtNTP2-T or NtNTP2-T and NtNTP2-S with respect to substrates or regulatory proteins; (iv) gene edited NtNTP2-T or NtNTP2-T and NtNTP
  • the NtNTP2-T or NtNTP2-T and NtNTP2-S is gene edited using the bacterial CRISPR/Cas system.
  • the at least one genetic alteration is at least one mutation.
  • the mutant, non-naturally occurring or transgenic plant or part of the plant comprises at least one nonsense mutation in the NtNTP2-T polynucleotide or the NtNTP2-T polypeptide or at least one nonsense mutation in the NtNTP2-T polynucleotide or NtNTP2-T polypeptide and at least one nonsense mutation in the NtNTP2-S polynucleotide or the NtNTP2-S polypeptide.
  • the mutant, non-naturally occurring or transgenic plant or part of the plant comprises a single nucleotide polymorphism in NtNTP2-S at nucleotide position 632 or 633 or 632 and 633 of SEQ ID NO: 3, suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 632 or 633 of SEQ I D NO: 3 or a ‘g ’ to ‘a’ mutation at nucleotide positions 632 and 633 of SEQ ID NO: 3.
  • the mutated NtNTP2-S polynucleotide sequence comprises, consists or consists essentially of SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6.
  • the mutant, non-naturally occurring or transgenic plant or part of the plant comprises a single nucleotide polymorphism in NtNTP2-T at nucleotide position 636 of SEQ ID NO: 11 , suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 635 or 636 of SEQ ID NO: 11 or a ‘g’ to ‘a’ mutation at nucleotide positions 635 and 636 of SEQ ID NO: 11.
  • the mutated NtNTP2-T polynucleotide sequence comprises, consists or consists essentially of SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15.
  • the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide each have at least one nonsense mutation at position W212 and W211 , respectively.
  • the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide comprise(s), consist(s) or consist(s) essentially of either SEQ ID NO: 16 or SEQ ID NO: 8 and SEQ ID NO: 16, respectively; optionally, wherein the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and the mutated NtNTP2-S polypeptide are truncated.
  • the plant part is selected from: (i) green leaf or part thereof; or (ii) dried leaf or part thereof, suitably, wherein the dried leaf or part thereof is air dried, suitably, sun dried or fire dried, flue cured; or (iii) cured leaf or part thereof, suitably, wherein the cured leaf is air cured, more suitably, sun cured or fire cured, flue cured.
  • a method of preparing a plant or a part of the plant comprising: (a) providing a plant comprising: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2- S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or (v) a NtNTP2-T polypeptide having at least 77 % sequence identity to SEQ ID NO: 12; (b) reducing the expression or activity of the NtNTP2-T or
  • the plant or the part of the plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • a mutant, non-naturally occurring or transgenic plant or a part thereof obtained or obtainable by the method of the present invention.
  • a mutant, non-naturally occurring or transgenic plant or a part thereof in which there is no significant difference in nitrate levels as compared to a control plant grown in the same fertilization conditions, wherein biomass yield is higher as compared to the control plant grown in the same fertilization conditions, and wherein the NUE of the plant is higher as compared to the control plant grown in the same fertilization conditions.
  • the mutant, non-naturally occurring or transgenic plant or part thereof can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • this mutant, non-naturally occurring or transgenic plant or part of the plant has reduced or inhibited expression or activity of NtNTP2-T, or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S
  • said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO: 7; or (v) a NtNT
  • a tobacco product or a smoking article comprising the mutant, non-naturally occurring or transgenic plant or part of the plant according to the present invention.
  • a method of improving an agronomic characteristic of a plant comprising reducing or inhibiting the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or (v) a
  • the agronomic characteristic is: (i) nitrate levels are not decreased; (ii) biomass (for example, leaf biomass) is increased in both standard and nitrogen starvation conditions; and (iii) NUE response, intended as biomass per unit of nitrogen fertilization applied, is increased.
  • Figure 1 is a photograph showing the morphology of AA37 ntp2-S W211stop and ntp2-T W212stop double mutant lines in field at harvest time. Representative pictures of two topped plants in field at harvest time. Double wild type indicates an example of an outsegregant wild type plant; Double mutant indicates an example of ntp2-S W211stop/ntp2-T W212stop double mutant plant.
  • Figure 3 is a graph showing the morphology of AA37 ntp2-S W211stop and ntp2-T W212stop double mutant lines in the field. Representative pictures plants growing in the field 3 months after transplant. Wild type indicates an example of out-segregant wild type 10 plant plot; Double mutant indicates an example of 10 plant plots for ntp2-S W211stop/ntp2-T W212stop double mutant; AA37 indicates the control AA37 non mutated plots. Upper panel (Burley regime) indicates the field part fertilized with 254 units of nitrogen; lower panel (Virginia regime) indicates the field part fertilized with 55 units of nitrogen.
  • Figure 5 is a graph showing segregation of the mutant phenotype. Indicated is the cured biomass (expressed as grams per plant) of total leaf harvest from plants grown in standard conditions during a second year trial.
  • SSTT indicates the outsegregant wild type plants
  • sstt indicates the ntp2 double mutant homozygous genotype
  • ssTT and SStt indicate the homozygoyus single mutants of the -S and -T forms respectively. Error bars indicate standard errors.
  • N 8 to 10 plots of 8 plants each (the two edge plants were discarded to minimize positional effects).
  • Figure 6 is a graph showing the NUE index, intended as biomass per unit of nitrogen fertilization applied, of AA37 ntp2 double stop mutant lines grown in different nitrogen regimes. Indicated is the NUE index (expressed as kilograms of cured leaf biomass produced per kilogram of N input per hectare, assuming a number of 12 thousand plants per hectare) of total leaf harvest from plants grown in standard conditions (Burley regime) or Flue cured nitrogen fertilization (Virginia regime) during growing season 2019.
  • sstt indicates the ntp2 double mutant homozygous genotype
  • SSTT indicates the ntp2 double mutant out-segregant wild type genotype. Error bars indicate confidence intervals at 95%.
  • N 19 plots of 8 plants each (the two edge plants were discarded to minimize positional effects).
  • Figure 7 is a graph showing the expression level of NtNTP2 genes in tobacco leaves from different varieties before and shortly after harvesting.
  • Microarray expression profiles of NtNTP2 during plant growth (green mature), at harvest time (harvest) and during early curing (7h curing and 15h curing) of Swiss-Burley (Burley) and Swiss-Flue-cured (Virginia) are shown. Expression levels are indicated as fold changes in a Iog2 scale normalized on each total transcript level.
  • Figure 8 is two graphs showing how NtNTP2 mutation effects root development.
  • Figure 8(A) shows the number of lateral roots in seedlings grown for 13 days on agar plates.
  • Figure 8(B) shows the maximum length of aquatic root in 6 week old young plants grown in hydroponic conditions.
  • SSTT, sstt, ssTT and SStt indicate respectively the outsegregant wild type, the homozygous double -S/-T mutant, and the -S and -T single mutants.
  • Letters on top of the columns in Figure 8(B) indicates statistical groups in a one-way ANOVA test. Error bars indicate confidence intervals at 95%. P value is indicated.
  • Figure 9 is two graphs of average root number per plant of Ntntp2-S W211stop/Ntntp2-T W212stop BC2S2 TN90 (A) and K326 (B) mutant plants and their wild type outsegregant controls.
  • SSTT indicates wild type plants
  • sstt indicates the double mutant Ntntp2-S W211stop/Ntntp2-T W212stop genetic background.
  • TN90 and K326 BC2S2 plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions. Between 4 to 6 weeks after transplant, the primary roots sprouting from the stele were counted for a minimum of 18 plants per genotype for the TN90 variety and a minimum of 25 plants for K326.
  • FIG. 10 is a graph showing average root diameter for TN90 BC2S2 plants.
  • SSTT indicates wild type plants
  • sstt indicates the double mutant Ntntp2-S W211stop/Ntntp2-T W212stop genetic background.
  • TN90 BC2S2 plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions. Between 4 to 6 weeks after transplant, the diameter of the primary roots sprouting from the stele is measured for a minimum of 655 roots per genotype.
  • the statistical validity of the data was tested in t-Student test and the p value is reported in figure. Error bars indicate the confidence interval at 95%.
  • Figure 11 is a graph showing total root diameter distribution in BC2S2 TN90 plants grown in 50% N fertilization.
  • TN90 BC2S2 plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions.
  • the primary roots sprouting from the stele are counted and their diameter is measured with a thickness gage instrument.
  • the distribution of the root diameter measured for 15 plants per genotype plotted against the number of roots measured is reported.
  • SSTT indicates wild type plants
  • sstt indicates the double mutant Ntntp2-S W211stop/Ntntp2-T W212stop genetic background.
  • Figure 12 is a diagram showing a synthetized RNAi loop with 35S CaMV terminator, having the DNA sequence shown in SEQ ID NO: 45.
  • Figure 13 is a diagram showing the RNAi loop from Figure 12 cloned via Hindi I l-Avrl I into a binary vector carrying an MMV promoter and translator enhancer.
  • Figure 14 is a graph showing average root number per plant in TN90 transgenic plants grown in 50% N fertilization. TN90 transgenic plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions. Between 4 to 6 weeks after transplant, the primary roots sprouting from the stele are counted for a minimum of 24 plants per genotype.
  • CT-T2 indicates control plants transformed with an empty binary vector
  • RNAi-T2 indicates the plants transformed with an RNAi vector. The statistical validity of the data is tested in t-Student test and p value is reported together with the percentage of increase of the RNAi lines compared to the control. Error bars report the confidence interval at 95%.
  • Figure 15 is a graph of total root diameter distribution in TN90 transgenic plants grown in 50% N fertilization.
  • TN90 transgenic plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions. Between 4 to 6 weeks after transplant, the primary roots sprouting from the stele are counted and their diameter is measured. The distribution of the root diameter measured for 7 plants per construct plotted against the number of roots measured is reported.
  • CT-T2 indicates control plants transformed with an empty binary vector
  • RNAi-T2 indicates the plants transformed with an RNAi vector.
  • Plants that have increased yield with lower fertilization input and therefore an improved NUE, intended as biomass per unit of nitrogen fertilization applied, can be obtained.
  • the phenotype can be achieved via a non-transgenic approach to develop non-genetically modified (non-GM) plants. This is highly desirable due to the difficulties of growing and commercialising GM plants in various countries, including Europe. Mutant plants featuring one or more single nucleotide polymorphisms are not considered to be GM plants. In the EU for example, there are no special regulations for plants derived from mutation breeding. Thus, in certain embodiments, it is preferred that the plant contains only one or more single nucleotide polymorphisms (that is, one or more mutations) to result in a non-genetically modified plant.
  • a further advantage is that the present invention can be applied to green leaves or dried leaves.
  • green tobacco or dried tobacco can be obtained in greater yield.
  • This can be used in various tobacco applications that can include: (i) smoking products; (ii) biofuel production; (iii) producing recombinant products; and (iv) extraction of bioactive compounds.
  • the present invention can also be applied to cured leaves.
  • Section headings as used in this disclosure are for organisation purposes and are not intended to be limiting.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
  • Coding sequence or “polynucleotide encoding” means the nucleotides (RNA or DNA molecule) that comprise a polynucleotide which encodes a polypeptide.
  • the coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered.
  • the coding sequence may be codon optimized.
  • “Complement” or “complementary” can mean Watson-Crick (for example, A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs. “Complementarity” refers to a property shared between two polynucleotides, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary. "Construct” refers to a double-stranded, recombinant polynucleotide fragment comprising one or more polynucleotides.
  • the construct comprises a "template strand" base-paired with a complementary "sense or coding strand.”
  • a given construct can be inserted into a vector in two possible orientations, either in the same (or sense) orientation or in the reverse (or antisense) orientation with respect to the orientation of a promoter positioned within a vector - such as an expression vector.
  • control in the context of a control plant or control plant cells means a plant or plant cells in which the expression, function or activity of one or more NtNTP2 genes or NtNTP2 polypeptides has not been modified (for example, increased or reduced or inhibited) and so it can provide a comparison with a plant in which the expression, function or activity of the same one or more NtNTP2 genes or NtNTP2 polypeptides has been modified.
  • a “control plant” is a plant that is substantially equivalent to a test plant or modified plant in all parameters with the exception of the test parameters.
  • a control plant is an equivalent plant into which no such polynucleotide has been introduced or no such polynucleotide has been modified.
  • a control plant can be an equivalent plant into which a control polynucleotide has been introduced.
  • the control polynucleotide is one that is expected to result in little or no phenotypic effect on the plant.
  • the control plant may comprise an empty vector.
  • the control plant may correspond to a wildtype (WT) plant.
  • WT wildtype
  • the control plant may be a null segregant wherein the T 1 segregant no longer possesses the transgene. For comparison purposed, the control plant and the plant which is being compared to the control plant will be grown under the same conditions.
  • Donor DNA or “donor template” refers to a double-stranded DNA fragment or molecule that includes at least a portion of the gene of interest.
  • the donor DNA may encode a fully- functional polypeptide or a partially-functional polypeptide.
  • Endogenous gene or polypeptide refers to a gene or polypeptide that originates from the genome of an organism and has not undergone a change, such as a loss, gain, or exchange of genetic material. An endogenous gene undergoes normal gene transmission and gene expression. An endogenous polypeptide undergoes normal expression.
  • Enhancer sequences refer to the sequences that can increase gene expression. These sequences can be located upstream, within introns or downstream of the transcribed region. The transcribed region is comprised of the exons and the intervening introns, from the promoter to the transcription termination region. The enhancement of gene expression can be through various mechanisms including increasing transcriptional efficiency, stabilization of mature mRNA and translational enhancement.
  • “Expression” refers to the production of a functional product.
  • expression of a polynucleotide fragment may refer to transcription of the polynucleotide fragment (for example, transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature polypeptide.
  • “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in a null segregating (or non- transgenic) organism from the same experiment.
  • “Functional” and “full-functional” describes a polypeptide that has biological function or activity.
  • a “functional gene” refers to a gene transcribed to mRNA, which is translated to a functional or active polypeptide.
  • Geneetic construct refers to DNA or RNA molecules that comprise a polynucleotide that encodes a polypeptide.
  • the coding sequence can include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression.
  • Genome editing refers to changing an endogenous gene that encodes an endogenous polypeptide, such that polypeptide expression of a truncated endogenous polypeptide or an endogenous polypeptide having an amino acid substitution is obtained.
  • Genome editing can include replacing the region of the endogenous gene to be targeted or replacing the entire endogenous gene with a copy of the gene that has a truncation or an amino acid substitution with a repair mechanism - such as HDR.
  • Genome editing may also include generating an amino acid substitution in the endogenous gene by generating a double stranded break in the endogenous gene that is then repaired using NHEJ. NHEJ may add or delete at least one base pair during repair which may generate an amino acid substitution.
  • Genome editing may also include deleting a gene segment by the simultaneous action of two nucleases on the same DNA strand in order to create a truncation between the two nuclease target sites and repairing the DNA break by NHEJ.
  • Heterologous with respect to a sequence means a sequence 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.
  • “Homology-directed repair” or “HDR” refers to a mechanism in cells to repair double strand DNA lesions when a homologous piece of DNA is present in the nucleus, mostly in G2 and S phase of the cell cycle. HDR uses a donor DNA or donor template to guide repair and may be used to create specific sequence changes to the genome, including the targeted addition of whole genes. If a donor template is provided along with the site-specific nuclease, then the cellular machinery will repair the break by homologous recombination, which is enhanced several orders of magnitude in the presence of DNA cleavage. When the homologous DNA piece is absent, NHEJ may take place instead.
  • homology refers to the degree of sequence similarity between two polypeptides or between two polynucleotide molecules compared by sequence alignment.
  • degree of homology between two discrete polynucleotides being compared is a function of the number of identical, or matching, nucleotides at comparable positions.
  • Identity in the context of two or more polynucleotides or polypeptides means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
  • Percentage identity can be determined over the full length of a sequence. When comparing DNA and RNA, thymine (T) and uracil (II) may be considered equivalent. Identity may be determined manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA or Smith-Waterman.
  • ClustalW Nucleic Acids Research (1994) 22, 4673-4680; Nucleic Acids Research (1997), 24, 4876-4882) is a suitable way for generating multiple alignments of polypeptides or polynucleotides.
  • N is the number of positions at which the sequences share an identical residue
  • T is the total number of positions compared including gaps but excluding overhangs.
  • the term "increase” or “increased” refers to an increase of from about 5% to about 99%, or an increase of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, at least about 150%, or at least about 200% or more or more of a quantity or a function or an activity, such as but not limited to polypeptide function or activity, transcriptional function or activity, and/or polypeptide expression.
  • the term “increased,” or the phrase “an increased amount” can refer to a quantity or a function or an activity in a modified plant or a product generated from the modified plant that is more than what would be found in a plant or a product from the same variety of plant processed in the same manner, which has not been modified.
  • a wild- type plant of the same variety that has been processed in the same manner is used as a control by which to measure whether an increase in quantity is obtained.
  • reduce refers to a reduction of from about 5% to about 99%, or a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100% or, or at least about 150%, or at least about 200% more of a quantity or a function - such as polypeptide function, transcriptional function, or gene expression.
  • a quantity or a function - such as polypeptide function, transcriptional function, or gene expression.
  • reduced can refer to a quantity or a function in a modified plant or a product generated from the modified plant that is less than what would be found in a plant or a product from the same plant processed in the same manner, which has not been modified.
  • a wild-type plant of the same species or variety that has been processed in the same manner is used as a control by which to measure whether a reduction in quantity is obtained.
  • inhibitor refers to a reduction of from about 98% to about 100%, or a reduction of at least about 98%, at least about 99%, but particularly about 100%, of a quantity or a function or an activity, such as but not limited to polypeptide function or activity, transcriptional function or activity, and/or polypeptide expression.
  • the term “introduced” means providing a polynucleotide (for example, a construct) or polypeptide into a cell. Introduced includes reference to the incorporation of a polynucleotide into a eukaryotic cell where the polynucleotide may be incorporated into the genome of the cell, and includes reference to the transient provision of a polynucleotide or polypeptide to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing.
  • "introduced” in the context of inserting a polynucleotide (for example, a recombinant construct/expression construct) into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a polynucleotide into a eukaryotic cell where the polynucleotide may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).
  • a polynucleotide for example, a recombinant construct/expression construct
  • transduction includes reference to the incorporation of a polynucleotide into a eukaryotic cell where the polynucleotide may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon,
  • isolated or “purified” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. In particular, an isolated polynucleotide is separated from open reading frames that flank the desired gene and encode polypeptides other than the desired polypeptide.
  • purified denotes that a polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel.
  • polynucleotide or polypeptide is at least 85% pure, more suitably at least 95% pure, and most suitably at least 99% pure.
  • Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional polynucleotide purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
  • level refers to an amount and is used interchangeably with “content”.
  • Modulate refers to causing or facilitating a qualitative or quantitative change, alteration, or modification in a process, pathway, function or activity of interest. Without limitation, such a change, alteration, or modification may be an increase or a reduction in the relative process, pathway, function or activity of interest. For example, NtNTP2 gene expression or NtNTP2 polypeptide expression or NtNTP2 polypeptide function or activity can be modulated. Typically, the relative change, alteration, or modification will be determined by comparison to a control.
  • Non-homologous end joining (NHEJ) pathway refers to a pathway that repairs double-strand breaks in DNA by directly ligating the break ends without the need for a homologous template.
  • the template-independent re-ligation of DNA ends by NHEJ is a stochastic, error-prone repair process that introduces random micro-insertions and microdeletions (indels) at the DNA breakpoint. This method may be used to intentionally disrupt, delete, or alter the reading frame of targeted gene sequences.
  • NHEJ typically uses short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the end of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately, yet imprecise repair leading to loss of nucleotides may also occur, but is much more common when the overhangs are not compatible.
  • non-naturally occurring describes an entity - such as a polynucleotide, a genetic mutation, a polypeptide, a plant, a plant cell and plant material - that is not formed by nature or that does not exist in nature.
  • entity - such as a polynucleotide, a genetic mutation, a polypeptide, a plant, a plant cell and plant material - that is not formed by nature or that does not exist in nature.
  • Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by methods described herein or that are known in the art.
  • Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by man.
  • a non-naturally occurring plant a non-naturally occurring plant cell or non- naturally occurring plant material may be made using traditional plant breeding techniques - such as backcrossing - or by genetic manipulation technologies - such as antisense RNA, interfering RNA, meganuclease and the like.
  • a non-naturally occurring plant, a non-naturally occurring plant cell or non-naturally occurring plant material may be made by introgression of or by transferring one or more genetic mutations (for example one or more polymorphisms) from a first plant or plant cell into a second plant or plant cell (which may itself be naturally occurring), such that the resulting plant, plant cell or plant material or the progeny thereof comprises a genetic constitution (for example, a genome, a chromosome or a segment thereof) that is not formed by nature or that does not exist in nature.
  • the resulting plant, plant cell or plant material is thus artificial or non-naturally occurring.
  • an artificial or non-naturally occurring plant or plant cell may be made by modifying a genetic sequence in a first naturally occurring plant or plant cell, even if the resulting genetic sequence occurs naturally in a second plant or plant cell that comprises a different genetic background from the first plant or plant cell.
  • a mutation is not a naturally occurring mutation that exists naturally in a polynucleotide or a polypeptide. Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art - such as polynucleotide sequencing, presence or absence of genetic markers (for example, microsatellite RNA markers).
  • “Oligonucleotide” or “polynucleotide” means at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a polynucleotide also encompasses the complementary strand of a depicted single strand.
  • Many variants of a polynucleotide may be used for the same purpose as a given polynucleotide.
  • a polynucleotide also encompasses substantially identical polynucleotides and complements thereof.
  • a single strand provides a probe that may hybridize to a given sequence under stringent hybridization conditions.
  • a polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Polynucleotides may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence.
  • the polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods.
  • the specificity of singlestranded DNA to hybridize complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)).
  • Hybridization stringency increases as the propensity to form DNA duplexes reduces.
  • the stringency can be chosen to favor specific hybridizations (high stringency), which can be used to identify, for example, full- length clones from a library. Less-specific hybridizations (low stringency) can be used to identify related, but not exact (homologous, but not identical), DNA molecules or segments.
  • DNA duplexes are stabilised by: (1) the number of complementary base pairs; (2) the type of base pairs; (3) salt concentration (ionic strength) of the reaction mixture; (4) the temperature of the reaction; and (5) the presence of certain organic solvents, such as formamide, which reduces DNA duplex stability.
  • the longer the probe the higher the temperature required for proper annealing.
  • a common approach is to vary the temperature; higher relative temperatures result in more stringent reaction conditions.
  • stringent conditions describes hybridization protocols in which polynucleotides at least 60% homologous to each other remain hybridized.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to the given sequence hybridize to the given sequence at equilibrium. Since the given sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • NtNTP2 is used herein to refer to: (i) a NtNTP2-S polynucleotide or a NtNTP2-T polynucleotide or a combination of NtNTP2-S and NtNTP2-T polynucleotides; or (ii) a NtNTP2- S polypeptide or a NtNTP2-T polypeptide or a combination of NtNTP2-S and NtNTP2-T polypeptides.
  • Stringent hybridisation conditions are conditions that enable a probe, primer, or oligonucleotide to hybridize only to its specific sequence. Stringent conditions are sequencedependent and will differ. Stringent conditions typically comprise: (1) low ionic strength and high temperature washes, for example 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1 % sodium dodecyl sulfate, at 50°C; (2) a denaturing agent during hybridization, for example, 50% (v/v) formamide, 0.1 % bovine serum albumin, 0.1% Ficoll, 0.1 % polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate; pH 6.5), at 42°C; or (3) 50% formamide.
  • low ionic strength and high temperature washes for example 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1 % sodium dodecyl sulfate, at 50°C
  • Washes typically also comprise 5xSSC (0.75 M NaCI, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xDenhardt's solution, sonicated salmon sperm DNA (50 pg/mL), 0.1% SDS, and 10% dextran sulfate at 42°C, with a wash at 42°C in 0.2xSSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of O.IxSSC containing EDTA at 55°C.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • Modely stringent conditions use washing solutions and hybridization conditions that are less stringent, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide.
  • One example comprises hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 pg/mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1xSSC, 0.1% SDS at 37°C.
  • the temperature, ionic strength, etc. can be adjusted to accommodate experimental factors such as probe length.
  • Low stringent conditions use washing solutions and hybridization conditions that are less stringent than those for moderate stringency, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide.
  • a non-limiting example of low stringency hybridization conditions includes hybridization in 35% formamide, 5xSSC, 50 mM Tris HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/mL denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2xSSC, 25 mM Tris HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • Other conditions of low stringency such as those for cross-species hybridizations, are well-described (see Ausubel et al., 1993; Kriegler, 1990).
  • “Operably linked” means that expression of a gene is under the control of a promoter with which it is spatially connected.
  • a promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control.
  • the distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
  • “Operably linked” refers to the association of polynucleotide fragments in a single fragment so that the function of one is regulated by the other.
  • a promoter is operably linked with a polynucleotide fragment when it is capable of regulating the transcription of that polynucleotide fragment.
  • plant refers to any plant at any stage of its life cycle or development, and its progenies.
  • the term includes reference to whole plants, plant organs, plant tissues - such as leaf, plant propagules, plant seeds, plant cells and progeny of same.
  • Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Suitable species, cultivars, hybrids and varieties of tobacco plant are described herein.
  • Polynucleotide refers to a polymer of RNA or DNA that is single- or doublestranded, optionally containing synthetic, non-natural or altered nucleotide bases.
  • Nucleotides are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), "K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
  • a polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) thereof.
  • a polynucleotide can be single-stranded or double-stranded, a mixture of single-stranded and double-stranded regions, a hybrid molecule comprising DNA and RNA, or a hybrid molecule with a mixture of single-stranded and double-stranded regions or a fragment(s) thereof.
  • the polynucleotide can be composed of triple-stranded regions comprising DNA, RNA, or both or a fragment(s) thereof.
  • a polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • polynucleotides can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or a combination of the foregoing.
  • the polynucleotides described herein are shown as DNA sequences, the polynucleotides include their corresponding RNA sequences, and their complementary (for example, completely complementary) DNA or RNA sequences, including the reverse complements thereof.
  • the polynucleotides of the present disclosure are set forth in the accompanying sequence listing.
  • Polypeptide or “polypeptide sequence” refer to a polymer of amino acids in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring polymers of amino acids. The terms are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • the polypeptides of the present disclosure are set forth in the accompanying sequence listing.
  • “Promoter” means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a polynucleotide in a cell.
  • Promoters can be derived entirely from regions proximate to a native gene of interest, or can be composed of different elements derived from different native promoters or synthetic polynucleotide segments.
  • a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same.
  • a promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription.
  • a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter may regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
  • tissue-specific promoter and “tissue-preferred promoter” as used interchangeably herein refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell.
  • a “developmentally regulated promoter” refers to a promoter whose function is determined by developmental events.
  • a “constitutive promoter” refers to a promoter that causes a gene to be expressed in most cell types at most times.
  • An “inducible promoter” selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Examples of inducible or regulated promoters include promoters regulated by light, heat, stress, flooding or drought, pathogens, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
  • Recombinant refers to an artificial combination of two otherwise separated segments of sequence - such as by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques.
  • the term also includes reference to a cell or vector, that has been modified by the introduction of a heterologous polynucleotide or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (for example, spontaneous mutation, natural transformation or transduction or transposition) such as those occurring without deliberate human intervention.
  • Recombinant construct refers to a combination of polynucleotides that are not normally found together in nature. Accordingly, a recombinant construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature. The recombinant construct can be a recombinant DNA construct.
  • regulatory sequences and “regulatory elements” as used interchangeably herein refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms “regulatory sequence” and “regulatory element” are used interchangeably herein.
  • Site-specific nuclease refers to an enzyme capable of specifically recognizing and cleaving DNA sequences.
  • the site-specific nuclease may be engineered.
  • engineered sitespecific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), CRISPR/Cas9-based systems, and meganucleases.
  • tobacco is used in a collective sense to refer to tobacco crops (for example, a plurality of tobacco plants grown in the field and not hydroponically grown tobacco), tobacco plants and parts thereof, including but not limited to, root, stem, leaf, flower, and seed prepared and/or obtained, as described herein. It is understood that “tobacco” includes Nicotiana tabacum plants and parts thereof.
  • tobacco products refers to consumer tobacco products, including but not limited to, smoking materials (for example, cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials and ingredients for manufacture of consumer tobacco products. Suitably, these tobacco products are manufactured from tobacco leaves and stems harvested from tobacco and cut, dried, cured, and/or fermented according to conventional techniques in tobacco preparation. The tobacco in the tobacco products may be combined with a binder, as described herein.
  • Transcription terminator refers to DNA sequences located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • Transgenic refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event.
  • a heterologous polynucleotide such as a recombinant construct
  • the term does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events - such as random cross-fertilization, nonrecombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
  • Transgenic plant refers to a plant which comprises within its genome one or more heterologous polynucleotides, that is, a plant that contains recombinant genetic material not normally found therein and which has been introduced into the plant in question (or into progenitors of the plant) by human manipulation.
  • the heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant construct.
  • the commercial development of genetically improved germplasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking approach.
  • transgenic plant encompasses the entire plant or tree and parts of the plant or tree, for instance grains, seeds, flowers, leaves, roots, fruit, pollen, stems and the like. Each heterologous polynucleotide may confer a different trait to the transgenic plant.
  • TALE Transcription activator-like effector
  • the “TALE DNA-binding domain” refers to a DNA-binding domain that includes an array of tandem 33-35 amino acid repeats, also known as RVD modules, each of which specifically recognizes a single base pair of DNA. RVD modules may be arranged in any order to assemble an array that recognizes a defined sequence. A binding specificity of a TALE DNA-binding domain is determined by the RVD array followed by a single truncated repeat of 20 amino acids.
  • a TALE DNA-binding domain may have 12 to 27 RVD modules, each of which contains an RVD and recognizes a single base pair of DNA.
  • TALE DNA-binding domains are modular, repeats that recognize the four different DNA nucleotides may be linked together to recognize any particular DNA sequence. These targeted DNA-binding domains may then be combined with catalytic domains to create functional enzymes, including artificial transcription factors, methyltransferases, integrases, nucleases, and recombinases.
  • Transcription activator-like effector nucleases or “TALENs” as used interchangeably herein refers to engineered fusion polypeptides of the catalytic domain of a nuclease, such as endonuclease Fokl, and a designed TALE DNA-binding domain that may be targeted to a custom DNA sequence.
  • a “TALEN monomer” refers to an engineered fusion polypeptide with a catalytic nuclease domain and a designed TALE DNA-binding domain. Two TALEN monomers may be designed to target and cleave a TALEN target region.
  • Transgene refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or polypeptide in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code. The introduction of a transgene has the potential to change the phenotype of an organism.
  • “Variant” or “mutant” with respect to a polynucleotide refers to a polynucleotide that differs from the wild-type polynucleotide (such as SEQ ID NO: 3 or SEQ ID NO: 11) by one or more nucleic acid deletions, additions, substitutions or side-chain modifications. Exemplary polynucleotide variants or mutants are shown in SEQ ID NOs: 4, 5, 6, 13, 14 and 15. “Variant” or “mutant” with respect to a polypeptide means a polypeptide that differs in sequence by the insertion, deletion, or conservative substitution of one or more amino acids.
  • the variant or mutant may retain all or some or no biological function or activity as compared to the polypeptide that does not contain the insertion, deletion, or conservative substitution of the one or more amino acids.
  • a conservative substitution of an amino acid that is, replacing an amino acid with a different amino acid of similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change.
  • Exemplary polypeptide variants or mutants are shown in SEQ ID NOs: 8 and 16.
  • variety in the context of a plant refers to a population of plants that share constant characteristics which separate them from other plants of the same species. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individuals within that variety. A variety is often sold commercially.
  • Vector refers to a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the transport of polynucleotides, polynucleotide constructs and polynucleotide conjugates and the like.
  • a vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector may be a DNA or RNA vector. Suitable vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other vectors of any origin.
  • an "expression vector” as used herein is a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the expression of polynucleotide(s), polynucleotide constructs and polynucleotide conjugates and the like.
  • Suitable expression vectors include episomes capable of extra- chromosomal replication such as circular, double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other functionally equivalent expression vectors of any origin.
  • An expression vector comprises at least a promoter positioned upstream and operably-linked to a polynucleotide, polynucleotide constructs or polynucleotide conjugate, as defined below.
  • Zinc finger refers to a polypeptide structure that recognizes and binds to DNA sequences.
  • the zinc finger domain is the most common DNA-binding motif in the human proteome.
  • a single zinc finger contains approximately 30 amino acids and the domain typically functions by binding 3 consecutive base pairs of DNA via interactions of a single amino acid side chain per base pair.
  • Zinc finger nuclease or “ZFN” refers to a chimeric polypeptide molecule comprising at least one zinc finger DNA binding domain effectively linked to at least one nuclease or part of a nuclease capable of cleaving DNA when fully assembled.
  • an isolated polynucleotide comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to any of the sequences described herein, including any of polynucleotides shown in the sequence listing.
  • the isolated polynucleotide comprises, consists or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity thereto.
  • the polynucleotide(s) described herein encode an active polypeptide that has at least about 45%, 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99%, 100% or more of the function or activity of the polypeptide(s) shown in the sequence listing.
  • an isolated polynucleotide comprising, consisting or consisting essentially of a polynucleotide having at least 70% sequence identity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO: 15 (provided that the recited mutation is maintained).
  • the isolated polynucleotide comprises, consists or consist essentially of a sequence having at least about 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or
  • polynucleotides comprising, consisting or consisting essentially of polynucleotides with substantial homology (that is, sequence similarity) or substantial identity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO:15 (provided that the recited mutation is maintained).
  • fragments of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO: 15 (provided that the recited mutation is maintained) with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.
  • polynucleotides comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO: 15 (provided that the recited mutation is maintained) that encode a polypeptide that functions as a NtNTP2.
  • a polymer of polynucleotides which comprises, consists or consists essentially of a polynucleotide designated herein as SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13, SEQ ID NO:14 or SEQ ID NO:15.
  • the NtNTP2 polynucleotide(s) described herein can encode a functional NtNTP2(s).
  • the NtNTP2 polynucleotide(s) described herein can contain at least one mutation, suitably at least one mutation encoding a stop codon that causes the encoded polypeptide to terminate or end its translation earlier than expected, which will result in a nonfunctional fragment of NtNTP2(s).
  • Such exemplary poly nucleotides are disclosed herein.
  • a polynucleotide as described herein can include a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingly, a polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) thereof. Moreover, a polynucleotide can be singlestranded or double-stranded DNA, DNA that is a mixture of single-stranded and doublestranded regions, a hybrid molecule comprising DNA and RNA, or a hybrid molecule with a mixture of single-stranded and double-stranded regions or a fragment(s) thereof.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • a polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or
  • polynucleotide can be composed of triple-stranded regions comprising DNA, RNA, or both or a fragment(s) thereof.
  • a polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid.
  • polynucleotides can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or a combination of the foregoing.
  • the polynucleotides described herein are shown as DNA sequences, they include their corresponding RNA sequences, and their complementary (for example, completely complementary) DNA or RNA sequences, including the reverse complements thereof.
  • a polynucleotide as described herein will generally contain phosphodiester bonds, although in some cases, polynucleotide analogues are included that may have alternate backbones, comprising, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O- methylphophoroamidite linkages; and peptide polynucleotide backbones and linkages.
  • polynucleotide analogues include those with positive backbones; non-ionic backbones, and non-ribose backbones.
  • Modifications of the ribose-phosphate backbone may be done for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip.
  • Mixtures of naturally occurring polynucleotides and analogues can be made; alternatively, mixtures of different polynucleotide analogues, and mixtures of naturally occurring polynucleotides and analogues may be made.
  • a variety of polynucleotide analogues are known, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages and peptide polynucleotide backbones and linkages.
  • Other analogue polynucleotides include those with positive backbones, non-ionic backbones and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also included.
  • analogues include peptide polynucleotides which are peptide polynucleotide analogues. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring polynucleotides. This may result in advantages.
  • the peptide polynucleotide backbone may exhibit improved hybridization kinetics.
  • Peptide polynucleotides have larger changes in the melting temperature for mismatched versus perfectly matched base pairs. DNA and RNA typically exhibit a 2-4 °C drop in melting temperature for an internal mismatch. With the non-ionic peptide polynucleotide backbone, the drop is closer to 7-9 °C.
  • peptide polynucleotides may not be degraded or degraded to a lesser extent by cellular enzymes, and thus may be more stable.
  • fragments as probes in hybridisation assays or primers for use in amplification assays.
  • Such fragments generally comprise at least about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more contiguous nucleotides of a DNA sequence.
  • a DNA fragment comprises at least about 10, 15, 20, 30, 40, 50 or 60 or more contiguous nucleotides of a DNA sequence.
  • a method for detecting a polynucleotide comprising the use of the probes or primers or both. Exemplary primers are described herein.
  • oligonucleotides are useful as primers, for example, in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified.
  • degenerate primers can be used as probes for genetic libraries.
  • libraries include cDNA libraries, genomic libraries, and even electronic express sequence tag or DNA libraries. Homologous sequences identified by this method would then be used as probes to identify homologues of the sequences identified herein.
  • polynucleotides and oligonucleotides for example, primers or probes
  • hybridize under reduced stringency conditions typically moderately stringent conditions, and commonly highly stringent conditions to the polynucleotide(s), as described herein.
  • the basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and can be readily determined by those having ordinary skill in the art based on, for example, the length or base composition of the polynucleotide.
  • wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, for example, Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y).
  • the hybrid length is assumed to be that of the hybridizing polynucleotide.
  • the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
  • each such hybridizing polynucleotide has a length that is at least 25% (commonly at least 50%, 60%, or 70%, and most commonly at least 80%) of the length of a polynucleotide to which it hybridizes, and has at least 60% sequence identity (for example, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) with a polynucleotide to which it hybridizes.
  • a linear DNA has two possible orientations: the 5'-to-3' direction and the 3'-to-5' direction. For example, if a first sequence is positioned in the 5'-to-3' direction, and if a second sequence is positioned in the 5'-to-3' direction within the same polynucleotide molecule/strand, then the first sequence and the second sequence are orientated in the same direction, or have the same orientation.
  • a promoter sequence and a gene of interest under the regulation of the given promoter are positioned in the same orientation.
  • first sequence positioned in the 5'-to-3' direction if a second sequence is positioned in the 3'-to-5' direction within the same polynucleotide molecule/strand, then the first sequence and the second sequence are orientated in anti-sense direction, or have anti-sense orientation.
  • Two sequences having antisense orientations with respect to each other can be alternatively described as having the same orientation, if the first sequence (5'-to-3' direction) and the reverse complementary sequence of the first sequence (first sequence positioned in the 5'-to-3') are positioned within the same polynucleotide molecule/strand.
  • the sequences set forth herein are shown in the 5'- to-3' direction.
  • Fragments of the polynucleotides described herein are also disclosed and may range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides or about 1400 nucleotides and up to the full-length polynucleotide.
  • an isolated polypeptide comprising, consisting or consisting essentially of a polypeptide having at least 77 % sequence identity to any of the polypeptide described herein, including any of the polypeptides shown in the sequence listing.
  • the isolated polypeptide comprises, consists or consists essentially of a sequence having at least 77%, 78%, 79%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity thereto.
  • an isolated polypeptide comprising, consisting or consisting essentially of a sequence having at least 77%, 78%, 79%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:16.
  • the polypeptide can include sequences comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 12 or SEQ ID NO:16to function as a NtNTP2.
  • the fragments of the polypeptide(s) typically retain some or all of the NtNTP2 function or activity of the full-length sequence.
  • the polypeptides can include mutations - such as mutations encoding a stop codon as in SEQ ID NOs:8 and 16.
  • SEQ ID NO: 8 has a mutation at amino acid position 211 which mutates amino acid W211 of NtNtp2-S into a stop codon.
  • SEQ ID NO: 16 has a mutation at amino acid position 212 which mutates amino acid W212 of NtNtp2-T into a stop codon.
  • the advantageous phenotype can be obtained by other mutations in the NtNtp2 polynucleotide sequence(s) described herein that cause a truncation of the expressed polypeptide.
  • other genetic techniques as described herein can be used to obtain the advantageous phenotype by modulating (for example, reducing or inhibiting) the expression or activity of NtNtp2.
  • Mutants can be produced by introducing any type of alterations (for example, insertions, deletions, or substitutions of amino acids; changes in glycosylation states; changes that affect refolding or isomerizations, three-dimensional structures, or self-association states), which can be deliberately engineered or isolated naturally provided that they still have some or all of their function or activity.
  • this function or activity is modulated, more suitably, reduced or lost.
  • a deletion refers to removal of one or more amino acids from a polypeptide.
  • An insertion refers to one or more amino acid residues being introduced into a predetermined site in a polypeptide. Insertions may comprise intra-sequence insertions of single or multiple amino acids.
  • a substitution refers to the replacement of amino acids of the polypeptide with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or p-sheet structures).
  • Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from about 1 to about 10 amino acids. The amino acid substitutions are suitably conservative amino acid substitutions as described below.
  • Amino acid substitutions, deletions and/or insertions can be made using peptide synthetic techniques - such as solid phase peptide synthesis or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion mutations of a polypeptide are well known in the art.
  • the mutant may have alterations which produce a silent change and result in a functionally equivalent polypeptide.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and the amphipathic nature of the residues as long as the secondary binding of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and suitably in the same line in the third column may be substituted for each other:
  • the polypeptide may be a mature polypeptide or an immature polypeptide or a polypeptide derived from an immature polypeptide.
  • Polypeptides may be in linear form or cyclized using known methods. Polypeptides typically comprise at least 10, at least 20, at least 30, or at least 40 contiguous amino acids.
  • Fragments of the disclosed polypeptides are also disclosed. Fragments of a polypeptide may range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, and up to the full-length polypeptide described herein.
  • the encoded polypeptide fragment may retain all or a portion or none of the biological activity of the full-length polypeptide.
  • Recombinant constructs can be used to transform plants or plant cells to modulate NtNTP2 polypeptide expression, function or activity.
  • a recombinant NtNTP2 polynucleotide construct can comprise a NtNTP2 polynucleotide encoding one or more NtNTP2 polynucleotides as described herein, operably linked to a regulatory region suitable for expressing the NtNTP2 polypeptide.
  • a NtNTP2 polynucleotide can comprise a coding sequence that encodes the NtNTP2 polypeptide as described herein.
  • Plants or plant cells in which NtNTP2 polypeptide expression, function or activity are modulated can include mutant, non-naturally occurring, transgenic, man-made or genetically engineered plants or plant cells.
  • the transgenic plant or plant cell comprises a genome that has been altered by the stable integration of recombinant DNA.
  • Recombinant DNA includes DNA which has been genetically engineered and constructed outside of a cell and includes DNA containing naturally occurring DNA or cDNA or synthetic DNA.
  • a transgenic plant can include a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant.
  • the transgenic modification alters the expression or function or activity of the NtNTP2 polynucleotide or the NtNTP2 polypeptide described herein as compared to a control plant.
  • the NtNTP2 polypeptide encoded by a recombinant NtNTP2 polynucleotide can be a native NtNTP2 polypeptide or it can be heterologous to the cell.
  • the recombinant construct contains a NtNTP2 polynucleotide that modulates expression, operably linked to a regulatory region. Examples of suitable regulatory regions are described herein.
  • Vectors containing recombinant polynucleotide constructs, including recombinant NtNTP2 polynucleotide constructs are also provided.
  • Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available.
  • the vectors can include, for example, origins of replication, scaffold attachment regions or markers.
  • a marker gene can confer a selectable phenotype on a plant cell.
  • a marker can confer biocide resistance, such as resistance to an antibiotic (for example, kanamycin, G418, bleomycin, or hygromycin), or an herbicide (for example, glyphosate, chlorsulfuron or phosphinothricin).
  • an expression vector can include a tag sequence designed to facilitate manipulation or detection (for example, purification or localization) of the expressed polypeptide.
  • Tag sequences such as luciferase, betaglucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc or hemagglutinin sequences typically are expressed as a fusion with the encoded polypeptide.
  • Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.
  • a plant or plant cell can be transformed by having the recombinant NtNTP2 polynucleotide integrated into its genome to become stably transformed.
  • the plant or plant cell described herein can be stably transformed.
  • Stably transformed cells typically retain the introduced NtNTP2 polynucleotide with each cell division.
  • a plant or plant cell can be transiently transformed such that the recombinant NtNTP2 polynucleotide is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced recombinant NtNTP2 polynucleotide with each cell division such that the introduced recombinant NtNTP2 polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions.
  • a number of methods are available in the art for transforming a plant cell including biolistics, gene gun techniques, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw method, microparticle bombardment, direct DNA uptake, sonication, microinjection, plant virus-mediated transfer, and electroporation.
  • the Agrobacterium system for integration of foreign DNA into plant chromosomes has been extensively studied, modified, and exploited for plant genetic engineering. Naked recombinant DNA molecules comprising DNA sequences corresponding to the subject purified polypeptide operably linked, in the sense or antisense orientation, to regulatory sequences are joined to appropriate T-DNA sequences by conventional methods.
  • plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
  • regulatory regions to be included in a recombinant construct depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue- preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. Transcription of a NtNTP2 polynucleotide can be modulated in a similar manner. Some suitable regulatory regions initiate transcription only, or predominantly, in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known in the art.
  • Suitable promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (for example, root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present during different developmental stages, or present in response to different environmental conditions. Suitable promoters include constitutive promoters that can be activated in most cell types without requiring specific inducers. Examples of suitable promoters for controlling RNAi polypeptide production include the cauliflower mosaic virus 35S (CaMV/35S), SSU, OCS, Iib4, usp, STLS1 , B33, nos or ubiquitin- or phaseolin-promoters. Persons skilled in the art are capable of generating multiple variations of recombinant promoters.
  • Tissue-specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues. Tissue-specific expression can be advantageous, for example, when the expression of polynucleotides in certain tissues is preferred.
  • tissue-specific promoters under developmental control include promoters that can initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, for example, roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue.
  • Reproductive tissue-specific promoters may be, for example, anther-specific, ovulespecific, embryo-specific, endosperm-specific, integument-specific, seed and seed coatspecific, pollen-specific, petal-specific, sepal-specific, or combinations thereof.
  • Suitable leaf-specific promoters include pyruvate, orthophosphate dikinase (PPDK) promoter from C4 plant (maize), cab-m1Ca+2 promoter from maize, the Arabidopsis thaliana myb- related gene promoter (Atmyb5), the ribulose biphosphate carboxylase (RBCS) promoters (for example, the tomato RBCS 1 , RBCS2 and RBCS3A genes expressed in leaves and light- grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruits or ribulose bisphosphate carboxylase promoter expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels).
  • PPDK orthophosphate dikinase
  • Atmyb5 the Arabidopsis thaliana myb- related gene promoter
  • RBCS ribulose biphosphate carboxylase
  • Suitable senescence-specific promoters include a tomato promoter active during fruit ripening, senescence and abscission of leaves, a maize promoter of gene encoding a cysteine protease, the promoter of 82E4 and the promoter of SAG genes. Suitable anther-specific promoters can be used. Suitable root- preferred promoters known to persons skilled in the art may be selected. Suitable seed-preferred promoters include both seed-specific promoters (those promoters active during seed development such as promoters of seed storage polypeptides) and seed-germinating promoters (those promoters active during seed germination).
  • Such seed-preferred promoters include Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1 -phosphate synthase); mZE40-2, also known as Zm-40; nuclc; and celA (cellulose synthase).
  • Gama-zein is an endosperm-specific promoter.
  • Glob-1 is an embryo-specific promoter.
  • seed-specific promoters include bean beta-phaseolin, napin, p-conglycinin, soybean lectin, cruciferin, and the like.
  • seed-specific promoters include a maize 15 kDa zein promoter, a 22 kDa zein promoter, a 27 kDa zein promoter, a g-zein promoter, a 27 kDa gamma-zein promoter (such as gzw64A promoter, see Genbank Accession number S78780), a waxy promoter, a shrunken 1 promoter, a shrunken 2 promoter, a globulin 1 promoter (see Genbank Accession number L22344), an Itp2 promoter, cim1 promoter, maize end1 and end2 promoters, nuc1 promoter, Zm40 promoter, eep1 and eep2; led , thioredoxin H promoter; mlip15 promoter, PCNA2 promoter; and the shrunken-2 promoter.
  • a maize 15 kDa zein promoter such as gzw64A promoter,
  • inducible promoters include promoters responsive to pathogen attack, anaerobic conditions, elevated temperature, light, drought, cold temperature, or high salt concentration.
  • Pathogen-inducible promoters include those from pathogenesis-related polypeptides (PR polypeptides), which are induced following infection by a pathogen (for example, PR polypeptides, SAR polypeptides, beta-1 , 3-glucanase, chitinase).
  • promoters may be derived from bacterial origin for example, the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from Ti plasmids, or may be derived from viral promoters (for example, 35S and 19S RNA promoters of cauliflower mosaic virus (CaMV), constitutive promoters of tobacco mosaic virus, cauliflower mosaic virus (CaMV) 19S and 35S promoters, or figwort mosaic virus 35S promoter).
  • CaMV cauliflower mosaic virus
  • CaMV cauliflower mosaic virus
  • CaMV constitutive promoters of tobacco mosaic virus
  • CaMV cauliflower mosaic virus
  • CaMV cauliflower mosaic virus
  • figwort mosaic virus 35S promoter figwort mosaic virus 35S promoter
  • Suitable methods of introducing polynucleotides into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al., Biotechniques 4:320-334 (1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602-5606 (1986)), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,981 ,840 and 5,563,055), direct gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)), and ballistic particle acceleration (see, for example, U.S. Pat. Nos.
  • a plant or plant cell comprising a mutation in one or more NtNTP2 polynucleotides or NtNTP2 polypeptides described herein is disclosed, wherein said mutation results in modulated function or activity of NtNTP2 (including total loss of function or activity).
  • the mutation reduces or inhibits the expression or activity of NtNTP2.
  • the mutant plants or plant cells can have one or more further mutations either in the same NtNTP2 polynucleotides or NtNTP2 polypeptides as described herein or in one or more other polynucleotides or polypeptides within the genome.
  • other techniques can be utilised by the skilled person to modulate the function or activity of NtNTP2, as described in detail herein.
  • NtNTP2 polypeptide in a plant or in plant material said method comprising introducing into the genome of said plant one or more mutations that modulate expression of at least one NtNTP2 gene, wherein said at least one NtNTP2 gene is selected from the sequences according to the present disclosure.
  • a method for identifying a plant that does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and has increased biomass as compared to the control plant grown in the same fertilization conditions; and has increased NUE response as compared to the control plant grown in the same fertilization conditions comprising screening a polynucleotide sample from a plant of interest for the presence of one or more mutations in the NtNTP2 sequences described herein.
  • the plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • a number of approaches can be used to combine mutations in one plant including sexual crossing.
  • a plant having one or more favourable heterozygous or homozygous mutations in a NtNTP2 gene that modulates expression of the NtNTP2 gene or the function or activity of the NtNTP2 polypeptide encoded thereby can be crossed with a plant having one or more favourable heterozygous or homozygous mutations in one or more other genes that modulate expression thereof or the function or activity of the polypeptide encoded thereby.
  • crosses are made to introduce one or more favourable heterozygous or homozygous mutations within the NtNTP2 gene(s) within the same plant.
  • the function or activity of one or more NtNTP2 polypeptides of the present disclosure in a plant is increased or reduced if the function or activity is lower or higher than the function or activity of the same polypeptide(s) in a plant that has not been modified to modulate the function or activity of that polypeptide and which has been cultured, harvested and optionally cured using the same protocols.
  • the mutation(s) is introduced into a plant or plant cell using a mutagenesis approach, and the introduced mutation is identified or selected using methods known to those of skill in the art - such as Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that impact NtNTP2 gene expression or that interfere with the function of the encoded NtNTP2 polypeptide can be determined using methods that are well known in the art. Insertional mutations in NtNTP2 gene exons usually result in null-mutants. Mutations in conserved residues can be particularly effective in reducing or inhibiting the metabolic function of the encoded NtNTP2 polypeptide.
  • a mutation in one or more of the highly conserved regions would likely alter NtNTP2 polypeptide function, while a mutation outside of those highly conserved regions would likely have little to no effect on NtNTP2 polypeptide function.
  • a mutation in a single nucleotide can create a stop codon, which would result in a truncated NtNTP2 polypeptide and, depending on the extent of truncation, reduction or complete loss of function.
  • the mutation(s) results in reduced expression of NtNTP2 and reduced biological function of NtNTP2, more suitably, the mutation(s) results in complete loss of expression of NtNTP2 and complete loss of biological function of NtNTP2.
  • Any plant of interest including a plant cell or plant material or plant leaf can be genetically modified by various methods known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically-induced mutagenesis, irradiation-induced mutagenesis, mutagenesis utilizing modified bases, mutagenesis utilizing gapped duplex DNA, double-strand break mutagenesis, mutagenesis utilizing repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling and other equivalent methods.
  • Mutant NtNTP2 polypeptides can be used to create mutant, non-naturally occurring or transgenic plants (for example, mutant, non-naturally occurring, transgenic, man-made or genetically engineered plants) or plant cells comprising one or more mutant polypeptides.
  • mutant NtNTP2 polypeptides can retain all, some or no function of the unmutated polypeptide.
  • the function of the mutant NtNTP2 polypeptide may be higher, lower or about the same as the unmutated polypeptide.
  • it is preferred that the mutant NtNTP2 polypeptide has reduced function or does not retain any function.
  • Mutations in the NtNTP2 polynucleotides and NtNTP2 polypeptides can include man-made mutations or synthetic mutations or genetically engineered mutations. Mutations in the NtNTP2 polynucleotides and NtNTP2 polypeptides described herein can be mutations that are obtained or obtainable via a process which includes an in vitro or an in vivo manipulation step. Mutations in the NtNTP2 polynucleotides and NtNTP2 polypeptides described herein can be mutations that are obtained or obtainable via a process which includes intervention by man. Methods that introduce a mutation randomly in a polynucleotide can include chemical mutagenesis and radiation mutagenesis.
  • Chemical mutagenesis involves the use of exogenously added chemicals - such as mutagenic, teratogenic, or carcinogenic organic compounds - to induce mutations.
  • Mutagens that create primarily point mutations and short deletions, insertions, missense mutations, simple sequence repeats, transversions, and/or transitions, including chemical mutagens or radiation, may be used to create the mutations.
  • Mutagens include ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosurea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, and the like), 2-methoxy-6-chloro-9[3-(ethyl- 2-chloro-ethyl)aminopropylamino]acridine di
  • Suitable mutagenic agents can also include, for example, ionising radiation - such as X-rays, gamma rays, fast neutron irradiation and UV radiation.
  • the dosage of the mutagenic chemical or radiation is determined experimentally for each type of plant tissue such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility. Any method of plant polynucleotide preparation known to those of skill in the art may be used to prepare the plant polynucleotide for mutation screening.
  • the mutation process may include one or more plant crossing steps.
  • screening can be performed to identify mutations that create premature stop codons or otherwise non-functional genes. After mutation, screening can be performed to identify mutations that create functional genes that are capable of being expressed at increased or reduced levels. Screening of mutants can be carried out by sequencing, or by the use of one or more probes or primers specific to the NtNTP2 gene or NtNTP2 polypeptide. Specific mutations in NtNTP2 polynucleotides can also be created that can result in modulated NtNTP2 gene expression, modulated stability of mRNA, or modulated stability of polypeptide. Such plants are referred to herein as "non-naturally occurring" or "mutant" plants.
  • the mutant or non-naturally occurring plants will include at least a portion of foreign or synthetic or man-made nucleotide (for example, DNA or RNA) that was not present in the plant before it was manipulated.
  • the foreign nucleotide may be a single nucleotide, two or more nucleotides, two or more contiguous nucleotides or two or more non-contiguous nucleotides - such as at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or more contiguous or non-contiguous nucleotides.
  • compositions that can modulate the expression or function or activity of one or more of the NtNTP2 polynucleotides or NtNTP2 polypeptides include sequencespecific polynucleotides that can interfere with the transcription of one or more endogenous gene(s); sequence-specific polynucleotides that can interfere with the translation of RNA transcripts (for example, double-stranded RNAs, siRNAs, ribozymes); sequence-specific polypeptides that can interfere with the stability of one or more polypeptides; sequencespecific polynucleotides that can interfere with the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides with respect to substrates or regulatory polypeptides; antibodies that exhibit specificity for one or more polypeptides; small molecule compounds that can interfere with the stability of one or more polypeptides or the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides; zinc finger polypeptid
  • Zinc finger polypeptides can be used to modulate the expression or function or activity of one or more of the NtNTP2 polynucleotides described herein.
  • a genomic DNA sequence comprising a part of or all of the coding sequence of the NtNTP2 polynucleotide is modified by zinc finger nuclease-mediated mutagenesis.
  • the genomic DNA sequence is searched for a unique site for zinc finger polypeptide binding.
  • the genomic DNA sequence is searched for two unique sites for zinc finger polypeptide binding wherein both sites are on opposite strands and close together, for example, 1 , 2, 3, 4, 5, 6 or more base pairs apart.
  • a zinc finger polypeptide that bind to NtNTP2 polynucleotides.
  • a zinc finger polypeptide may be engineered to recognize a selected target site in a gene.
  • a zinc finger polypeptide can comprise any combination of motifs derived from natural zinc finger DNA-binding domains and non-natural zinc finger DNA- binding domains by truncation or expansion or a process of site-directed mutagenesis coupled to a selection method such as, but not limited to, phage display selection, bacterial two-hybrid selection or bacterial one-hybrid selection.
  • non-natural zinc finger DNA-binding domain refers to a zinc finger DNA-binding domain that binds a three-base pair sequence within the polynucleotide target and that does not occur in the cell or organism comprising the polynucleotide which is to be modified.
  • Methods for the design of zinc finger polypeptides which bind specific polynucleotides which are unique to a target gene are known in the art.
  • a zinc finger polypeptide may be selected to bind to a regulatory sequence of a NtNTP2 polynucleotide. More specifically, the regulatory sequence may comprise a transcription initiation site, a start codon, a region of an exon, a boundary of an exon-intron, a terminator, or a stop codon. Accordingly, the disclosure provides a mutant, non- naturally occurring or transgenic plant or plant cells, produced by zinc finger nuclease- mediated mutagenesis in the vicinity of or within one or more NtNTP2 polynucleotides described herein, and methods for making such a plant or plant cell by zinc finger nuclease- mediated mutagenesis. Methods for delivering zinc finger polypeptide and zinc finger nuclease to a plant are similar to those described below for delivery of meganuclease.
  • meganucleases such as l-Crel
  • Naturally occurring meganucleases as well as recombinant meganucleases can be used to specifically cause a double-stranded break at a single site or at relatively few sites in the genomic DNA of a plant to allow for the disruption of one or more NtNTP2 polynucleotides described herein.
  • the meganuclease may be an engineered meganuclease with altered DNA- recognition properties. Meganuclease polypeptides can be delivered into plant cells by a variety of different mechanisms known in the art.
  • the disclosure encompasses the use of meganucleases to inactivate a NtNTP2 polynucleotide(s) described herein (or any combination thereof as described herein) in a plant cell or plant.
  • the disclosure provides a method for inactivating a NtNTP2 polynucleotide in a plant using a meganuclease comprising: a) providing a plant cell comprising a NtNTP2 polynucleotide as described herein; (b) introducing a meganuclease or a construct encoding a meganuclease into said plant cell; and (c) allowing the meganuclease to substantially inactivate the NtNTP2 polynucleotide(s)
  • Meganucleases can be used to cleave meganuclease recognition sites within the coding regions of a NtNTP2 polynucleotide. Such cleavage frequently results in the deletion of DNA at the meganuclease recognition site following mutagenic DNA repair by non-homologous end joining. Such mutations in the gene coding sequence are typically sufficient to inactivate the gene.
  • This method to modify a plant cell involves, first, the delivery of a meganuclease expression cassette to a plant cell using a suitable transformation method. For highest efficiency, it is desirable to link the meganuclease expression cassette to a selectable marker and select for successfully transformed cells in the presence of a selection agent.
  • the meganuclease expression cassette (and linked selectable marker gene) may be segregated away in subsequent plant generations using conventional breeding techniques.
  • plant cells are grown, initially, under conditions that are typical for the particular transformation procedure that was used. This may mean growing transformed cells on media at temperatures below 26°C, frequently in the dark. Such standard conditions can be used for a period of time, suitably 1-4 days, to allow the plant cell to recover from the transformation process. At any point following this initial recovery period, growth temperature may be raised to stimulate the function of the engineered meganuclease to cleave and mutate the meganuclease recognition site.
  • TALENs transcription activator-like effector nucleases
  • NHEJ reconnects DNA from either side of a double-strand break where there is very little or no sequence overlap for annealing.
  • This repair mechanism induces errors in the genome via insertion or deletion, or chromosomal rearrangement. Any such errors may render the gene products coded at that location non-functional.
  • Such applications are possible using a pair of engineered meganucleases, each of which cleaves a meganuclease recognition site on either side of the intended deletion.
  • TALENs that are able to recognize and bind to a gene and introduce a double-strand break into the genome can also be used.
  • methods for producing mutant, non-naturally occurring or transgenic or otherwise genetically-modified plants as described herein using TAL Effector Nucleases are contemplated.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • crRNAs CRISPR RNAs
  • tracrRNA trans-activating crRNA
  • Cas CRISPR-associated polypeptides
  • RNAs9 is normally programmed by a dual RNA consisting of the crRNA and tracrRNA. However, the core components of these RNAs can be combined into a single hybrid ‘guide RNA’ for Cas9 targeting.
  • guide RNA The use of a noncoding RNA guide to target DNA for site-specific cleavage promises to be significantly more straightforward than existing technologies - such as TALENs.
  • CRISPR/Cas strategy retargeting the nuclease complex only requires introduction of a new RNA sequence and there is no need to reengineer the specificity of polypeptide transcription factors.
  • CRISPR/Cas technology was implemented in plants in the method of international application WO 2015/189693 A1 , which discloses a viral-mediated genome editing platform that is broadly applicable across plant species.
  • the RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and deliver guide RNA into Nicotiana benthamiana plants overexpressing Cas9 endonuclease.
  • a guide RNA may be derived from any of the sequences disclosed herein and the teaching of WO 2015/189693 A1 applied to edit the genome of a plant cell and obtain a desired mutant plant.
  • the fast pace of the development of the technology has generated a great variety of protocols with broad applicability in plantae, which have been well catalogued in a number of recent scientific review articles (for example, Schiml et al. Plant Methods 2016 12:8; and Khatodia et al. Front Plant Sci. 2016; 7: 506).
  • a review of CRISPR/Cas systems with a particular focus on its application in plants is given by Bortesi and Fischer (Biotechnology Advances (2015) 33, 1 , 41-52).
  • CRISPR/Cas9 plasmids for use in plants are listed in “addgene”, the nonprofit plasmid repository (addgene.org), and CRISPR/Cas plasmids are commercially available.
  • Antisense technology is another well-known method that can be used to modulate the expression or activity of a NtNTP2 polypeptide.
  • a polynucleotide of the gene to be repressed is cloned and operably linked to a regulatory region and a transcription termination sequence so that the antisense strand of RNA is transcribed.
  • the recombinant construct is then transformed into a plant cell and the antisense strand of RNA is produced.
  • the polynucleotide need not be the entire sequence of the gene to be repressed, but typically will be substantially complementary to at least a portion of the sense strand of the gene to be repressed.
  • a polynucleotide may be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA.
  • Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA.
  • Heterologous polynucleotides can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide.
  • Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site-specific recognition sequences can be used.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target RNA contains a 5'-UG-3' polynucleotide.
  • the construction and production of hammerhead ribozymes is known in the art.
  • Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo.
  • tRNA transfer RNA
  • the sequence-specific polynucleotide that can interfere with the translation of RNA transcript(s) is interfering RNA.
  • RNA interference or RNA silencing is an evolutionarily conserved process by which specific mRNAs can be targeted for enzymatic degradation.
  • a double-stranded RNA (double-stranded RNA) is introduced or produced by a cell (for example, double-stranded RNA virus, or interfering RNA polynucleotides) to initiate the interfering RNA pathway.
  • the double-stranded RNA can be converted into multiple small interfering RNA (siRNA) duplexes of 21-24 bp length by RNases III, which are double-stranded RNA-specific endonucleases.
  • the siRNAs can be subsequently recognized by RNA-induced silencing complexes that promote the unwinding of siRNA through an ATP-dependent process.
  • the unwound antisense strand of the siRNA guides the activated RNA-induced silencing complexes to the targeted mRNA comprising a sequence complementary to the siRNA anti-sense strand.
  • the targeted mRNA and the anti-sense strand can form an A-form helix, and the major groove of the A-form helix can be recognized by the activated RNA- induced silencing complexes.
  • the target mRNA can be cleaved by activated RNA-induced silencing complexes at a single site defined by the binding site of the 5'-end of the siRNA strand.
  • the activated RNA-induced silencing complexes can be recycled to catalyze another cleavage event.
  • Interfering RNA expression vectors may comprise interfering RNA constructs encoding interfering RNA polynucleotides that exhibit RNA interference by reducing the expression level of mRNAs, pre-mRNAs, or related RNA variants.
  • the expression vectors may comprise a promoter positioned upstream and operably-linked to an Interfering RNA construct, as further described herein.
  • Interfering RNA expression vectors may comprise a suitable minimal core promoter, a Interfering RNA construct of interest, an upstream (5') regulatory region, a downstream (3') regulatory region, including transcription termination and polyadenylation signals, and other sequences known to persons skilled in the art, such as various selection markers.
  • the double-stranded RNA molecules may include siRNA molecules assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a polynucleotide based or non- polynucleotide-based linker(s), as well as circular single-stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating interfering RNA.
  • small hairpin RNA molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a singlestranded RNA molecule and its reverse complement, such that they may anneal to form a double-stranded RNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3' end or the 5' end of either or both strands).
  • the spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double-stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end or the 5' end of either or both strands).
  • the spacer sequence is typically an unrelated polynucleotide that is situated between two complementary polynucleotides regions which, when annealed into a doublestranded polynucleotide, comprise a small hairpin RNA.
  • the spacer sequence generally comprises between about 3 and about 100 nucleotides.
  • RNA polynucleotide of interest can be produced by selecting a suitable sequence composition, loop size, and stem length for producing the hairpin duplex.
  • a suitable range for designing stem lengths of a hairpin duplex includes stem lengths of at least about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides - such as about 14-30 nucleotides, about 30-50 nucleotides, about 50-100 nucleotides, about 100-150 nucleotides, about 150-200 nucleotides, about 200-300 nucleotides, about 300-400 nucleotides, about 400-500 nucleotides, about 500-600 nucleotides, and about 600-700 nucleotides.
  • a suitable range for designing loop lengths of a hairpin duplex includes loop lengths of about 4-25 nucleotides, about 25-50 nucleotides, or longer if the stem length of the hair duplex is substantial.
  • a double-stranded RNA or ssRNA molecule is between about 15 and about 40 nucleotides in length.
  • the siRNA molecule is a double-stranded RNA or ssRNA molecule between about 15 and about 35 nucleotides in length.
  • the siRNA molecule is a double-stranded RNA or ssRNA molecule between about 17 and about 30 nucleotides in length.
  • the siRNA molecule is a double-stranded RNA or ssRNA molecule between about 19 and about 25 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule between about 21 to about 23 nucleotides in length. In certain embodiments, hairpin structures with duplexed regions longer than 21 nucleotides may promote effective siRNA- directed silencing, regardless of loop sequence and length. Exemplary sequences for RNA interference are described herein.
  • the target mRNA sequence is typically between about 14 to about 50 nucleotides in length.
  • the target mRNA can, therefore, be scanned for regions between about 14 and about 50 nucleotides in length that suitably meet one or more of the following criteria: an A+T/G+C ratio of between about 2:1 and about 1 :2; an AA dinucleotide or a CA dinucleotide at the 5' end; a sequence of at least 10 consecutive nucleotides unique to the target mRNA (that is, the sequence is not present in other mRNA sequences from the same plant); and no "runs" of more than three consecutive guanine (G) nucleotides or more than three consecutive cytosine (C) nucleotides.
  • G guanine
  • C cytosine
  • BLAST can be used to search publicly available databases to determine whether the selected sequence is unique to the target mRNA.
  • a sequence can be selected (and a siRNA sequence designed) using computer software available commercially (for example, OligoEngine, Target Finder and the siRNA Design Tool which are commercially available).
  • target mRNA sequences are selected that are between about 14 and about 30 nucleotides in length that meet one or more of the above criteria. In another embodiment, sequences are selected that are between about 16 and about 30 nucleotides in length that meet one or more of the above criteria. In a further embodiment, sequences are selected that are between about 19 and about 30 nucleotides in length that meet one or more of the above criteria. In another embodiment, sequences are selected that are between about 19 and about 25 nucleotides in length that meet one or more of the above criteria.
  • the siRNA molecules comprise a specific antisense sequence that is complementary to at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more contiguous nucleotides of any one of the polynucleotides described herein.
  • the specific antisense sequence comprised by the siRNA molecule can be identical or substantially identical to the complement.
  • the specific antisense sequence comprised by the siRNA molecule is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the complement of the target mRNA sequence.
  • Methods of determining sequence identity are known in the art and can be determined, for example, by using the BLASTN program of the University of Wisconsin Computer Group (GCG) software or provided on the NCBI website.
  • RNA-silencing One method for inducing double stranded RNA-silencing in plants is transformation with a gene construct producing hairpin RNA (see Nature (2000) 407, 319-320).
  • Such constructs comprise inverted regions of the target gene sequence, separated by an appropriate spacer.
  • the insertion of a functional plant intron region as a spacer fragment additionally increases the efficiency of the gene silencing induction, due to generation of an intron spliced hairpin RNA (Plant J. (2001), 27, 581-590).
  • the stem length is about 50 nucleotides to about 1 kilobases in length.
  • Interfering RNA molecules having a duplex or double-stranded structure can have blunt ends, or can have 3' or 5' overhangs.
  • overhang refers to the unpaired nucleotide or nucleotides that protrude from a duplex structure when a 3'-terminus of one RNA strand extends beyond the 5'-terminus of the other strand (3' overhang), or vice versa (5' overhang).
  • the nucleotides comprising the overhang can be ribonucleotides, deoxyribonucleotides or modified versions thereof.
  • at least one strand of the interfering RNA molecule has a 3' overhang from about 1 to about 6 nucleotides in length.
  • the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4 nucleotides in length.
  • the interfering RNA molecule comprises a 3' overhang at one end of the molecule, the other end can be blunt-ended or have also an overhang (5' or 3').
  • the interfering RNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be the same or different.
  • the interfering RNA molecule comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule.
  • the interfering RNA molecule is a double-stranded RNA having a 3' overhang of 2 nucleotides at both ends of the molecule.
  • the nucleotides comprising the overhang of the interfering RNA are TT dinucleotides or ULI dinucleotides.
  • the interfering RNA molecules can comprise one or more 5' or 3'-cap structures.
  • the term "cap structure” refers to a chemical modification incorporated at either terminus of an oligonucleotide, which protects the molecule from exonuclease degradation, and may also facilitate delivery or localisation within a cell.
  • Another modification applicable to interfering RNA molecules is the chemical linkage to the interfering RNA molecule of one or more moieties or conjugates which enhance the function, cellular distribution, cellular uptake, bioavailability or stability of the interfering RNA molecule.
  • the polynucleotides may be synthesized or modified by methods well established in the art. Chemical modifications include 2' modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and typically two, chemical linkages.
  • the nucleotides at one or both of the two single strands may be modified to modulate the activation of cellular enzymes, such as, for example, without limitation, certain nucleases.
  • cellular enzymes such as, for example, without limitation, certain nucleases.
  • Techniques for reducing or inhibiting the activation of cellular enzymes are known in the art including, but not limited to, 2'-amino modifications, 2'-fluoro modifications, 2'-alkyl modifications, uncharged backbone modifications, morpholino modifications, 2'-O-methyl modifications, and phosphoramidate.
  • Ligands may be conjugated to an interfering RNA molecule, for example, to enhance its cellular absorption.
  • a hydrophobic ligand is conjugated to the molecule to facilitate direct permeation of the cellular membrane.
  • conjugation of a cationic ligand to oligonucleotides often results in improved resistance to nucleases.
  • TILLING is another mutagenesis technology that can be used to generate and/or identify NtNTP2 polynucleotides encoding NtNTP2 polypeptides with modified expression, function or activity. TILLING also allows selection of plants carrying such mutants. TILLING combines high-density mutagenesis with high- throughput screening methods. Methods for TILLING are well known in the art (see McCallum et al., (2000) Nat Biotechnol 18: 455-457 and Stemple (2004) Nat Rev Genet 5(2): 145-50). Various embodiments are directed to expression vectors comprising one or more of the NtNTP2 polynucleotides or interfering RNA constructs that comprise one or more NtNTP2 polynucleotides described herein.
  • Various embodiments are directed to expression vectors comprising one or more of the NtNTP2 polynucleotides or one or more interfering RNA constructs described herein.
  • Various embodiments are directed to expression vectors comprising one or more NtNTP2 polynucleotides or one or more interfering RNA constructs encoding one or more interfering RNA polynucleotides described herein that are capable of self-annealing to form a hairpin structure, in which the construct comprises (a) one or more of the NtNTP2 polynucleotides described herein; (b) a second sequence encoding a spacer element that forms a loop of the hairpin structure; and (c) a third sequence comprising a reverse complementary sequence of the first sequence, positioned in the same orientation as the first sequence, wherein the second sequence is positioned between the first sequence and the third sequence, and the second sequence is operably-linked to the first sequence and to the third sequence.
  • RNA can be formed by (1) transcribing a first strand of the DNA by operably-linking to a first promoter, and (2) transcribing the reverse complementary sequence of the first strand of the DNA fragment by operably- linking to a second promoter.
  • Each strand of the polynucleotide can be transcribed from the same expression vector, or from different expression vectors.
  • the RNA duplex having RNA interference can be enzymatically converted to siRNAs to modulate RNA levels.
  • various embodiments are directed to expression vectors comprising one or more NtNTP2 polynucleotides or interfering RNA constructs described herein encoding interfering RNA polynucleotides capable of self-annealing, in which the construct comprises (a) one or more of the NtNTP2 polynucleotides described herein; and (b) a second sequence comprising a complementary (for example, reverse complementary) sequence of the first sequence, positioned in the same orientation as the first sequence.
  • compositions and methods are provided for modulating the endogenous expression levels of one or more of the NtNTP2 polypeptides described herein (or any combination thereof as described herein) by promoting co-suppression of gene expression.
  • compositions and methods are provided for modulating the endogenous gene expression level by modulating the translation of mRNA.
  • a host plant cell can be transformed with an expression vector comprising: a promoter operably-linked to a NtNTP2 polynucleotide, positioned in anti-sense orientation with respect to the promoter to enable the expression of RNA polynucleotides having a sequence complementary to a portion of mRNA.
  • RNA molecules for modulating the translation of mRNA may comprise: a promoter operably-linked to a NtNTP2 polynucleotide in which the sequence is positioned in anti-sense orientation with respect to the promoter.
  • the lengths of anti-sense RNA polynucleotides can vary, and may be from about 15-20 nucleotides, about 20-30 nucleotides, about 30-50 nucleotides, about 50-75 nucleotides, about 75-100 nucleotides, about 100-150 nucleotides, about 150-200 nucleotides, and about 200-300 nucleotides.
  • genes can be targeted for inactivation by introducing transposons (for example, IS elements) into the genomes of plants of interest.
  • transposons for example, IS elements
  • These mobile genetic elements can be introduced by sexual cross-fertilization and insertion mutants can be screened for loss in polypeptide function.
  • the disrupted gene in a parent plant can be introduced into other plants by crossing the parent plant with plant not subjected to transposon-induced mutagenesis by, for example, sexual cross-fertilization. Any standard breeding techniques known to persons skilled in the art can be utilized.
  • one or more genes can be inactivated by the insertion of one or more transposons.
  • Mutations can result in homozygous disruption of one or more genes, in heterozygous disruption of one or more genes, or a combination of both homozygous and heterozygous disruptions if more than one gene is disrupted.
  • Suitable transposable elements include retrotransposons, retroposons, and SI NE-like elements. Such methods are known to persons skilled in the art.
  • genes can be targeted for inactivation by introducing ribozymes derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. These RNAs can replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples of suitable RNAs include those derived from avocado sunblotch viroid and satellite RNAs derived from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus, and subterranean clover mottle virus. Various target RNA-specific ribozymes are known to persons skilled in the art.
  • the mutant or non-naturally occurring plants or plant cells can have any combination of one or more mutations in one or more genes which results in modulated expression or function or activity of those genes or their products.
  • the mutant or non-naturally occurring plants or plant cells may have a single mutation in a single gene; multiple mutations in a single gene; a single mutation in two or more or three or more or four or more genes; or multiple mutations in two or more or three or more or four or more genes. Examples of such mutations are described herein.
  • the mutant or non-naturally occurring plants or plant cells may have one or more mutations in a specific portion of the NtNTP2 gene(s) - such as in a region of the gene that encodes an active site of the NtNTP2 polypeptide or a portion thereof.
  • the mutant or non-naturally occurring plants or plant cells may have one or more mutations in a region outside of one or more NtNTP2 gene(s) - such as in a region upstream or downstream of the gene it regulates provided that they modulate the function or expression of the NtNTP2 gene(s).
  • Upstream elements can include promoters, enhancers or transcription factors.
  • Some elements - such as enhancers - can be positioned upstream or downstream of the gene it regulates.
  • the element(s) need not be located near to the gene that it regulates since some elements have been found located several hundred thousand base pairs upstream or downstream of the gene that it regulates.
  • the mutant or non-naturally occurring plants or plant cells may have one or more mutations located within the first 100 nucleotides of the gene(s), within the first 200 nucleotides of the gene(s), within the first 300 nucleotides of the gene(s), within the first 400 nucleotides of the gene(s), within the first 500 nucleotides of the gene(s), within the first 600 nucleotides of the gene(s), within the first 700 nucleotides of the gene(s), within the first 800 nucleotides of the gene(s), within the first 900 nucleotides of the gene(s), within the first 1000 nucleotides of the gene(s), within the first 1100 nucleotides of the gene(s), within the first 1200 nucleotides of the gene(s), within the first 1300 nucleotides of the gene(s), within the first 1400 nucleotides of the gene(s) or within the first 1500 nucleotides of the gene(s).
  • the mutant or non-naturally occurring plants or plant cells may have one or more mutations located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth set of 100 nucleotides of the gene(s) or combinations thereof.
  • Mutant or non-naturally occurring plants or plant cells comprising the mutant NtNTP2 polypeptides are disclosed.
  • seeds from plants are mutagenised and then grown into first generation mutant plants.
  • the first generation plants are then allowed to self-pollinate and seeds from the first generation plant are grown into second generation plants, which are then screened for mutations in their loci.
  • the mutagenized plant material can be screened for mutations, an advantage of screening the second generation plants is that all somatic mutations correspond to germline mutations.
  • plant materials including but not limited to, seeds, pollen, plant tissue or plant cells, may be mutagenised in order to create the mutant plants.
  • the type of plant material mutagenised may affect when the plant polynucleotide is screened for mutations.
  • the seeds resulting from that pollination are grown into first generation plants. Every cell of the first generation plants will contain mutations created in the pollen; thus these first generation plants may then be screened for mutations instead of waiting until the second generation.
  • Prepared NtNTP2 polynucleotide from individual plants, plant cells, or plant material can optionally be pooled in order to expedite screening for mutations in the population of plants originating from the mutagenized plant tissue, cells or material.
  • One or more subsequent generations of plants, plant cells or plant material can be screened.
  • the size of the optionally pooled group is dependent upon the sensitivity of the screening method used.
  • the samples can be subjected to NtNTP2 polynucleotidespecific amplification techniques, such as PCR.
  • Any one or more primers or probes specific to the NtNTP2 gene or the sequences immediately adjacent to the NtNTP2 gene may be utilized to amplify the sequences within the optionally pooled sample.
  • the one or more primers or probes are designed to amplify the regions of the locus where useful mutations are most likely to arise.
  • the primer is designed to detect mutations within regions of the NtNTP2 polynucleotide.
  • it is preferable for the primer(s) and probe(s) to avoid known polymorphic sites in order to ease screening for point mutations.
  • the one or more primers or probes may be labelled using any conventional labelling method. Primer(s) or probe(s) can be designed based upon the sequences described herein using methods that are well understood in the art.
  • the primer(s) or probe(s) may be labelled using any conventional labelling method. These can be designed based upon the sequences described herein using methods that are well understood in the art.
  • Polymorphisms may be identified by means known in the art and some have been described in the literature.
  • a plant may be regenerated or grown from the plant, plant tissue or plant cell. Any suitable methods for regenerating or growing a plant from a plant cell or plant tissue may be used, such as, without limitation, tissue culture or regeneration from protoplasts.
  • plants may be regenerated by growing transformed plant cells on callus induction media, shoot induction media and/or root induction media. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified.
  • transformed seeds refers to seeds that contain the nucleotide construct stably integrated into the plant genome.
  • a method of preparing a mutant plant involves providing at least one cell of a plant comprising a NtNTP2 gene encoding a functional NtNTP2 polypeptide.
  • the at least one cell of the plant is treated under conditions effective to modulate (reduce) the function of the NtNTP2 polynucleotide(s) described herein.
  • the at least one mutant plant cell is then propagated into a mutant plant, where the mutant plant has a modulated (reduced or inhibited) level of NtNTP2 polypeptide(s) as compared to that of a control plant.
  • the treating step involves subjecting the at least one cell to a chemical mutagenising agent as described above and under conditions effective to yield at least one mutant plant cell.
  • the treating step involves subjecting the at least one cell to a radiation source under conditions effective to yield at least one mutant plant cell.
  • mutant plant includes mutant plants in which the genotype is modified as compared to a control plant, suitably by means other than genetic engineering or genetic modification.
  • the mutant plant, mutant plant cell or mutant plant material may comprise one or more mutations that have occurred naturally in another plant, plant cell or plant material and confer a desired trait.
  • This mutation can be incorporated (for example, introgressed) into another plant, plant cell or plant material (for example, a plant, plant cell or plant material with a different genetic background to the plant from which the mutation was derived) to confer the trait thereto.
  • a mutation that occurred naturally in a first plant may be introduced into a second plant - such as a second plant with a different genetic background to the first plant.
  • the skilled person is therefore able to search for and identify a plant carrying naturally in its genome one or more mutant alleles of the genes described herein which confer a desired trait.
  • the mutant allele(s) that occurs naturally can be transferred to the second plant by various methods including breeding, backcrossing and introgression to produce a lines, varieties or hybrids that have one or more mutations in the genes described herein.
  • the same technique can also be applied to the introgression of one or more non-naturally occurring mutation(s) from a first plant into a second plant.
  • Plants showing a desired trait may be screened out of a pool of mutant plants.
  • the selection is carried out utilising the knowledge of the NtNTP2 polynucleotide as described herein. Consequently, it is possible to screen for a genetic trait as compared to a control.
  • Such a screening approach may involve the application of conventional amplification and/or hybridization techniques as discussed herein.
  • a further aspect of the present disclosure relates to a method for identifying a mutant plant comprising the steps of: (a) providing a sample comprising NtNTP2 polynucleotide from a plant; and (b) determining the sequence of the NtNTP2 polynucleotide, wherein a difference in the sequence of the NtNTP2 polynucleotide as compared to the NtNTP2 polynucleotide of a control plant is indicative that said plant is a mutant plant.
  • a method for identifying a mutant plant which: (i) does not have decreased nitrate levels as compared to a control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response as compared to the control plant grown in the same fertilization conditions comprising: (a) providing a sample from a plant to be screened; (b) determining if said sample comprises one or more mutations in one or more of the NtNTP2 polynucleotides described herein; and (c) determining the nitrate levels, the biomass and the NUE response as compared to the control plant grown in the same fertilization conditions.
  • a method for preparing a mutant plant which: (i) does not have decreased nitrate levels as compared to a control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response as compared to the control plant grown in the same fertilization conditions comprising: (a) providing a sample from a first plant;
  • the mutant plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • the mutation(s) can be transferred into the second plant using various methods that are known in the art - such as by genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing and the like.
  • the first plant is a naturally occurring plant.
  • the second plant has a different genetic background to the first plant.
  • a method for preparing a mutant plant which: (i) has decreased nitrate levels as compared to a control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response as compared to the control plant grown in the same fertilization conditions comprising: (a) providing a sample from a first plant; (b) determining if said sample comprises one or more mutations in one or more of the NtNTP2 polynucleotides described herein that results in decreased nitrate levels, increased biomass and increased NUE response as compared to the control plant grown in the same fertilization conditions; and (c) introgressing the one or more mutations from the first plant into a second plant.
  • the mutant plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • the step of introgressing comprises plant breeding, optionally including backcrossing and the like.
  • the first plant is a naturally occurring plant.
  • the second plant has a different genetic background to the first plant.
  • the first plant is not a cultivar or an elite cultivar.
  • the second plant is a cultivar or an elite cultivar.
  • a further aspect relates to a mutant plant (including a cultivar or elite cultivar mutant plant) obtained or obtainable by the methods described herein.
  • the “mutant plants” may have one or more mutations localised only to a specific region of the plant - such as within the sequence of the one or more NtNTP2 polynucleotide(s) described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be the same or substantially the same as the plant prior to the mutagenesis.
  • the mutant plants may have one or more mutations localised in more than one genomic region of the plant - such as within the sequence of one or more of the NtNTP2 polynucleotides described herein and in one or more further regions of the genome.
  • the mutant plants may not have one or more mutations in one or more, two or more, three or more, four or more or five or more exons of the polynucleotide(s) described herein; or may not have one or more mutations in one or more, two or more, three or more, four or more or five or more introns of the polynucleotide(s) described herein; or may not have one or more mutations in a promoter of the polynucleotide(s) described herein; or may not have one or more mutations in the 3’ untranslated region of the polynucleotide(s) described herein; or may not have one or more mutations in the 5’ untranslated region of the polynucleotide(s) described herein; or may not have one or more mutations in the coding region of the polynucle
  • a method of identifying a plant, a plant cell or plant material comprising a mutation in a gene encoding a NtNTP2 polynucleotide as described herein comprising: (a) subjecting a plant, a plant cell or plant material to mutagenesis; (b) obtaining a sample from said plant, plant cell or plant material or descendants thereof; and (c) determining the polynucleotide sequence of the NtNTP2 gene or a variant or a fragment thereof, wherein a difference in said sequence is indicative of one or more mutations therein.
  • This method also allows the selection of plants having mutation(s) that occur(s) in genomic regions that affect the expression of the NtNTP2 gene in a plant cell, such as a transcription initiation site, a start codon, a region of an intron, a boundary of an exon-intron, a terminator, or a stop codon.
  • the mutant, non-naturally occurring or transgenic plant or part thereof according to the present disclosure has an advantageous phenotype in which there is no significant difference in nitrate levels as compared to a control plant grown in the same fertilization conditions, leaf biomass yield is higher as compared to the control plant grown in the same fertilization conditions, and the NUE of the plant is higher as compared to the control plant grown in the same fertilization conditions.
  • a morphological difference observed between the mutant, non-naturally occurring or transgenic plants as compared to the control is an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • the mutant, non-naturally occurring or transgenic plant or part thereof according to the present disclosure generates more biomass - (for example, leaf biomass) as compared to a control.
  • leaf biomass is green leaves or dried leaves or cured leaves, more suitably, green leaves.
  • the increase in biomass can be at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29% or at least about 30% or at least about 31%, or at least about 34% or at least about 35%, or at least about 36%, or at least about 37% or more as compared to the control grown in the same conditions.
  • the amount of biomass that is generated can be higher in a nitrogen starvation regime as compared to a standard fertilization regime.
  • the increase in biomass in a standard fertilization regimeor in a nitrogen starvation regime, can be at least about 5% or more as compared to the control grown in the same conditions. In one embodiment, the increase in biomass in nitrogen starvation conditions is at least about 28% as compared to a control grown in the same conditions. Accordingly, NtNTP2 loss of activity results in an improved yield in different nitrogen regimes.
  • the NUE index (i.e. the units of biomass produced (expressed as kilograms per hectare, assuming a number of 12 thousand plants per hectare) per unit of nitrogen fertilization input (expressed as kilograms of nitrogen per hectare)) determined for the mutant, non-naturally occurring or transgenic plant or part thereof according to the present disclosure can be increased when grown in nitrogen starving conditions by at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21 %, or at least about 22% or more as compared to the control.
  • the NUE index can be increased by at least about 5%, at least about 9%, at least about 10%, at least about 13%, or at least about 14%, at least about 20%, a least about 25% or at least about 28% or more as compared to the control grown in the same conditions.
  • the increase in NUE index in nitrogen starvation conditions is at least about 28% as compared to the control grown in the same conditions. Accordingly, NtNTP2 loss of activity results in an improved NUE in different nitrogen regimes.
  • impairment in NTP2 protein activity can increase the plant’s ability to adjust to nitrogen starvation, thereby increasing the NUE of the plant, intended as biomass per unit of nitrogen fertilization applied.
  • the mutant, non-naturally occurring or transgenic plant or part thereof has an advantageous phenotype in which there is no significant difference in nitrate levels as compared to a control plant grown in the same fertilization conditions, biomass yield is increased by at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29% or at least about 30% or at least about 31%, or at least about 34% or at least about 35%, or at least about 36%, or at least about 37% or more as compared to the control and the NUE of the plant is increased by at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19
  • the plant or the part of the plant in standard fertilisation conditions: (i) does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and (ii) has at least a 5% biomass increase as compared to the control plant grown in the same fertilization conditions; and (iii) has at least a 5% increase in NUE response as compared to the control plant grown in the same fertilization conditions.
  • the plant or part thereof can have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • the plant or the part of the plant in nitrogen starvation conditions: (i) does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and (ii) has at least a 5% biomass increase as compared to the control plant grown in the same fertilization conditions; and (iii) has at least a 5% increase in NUE response as compared to the control plant grown in the same fertilization conditions.
  • the plant or part thereof can have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • the phenotype differs according to the fertilization input.
  • the plant or the part of the plant for example, leaf: (i) does not have decreased nitrate levels as compared to the control plant; and (ii) has at least a 5 % biomass increase as compared to the control plant; and (iii) has at least a 5% increase in NUE response as compared to the control plant.
  • the plant or the part of the plant in nitrogen starvation conditions: (i) does not have decreased nitrate levels as compared to the control plant; and (ii) has at least a 5% biomass increase as compared to the control plant; and (iii) has at least a 5% increase in NUE response as compared to the control plant.
  • the plant or part thereof can have an increase in root development as compared to the control plant grown in the same fertilization conditions.
  • the plants of the present disclosure therefore have increased yield with lower fertilization input and therefore an improved NUE.
  • the lower fertilization input can achieve a reduction in TSNAs.
  • ‘standard conditions’ are 254 nitrogen units (one unit expressed as kilograms per hectare) and ‘nitrogen starving conditions’ are 55 nitrogen units (one unit expressed as kilograms per hectare).
  • Plants according to the present disclosure include, but are not limited to, monocotyledonous and dicotyledonous plants and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae,
  • Suitable species may include members of the genera Abelmoschus, Abies, Acer, Agrostis,
  • Suitable species may include Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp.
  • Phleum pratense timothy
  • Panicum virgatum switchgrass
  • Sorghu56yclise56or sorghum, sudangrass
  • Miscanthus giganteus micanthus
  • Suitable species may include species of the genus Nicotiana, including N. rustica and N. tabacum (for example, LA B21 , LN KY171 , Tl 1406, Basma, Galpao, Perique, Beinhart 1000- 1 , and Petico).
  • Other Nicotiana species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, N. benavidesii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. Cleveland ii, N.
  • cordi folia N. corymbosa, N. debneyi, N. excelsior, N. forgetiana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. linearis, N. longiflora, N. maritime, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N. occidentalis subsp. hesperis, N. otophora, N. paniculata, N.
  • the plant is N. tabacum.
  • the transgenic, non-naturally occurring or mutant plant may therefore be a tobacco variety or elite tobacco cultivar that comprises one or more transgenes, or one or more genetic mutations or a combination thereof.
  • the genetic mutation(s) (for example, one or more polymorphisms) can be mutations that do not exist naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar) or can be genetic mutation(s) that do occur naturally provided that the mutation does not occur naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar).
  • Particularly useful N. tabacum varieties include Burley type, dark type, flue-cured type, and Oriental type tobaccos.
  • Non-limiting examples of varieties or cultivars are: BD 64, CC 101 , CC 200, CC 27, CC 301 , CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911 , DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171 , KY 907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McN
  • Embodiments are also directed to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that have been modified to modulate the expression or function of a NtNTP2 polynucleotide(s) described herein (or any combination thereof as described herein).
  • the mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that are obtained may be similar or substantially the same in overall appearance to control plants.
  • Various phenotypic characteristics such as degree of maturity, number of leaves per plant, stalk height, leaf insertion angle, leaf size (width and length), internode distance, and lamina-midrib ratio can be assessed by field observations.
  • One aspect relates to a seed of a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant described herein.
  • a further aspect relates to pollen or an ovule of a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant that is described herein.
  • a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant as described herein which further comprises a polynucleotide conferring male sterility.
  • the regenerable cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and a part thereof, ovules, shoots, stems, stalks, pith and capsules or callus or protoplasts derived therefrom.
  • the mutant, non-naturally occurring or transgenic plant leaf or part of the plant leaf obtained according to this disclosure can be similar or substantially the same in visual appearance to the corresponding control plant leaf or part of the plant leaf.
  • the leaf number is substantially the same as the control.
  • the chlorophyll content is substantially the same as the control plant grown in the same fertilization conditions.
  • the size or form or number or colouration of the leaf is substantially the same as the control plant grown in the same fertilization conditions.
  • Polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of the NtNTP2 polynucleotides or NtNTP2 polypeptides described herein.
  • a plant carrying a mutant allele of one or more NtNTP2 polynucleotides described herein (or any combination thereof as described herein) can be used in a plant breeding program to create useful lines, varieties and hybrids containing leaf of the desired genotype and phenotype.
  • the mutant allele is introgressed into the commercially important varieties described above.
  • methods for breeding plants that comprise crossing a mutant plant, a non-naturally occurring plant or a transgenic plant as described herein with a plant comprising a different genetic identity.
  • the method may further comprise crossing the progeny plant with another plant, and optionally repeating the crossing until a progeny with the desirable genotype and phenotype is obtained.
  • breeding methods One purpose served by such breeding methods is to introduce a desirable genetic trait into other varieties, breeding lines, hybrids or cultivars, particularly those that are of commercial interest. Another purpose is to facilitate stacking of genetic modifications of different genes in a single plant variety, lines, hybrids or cultivars. Intraspecific as well as interspecific matings are contemplated. The progeny plants that arise from such crosses, also referred to as breeding lines, are examples of non-naturally occurring plants of the disclosure.
  • a method for producing a non-naturally occurring plant comprising: (a) crossing a mutant or transgenic plant with a second plant to yield progeny tobacco seed; (b) growing the progeny tobacco seed, under plant growth conditions, to yield the non-naturally occurring plant.
  • the method may further comprise: (c) crossing the previous generation of non-naturally occurring plant with itself or another plant to yield progeny tobacco seed; (d) growing the progeny tobacco seed of step (c) under plant growth conditions, to yield additional non-naturally occurring plants; and repeating the crossing and growing steps of (c) and (d) multiple times to generate further generations of non-naturally occurring plants.
  • the method may optionally comprises prior to step (a), a step of providing a parent plant which comprises a genetic identity that is characterized and that is not identical to the mutant or transgenic plant.
  • the crossing and growing steps are repeated from 0 to 2 times, from 0 to 3 times, from 0 to 4 times, 0 to 5 times, from 0 to 6 times, from 0 to 7 times, from 0 to 8 times, from 0 to 9 times or from 0 to 10 times, in order to generate generations of non-naturally occurring plants.
  • Backcrossing is an example of such a method wherein a progeny is crossed with one of its parents or another plant genetically similar to its parent, in order to obtain a progeny plant in the next generation that has a genetic identity which is closer to that of one of the parents.
  • Techniques for plant breeding, particularly plant breeding, are well known and can be used in the methods of the disclosure.
  • the disclosure further provides non-naturally occurring plants produced by these methods. Certain embodiments exclude the step of selecting a plant.
  • leaf or a part of the leaf is harvested from the produced plant(s).
  • lines resulting from breeding and screening for variant NtNTP2 genes are evaluated in the field using standard field procedures.
  • Control genotypes including the original unmutagenised parent are included and entries are arranged in the field in a randomized complete block design or other appropriate field design.
  • standard agronomic practices are used, for example, the tobacco is harvested, weighed, and sampled for chemical and other common testing before and during curing.
  • Statistical analyses of the data are performed to confirm the similarity of the selected lines to the parental line. Cytogenetic analyses of the selected plants are optionally performed to confirm the chromosome complement and chromosome pairing relationships.
  • DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant alleles of a NtNTP2 gene into other tobaccos, as described herein.
  • MAS marker-assisted selection
  • a breeder can create segregating populations from hybridizations of a genotype containing a mutant NtNTP2 allele of interest with an agronomically desirable genotype. Plants in the F2 or backcross generations can be screened using a marker developed from a genomic sequence or a fragment thereof, using one of the techniques listed herein.
  • Plants identified as possessing the mutant NtNTP2 allele can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected inheritance pattern, it may be necessary to self-pollinate the selected plants before each cycle of backcrossing to aid identification of the desired individual plants. Backcrossing or other breeding procedure can be repeated until the desired phenotype of the recurrent parent is recovered.
  • successful crosses yield F1 plants that are fertile.
  • Selected F1 plants can be crossed with one of the parents, and the first backcross generation plants are self-pollinated to produce a population that is again screened for variant NtNTP2 gene expression (for example, the null version of the gene).
  • the process of backcrossing, self-pollination, and screening is repeated, for example, at least 4 times until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent.
  • This plant if desired, is self-pollinated and the progeny are subsequently screened again to confirm that the plant exhibits variant NtNTP2 gene expression.
  • a plant population in the F2 generation is screened for variant NtNTP2 gene expression, for example, a plant is identified that fails to express a NtNTP2 polypeptide due to the absence of the NtNTP2 gene according to standard methods, for example, by using a PCR method with primers based upon the polynucleotide sequence information for the NtNTP2 polynucleotide(s) described herein (or any combination thereof as described herein).
  • Hybrid tobacco varieties can be produced by preventing self-pollination of female parent plants (that is, seed parents) of a first variety, permitting pollen from male parent plants of a second variety to fertilize the female parent plants, and allowing F1 hybrid seeds to form on the female plants.
  • Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development.
  • pollen formation can be prevented on the female parent plants using a form of male sterility.
  • male sterility can be produced by cytoplasmic male sterility (CMS), or transgenic male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or self-incompatibility.
  • CMS cytoplasmic male sterility
  • transgenic male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or self-incompatibility.
  • Female parent plants containing CMS are particularly useful. In embodiments in which the female parent plants are CMS, pollen is harvested from male fertile plants and applied manually to the stigmas of CMS female parent plants, and the resulting F1 seed is harvested.
  • Varieties and lines described herein can be used to form single-cross tobacco F1 hybrids.
  • the plants of the parent varieties can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants.
  • the F1 seed formed on the female parent plants is selectively harvested by conventional means.
  • One also can grow the two parent plant varieties in bulk and harvest a blend of F1 hybrid seed formed on the female parent and seed formed upon the male parent as the result of self-pollination.
  • three-way crosses can be carried out wherein a single-cross F1 hybrid is used as a female parent and is crossed with a different male parent.
  • double-cross hybrids can be created wherein the F1 progeny of two different single-crosses are themselves crossed.
  • a population of mutant, non-naturally occurring or transgenic plants can be screened or selected for those members of the population that have a desired trait or phenotype.
  • a population of progeny of a single transformation event can be screened for those plants having a desired level of expression or function of the NtNTP2 polypeptide(s) encoded thereby.
  • Physical and biochemical methods can be used to identify expression or activity levels.
  • RNA transcripts include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, S1 RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme function of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides.
  • Other techniques such as in situ hybridization, enzyme staining, and immunostaining and enzyme assays also can be used to detect the presence or expression, function or activity of NtNTP2 polypeptides or NtNTP2 polynucleotides.
  • Mutant, non-naturally occurring or transgenic plant cells and plants are described herein comprising one or more recombinant NtNTP2 polynucleotides, one or more NtNTP2 polynucleotide constructs, one or more double-stranded RNAs, one or more conjugates or one or more vectors/expression vectors.
  • One or more of the following further genetic modifications can be present in the mutant, non- naturally occurring or transgenic plant leaf or part of the plant leaf.
  • One or more genes that are involved in the conversion of nitrogenous metabolic intermediates can be modified resulting in lower levels of at least one tobacco-specific nitrosamine (TSNA).
  • TSNA tobacco-specific nitrosamine
  • Non-limiting examples of such genes include those encoding nicotine demethylase - such as CYP82E4, CYP82E5 and CYP82E10 as described in W02006/091194, W02008/070274, W02009/064771 and WO2011/088180 - and nitrate reductase, as described in WO2016046288.
  • genes that are involved in heavy metal uptake or heavy metal transport can be modified resulting in lower heavy metal content.
  • Non-limiting examples include genes in the family of multidrug resistance associated polypeptides, the family of cation diffusion facilitators (CDF), the family of Zrt- 1 rt-like polypeptides (ZIP), the family of cation exchangers (CAX), the family of copper transporters (COPT), the family of heavy-metal ATPases (for example, HMAs, as described in W02009/074325 and WO2017/129739), the family of homologs of natural resistance-associated macrophage polypeptides (NRAMP), and other members of the family of ATP-binding cassette (ABC) transporters (for example, MRPs), as described in WO20 12/028309, which participate in transport of heavy metals - such as cadmium.
  • CDF family of cation diffusion facilitators
  • ZIP Zrt- 1 rt-like polypeptides
  • CAX family of
  • Glyphosate resistant transgenic plants have been developed by transferring the aroA gene (a glyphosate EPSP synthetase from Salmonella typhimurium and E.coli). Sulphonylurea resistant plants have been produced by transforming the mutant ALS (acetolactate synthetase) gene from Arabidopsis. OB polypeptide of photosystem II from mutant Amaranthus hybridus has been transferred in to plants to produce atrazine resistant transgenic plants; and bromoxynil resistant transgenic plants have been produced by incorporating the bxn gene from the bacterium Klebsiella pneumoniae.
  • aroA gene a glyphosate EPSP synthetase from Salmonella typhimurium and E.coli
  • Sulphonylurea resistant plants have been produced by transforming the mutant ALS (acetolactate synthetase) gene from Arabidopsis.
  • OB polypeptide of photosystem II from mutant Amaranthus hybridus has been transferred in to plants
  • Bacillus thuringiensis (Bt) toxins can provide an effective way of delaying the emergence of Bt-resistant pests, as recently illustrated in broccoli where pyramided crylAc and cry1C Bt genes controlled diamondback moths resistant to either single polypeptide and significantly delayed the evolution of resistant insects.
  • Another exemplary modification results in plants that are resistant to diseases caused by pathogens (for example, viruses, bacteria, fungi).
  • Plants expressing the Xa21 gene (resistance to bacterial blight) with plants expressing both a Bt fusion gene and a chitinase gene (resistance to yellow stem borer and tolerance to sheath) have been engineered.
  • Another exemplary modification results in altered reproductive capability, such as male sterility.
  • abiotic stress for example, drought, temperature, salinity
  • tolerant transgenic plants have been produced by transferring acyl glycerol phosphate enzyme from Arabidopsis; genes coding mannitol dehydrogenase and sorbitol dehydrogenase which are involved in synthesis of mannitol and sorbitol improve drought resistance.
  • Another exemplary modification results in plants in which the activity of one or more endogenous glycosyltransferases - such as N-acetylglucosaminyltransferase, (3(1 ,2)- xylosyltransferase and a(1 ,3)-fucosyl- transferase is modulated (see WO/2011/117249).
  • endogenous glycosyltransferases such as N-acetylglucosaminyltransferase, (3(1 ,2)- xylosyltransferase and a(1 ,3)-fucosyl- transferase is modulated (see WO/2011/117249).
  • Another exemplary modification results in plants in which the activity of one or more nicotine N-demethylases is modulated such that the levels of nornicotine and metabolites of nornicotine - that are formed during curing can be modulated (see WO2015169927).
  • SAM S-adenosyl-L-methionine
  • CGS cystathionine gamma-synthase
  • One or more genes that are involved in the nicotine synthesis pathway can be modified resulting in plants or parts of plants that when cured, produce modulated levels of nicotine.
  • the nicotine synthesis genes can be selected from the group consisting of: A622, BBLa, BBLb, JRE5L1 , JRE5L2, MATE1 , MATE 2, MPO1 , MPO2, MYC2a, MYC2b, NBB1 , nic1 , nic2, NUP1 , NLIP2, PMT 1 , PMT2, PMT3, PMT4 and QPT or a combination of one or more thereof.
  • Alkaloid level controlling genes can be selected from the group consisting of; BBLa, BBLb, JRE5L1 , JRE5L2, MATE1 , MATE 2, MYC2a, MYC2b, nic1 , nic2, NUP1 and NUP2 or a combination of two or more thereof.
  • Leaf material - such as lamina and midrib - can be incorporated into or used in making various consumable products including but not limited to aerosol forming materials, aerosol forming devices, smoking articles, smokable articles, smokeless products, medicinal or cosmetic products, intravenous preparations, tablets, powders, and tobacco products.
  • aerosol forming materials include tobacco compositions, tobaccos, tobacco extract, cut tobacco, cut filler, cured tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobaccos.
  • Smoking articles and smokable articles are types of aerosol forming devices. Examples of smoking articles or smokable articles include cigarettes, cigarillos, and cigars. Examples of smokeless products comprise chewing tobaccos, and snuffs.
  • a tobacco composition or another aerosol forming material is heated by one or more electrical heating elements to produce an aerosol.
  • an aerosol is produced by the transfer of heat from a combustible fuel element or heat source to a physically separate aerosol forming material, which may be located within, around or downstream of the heat source.
  • Smokeless tobacco products and various tobacco-containing aerosol forming materials may contain tobacco in any form, including as dried particles, shreds, granules, powders, or a slurry, deposited on, mixed in, surrounded by, or otherwise combined with other ingredients in any format, such as flakes, films, tabs, foams, or beads.
  • the term ‘smoke’ is used to describe a type of aerosol that is produced by smoking articles, such as cigarettes, or by combusting an aerosol forming material.
  • leaf material - such as lamina and midrib - can be processed according to the methods described in US20190142058A1 in which a cast sheet of homogenized tobacco material is prepared by pulping cellulose fibres with water; grinding a blend of tobacco of one or more tobacco types to tobacco particles; combining the pulped cellulose fibres with the tobacco particles and with a binder to form a slurry; homogenizing the slurry; casting the slurry to form a cast sheet of homogenized tobacco material from the slurry; discarding undesired portions of the cast sheet; and introducing the discarded undesired portions of the cast sheet into the slurry.
  • leaf material - such as lamina and midrib - can be combined with a binder - such as natural pectins, such as fruit, citrus or tobacco pectins; guar gums, such as hydroxyethyl guar and hydroxypropyl guar; locust bean gums, such as hydroxyethyl and hydroxypropyl locust bean gum; alginate; starches, such as modified or derivitized starches; celluloses, such as methyl, ethyl, ethyl hydroxymethyl and carboxymethyl cellulose; tamarind gum; dextran; pullalon; konjac flour; xanthan gum and the like.
  • a binder - such as natural pectins, such as fruit, citrus or tobacco pectins
  • guar gums such as hydroxyethyl guar and hydroxypropyl guar
  • locust bean gums such as hydroxyethyl and hydroxypropyl loc
  • tobacco material can comprise the leaf material as described herein and a binder.
  • cured plant leaf material there is also provided cured plant leaf material. Processes of curing green tobacco leaves are known by those having skills in the art and include without limitation air-curing, fire-curing, flue-curing and sun-curing as described herein.
  • tobacco products including tobacco-containing aerosol forming materials comprising plant leaf material, suitably cured leaf.
  • the tobacco products described herein can be a blended tobacco product which may further comprise unmodified tobacco.
  • mutant, non-naturally occurring or transgenic plant leaf or part of the plant leaf may have other uses in, for example, agriculture.
  • mutant, non-naturally occurring or transgenic plant leaf or part of the plant leaf described herein can be used to make animal feed and human food products.
  • the disclosure also provides methods for producing seeds comprising: cultivating the mutant plant, non-naturally occurring plant, or transgenic plant described herein, and collecting seeds from the cultivated plants.
  • Seeds from plants described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture.
  • Packaging material such as paper and cloth are well known in the art.
  • a package of seed can have a label, for example, a tag or label secured to the packaging material, a label printed on the package that describes the nature of the seeds therein.
  • compositions, methods and kits for genotyping plants for identification, selection, or breeding can comprise a means of detecting the presence of a NtNTP2 polynucleotide (or any combination thereof as described herein) in a sample of polynucleotide. Accordingly, a composition is described comprising one or more primers for specifically amplifying at least a portion of one or more of the NtNTP2 polynucleotides and optionally one or more probes and optionally one or more reagents for conducting the amplification or detection.
  • NtNTP2 gene specific oligonucleotide primers or probes comprising about 10 or more contiguous polynucleotides corresponding to the NtNTP2 polynucleotide(s) described herein are disclosed.
  • Said primers or probes may comprise or consist of about 15, 20, 25, 30, 40, 45 or 50 more contiguous polynucleotides that hybridise (for example, specifically hybridise) to the polynucleotide(s) described herein.
  • the primers or probes may comprise or consist of about 10 to 50 contiguous nucleotides, about 10 to 40 contiguous nucleotides, about 10 to 30 contiguous nucleotides or about 15 to 30 contiguous nucleotides that may be used in sequence-dependent methods of NtNTP2 gene identification (for example, Southern hybridization) or isolation (for example, in situ hybridization of bacterial colonies or bacteriophage plaques) or NtNTP2 gene detection (for example, as one or more amplification primers in amplification or detection).
  • the one or more specific primers or probes can be designed and used to amplify or detect a part or all of the polynucleotide(s).
  • two primers may be used in a PCR protocol to amplify a NtNTP2 polynucleotide fragment.
  • the PCR may also be performed using one primer that is derived from a NtNTP2 polynucleotide sequence and a second primer that hybridises to the sequence upstream or downstream of the polynucleotide sequence - such as a promoter sequence, the 3' end of the mRNA precursor or a sequence derived from a vector.
  • Examples of thermal and isothermal techniques useful for in vitro amplification of polynucleotides are well known in the art.
  • the sample may be or may be derived from a plant, a plant cell or plant material or a tobacco product made or derived from the plant, the plant cell or the plant material as described herein.
  • a method of detecting a NtNTP2 polynucleotide(s) described herein (or any combination thereof as described herein) in a sample comprising the step of: (a) providing a sample comprising, or suspected of comprising, a NtNTP2 polynucleotide; (b) contacting said sample with one or more primers or one or more probes for specifically detecting at least a portion of the NtNTP2 polynucleotide(s); and (c) detecting the presence of an amplification product, wherein the presence of an amplification product is indicative of the presence of the NtNTP2 polynucleotide(s) in the sample.
  • kits for detecting at least a portion of the NtNTP2 polynucleotide(s) which comprise one or more primers or probes for specifically detecting at least a portion of the NtNTP2 polynucleotide(s).
  • the kit may comprise reagents for NtNTP2 polynucleotide amplification - such as PCR - or reagents for probe hybridization-detection technology - such as Southern Blots, Northern Blots, in-situ hybridization, or microarray.
  • the kit may comprise reagents for antibody bindingdetection technology such as Western Blots, ELISAs, SELDI mass spectrometry or test strips.
  • the kit may comprise reagents for DNA sequencing.
  • the kit may comprise reagents and instructions for using the kit.
  • kits may comprise instructions for one or more of the methods described.
  • the kits described may be useful for genetic identity determination, phylogenetic studies, genotyping, haplotyping, pedigree analysis or plant breeding particularly with codominant scoring.
  • the present disclosure also provides a method of genotyping a plant, a plant cell or plant material comprising a NtNTP2 polynucleotide as described herein.
  • Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
  • Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance.
  • the specific method of genotyping may employ any number of molecular marker analytic techniques including amplification fragment length polymorphisms (AFLPs).
  • AFLPs amplification fragment length polymorphisms
  • AFLPs are the product of allelic differences between amplification fragments caused by polynucleotide variability.
  • the present disclosure further provides a means to follow segregation of one or more NtNTP2 genes or polynucleotides as well as chromosomal sequences genetically linked to these NtNTP2 genes or polynucleotides using such techniques as AFLP analysis.
  • the present disclosure also provides a method of improving an agronomic characteristic of a plant by reducing or inhibiting the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or (v) a NtNTP2-T poly
  • the agronomic characteristic is: (i) nitrate levels are not decreased; (ii) biomass (for example, leaf biomass) is increased in both standard and nitrogen starvation conditions; and (iii) NUE response, intended as biomass per unit of nitrogen fertilization applied, is increased.
  • the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S can be reduced or inhibited using any of the methods that are described herein.
  • one or more sequence-specific polynucleotides that can interfere with the transcription of NtNTP2-T or NtNTP2-T and NtNTP2-S can be used.
  • one or more sequencespecific polypeptides that can interfere with the stability of NtNTP2-T or NtNTP2-T and NtNTP2-S can be used.
  • gene edited NtNTP2-T or NtNTP2-T and NtNTP2-S can be used.
  • the NtNTP2-T or NtNTP2-T and NtNTP2-S are gene edited using the bacterial CRISPR/Cas system.
  • at least one genetic alteration in the NtNTP2-T polynucleotide sequence or at least one genetic alteration in the NtNTP2-T polynucleotide and the NtNTP2-S polynucleotide sequence or at least one genetic alteration in the NtNTP2-T polypeptide sequence or at least one genetic alteration in the NtNTP2-T polypeptide sequence and the NtNTP2-S polypeptide sequence can be used.
  • the at least one genetic alteration can be at least one genetic alteration that causes the encoded polypeptide(s) to terminate or end translation earlier than in the control plant.
  • the at least one genetic alteration can reduce or inhibit the expression or activity of NtNTP2-T or of NtNTP2-T and NtNTP2-S.
  • the at least one genetic alteration can comprise at least one nonsense mutation in the NtNTP2-T polynucleotide or the NtNTP2- T polypeptide or at least one nonsense mutation in the NtNTP2-T polynucleotide or NtNTP2- T polypeptide and at least one nonsense mutation in the NtNTP2-S polynucleotide or the NtNTP2-S polypeptide.
  • the mutation can be a single nucleotide polymorphism in NtNTP2-S at nucleotide position 632 or 633 or 632 and 633 of SEQ ID NO: 3, suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 632 or 633 of SEQ ID NO: 3 or a ‘g’ to ‘a’ mutation at nucleotide positions 632 and 633 of SEQ ID NO: 3.
  • the mutated NtNTP2-S polynucleotide sequence can comprise, consist or consist essentially of SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6.
  • the mutation can be a single nucleotide polymorphism in NtNTP2-T at nucleotide position 636 of SEQ ID NO: 11 , suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 635 or 636 of SEQ ID NO: 11 or a ‘g’ to ‘a’ mutation at nucleotide positions 635 and 636 of SEQ ID NO: 11.
  • the mutated NtNTP2-T polynucleotide sequence can comprise, consist or consist essentially of SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15.
  • the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide can each have at least one nonsense mutation at position W212 or position W212 and W211 , respectively.
  • the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide can comprise, consist or consist essentially of either SEQ ID NO: 16 or SEQ ID NO: 8 and SEQ ID NO: 16, respectively. Sequences deposited in databases are described herein and can change over time. Suitably the current version of sequence database(s) are relied upon. Alternatively, the release in force at the date of filing is relied upon.
  • accession numbers may be version/dated accession numbers.
  • the citeable accession numbers for the current database entry are the same as herein, but omitting the decimal point and any subsequent digits.
  • GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences (National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA; Nucleic Acids Research, 2013 Jan;41 (D1):D36-42) and accession numbers provided relate to this unless otherwise apparent.
  • the current release is relied upon. More suitably the release available at the effective filing date is relied upon.
  • GenBank database release referred to is NCBI-GenBank Release 241 : 15 December 2020.
  • the current version of sequence database(s) are relied upon.
  • the release in force at the date of filing is relied upon.
  • UniProt consortium European Bioinformatics Institute (EBI), SIB Swiss Institute of Bioinformatics and Protein Information Resource (PIR)’s UniProt Knowledgebase (UniProtKB) Release 2021_01 published 10 February 2021 is relied upon.
  • UniProt Universal Protein Resource
  • UniProt Universal Protein Resource
  • Example 1 - EMS mutant population screening for ntp2 mutations and ntp2-S W211stop/ntp2- T W212stop double mutant breeding
  • primers are designed covering part of the NtNtp2-Sar ⁇ d NtNtp2-T gene sequences. The resulting amplified fragments are sequenced. The primer pairs used to identify n/p2 stop mutants are reported in Table 1.
  • the list of stop mutants identified are reported in Table 2.
  • the Table reports details of stop codon mutations identified during EMS screening.
  • F seq and R seq columns indicate the SNP (single nucleotide polymorphism) mutation (in 5’ and 3’ to the mutation, respectively).
  • the original wild type codons and corresponding amino acids (Codon ori and AS ori columns) and the corresponding mutated versions (Codon mut and AS mut columns) are also indicated.
  • a TaqMan assay is used to screen for the different ntp2 mutations and genotypes.
  • Table 3 reports details of the primers and probes used for genotyping AA37 ntp2 W211stop and W212stop double mutant lines.
  • the identity column indicates the gene and phenotype identified (wt indicates wild type genotype, mut indicates the mutant genotype; tAg and tgA indicate the two different mutations identified in the NtNtp2-S gene).
  • F primer and R primer columns indicate the primer sequences of forward and reverse primers respectively.
  • Probe column reports the sequences of the probes used (underlined bases indicate the discriminants between wt and different mutations).
  • the double mutant Ntntp2-S W211stop/Ntntp2-T W212stop in the AA37 background is generated by crossing single mutant plants Ntntp2-S W211stop (tgA mutation) and single mutant plants Ntntp2-T W212stop.
  • Nitrite- nitrate contents are measured using the Cayman chemical Nitrate/Nitrite Colorimetric Assay Kit (Item No. 780001) according to supplier instructions and as described in FASEB Journal (1992) 6, 3051-3064; Anal. Biochem. (1982) 126, 131-138; and Methods (1995) 7, 48-54.
  • NtNtp2 genes are present in the tobacco genome, one on the chromosome 8 (-S form Ntab-TN90_AYMYSS948 in the publicly available TN90 genome) and the other on chromosome 21 (-T form Ntab-TN90_AYMYSS1317). Genomic, transcript, gene and protein sequences for the NtNtp2 genes are presented in the accompanying list of sequences.
  • the NtNTP2-T putative protein sequence is BLASTed in protein databases from different plant species (Solanum melongena, Arabidopsis thaliana, Nicotiana benthamiana and Solanum lycopersicum), and the highest homologies are reported in Table 4. Reported are the homologous proteins (predicted proteins) and the degree of identity expressed as percentage of identical residues. Table 5 reports the degree of identity with the respective predicted coding regions (CDS), expressed as percentage of identical residues.
  • AA37 tobacco plants are grown in a field under Burley regime (254 nitrogen units, noted above) during two consecutive growing seasons. At harvest time, no morphological difference is evident between the double mutant and the wild type out-segregant genotypes by observing the plants in the field, as shown in Figure 1.
  • ntp2 insertional mutants display 50-64% less nitrate content in petiole and midrib compared to wild type, and a statistically significant 13% more nitrate in the lamina (Chiu et al. (2004) supra).
  • ntp2 double stop mutant plants have a yield difference between standard and nitrogen starvation conditions that is reduced compared to the out-segregant wild type genotype (30.8% compared to 37.3%), with a statistically significant higher cured biomass for the mutants compared to the wild type plants in all conditions (see Figure 4).
  • NUE index i.e. the units of biomass produced (expressed as kilograms per hectare, assuming a number of 12 thousand plants per hectare) per unit of nitrogen fertilization input (expressed as kilograms of nitrogen per hectare), as indicated in Table 6.
  • Per ntp2 genotype are reported the total harvest cured biomass per hectare, considering 12000 plants per hectare, expressed in kilograms (Biomass), the nitrogen units available in the different fertilization regimes, expressed as kilograms of nitrogen per hectare (N units) and the NUE index calculated as Biomass per N unit applied.
  • ntp2 double stop mutant plants increase their NUE index 16% more (3.97 vs 4.61) compared to the out-segregant wild type genotype and normal nitrogen conditions ntp2 double stop mutant plants increase their NUE index 28% more (11 .49 vs 14.75) compared to the out-segregant wild type genotype. Impairment in NTP2 protein activity in tobacco therefore increases the plant’s ability to adjust to nitrogen starvation, increasing the NUE of the plant.
  • microarray data is obtained for the identification of gene candidates differentially expressed in green and ripe leaves in different tobacco varieties. Expression dynamics during air curing of Swiss Burley material, notoriously exhibiting low NUE, is compared to the corresponding profiles in the other tobacco varieties more Nitrogen Use Efficient, such as Virginia type tobacco, along their maturity and respective early curing processes. Gene candidates are selected based on a literature search and their implication in nitrogen assimilation pathways and transport. Genes differentially expressed between Virginia and Burley tobaccos are focussed upon. The NtNtp2 gene is identified as highly expressed in Burley green mature leaves and at harvesting time; in Virginia tobacco, expression levels increase during curing to reach Burley levels as shown in Figure 7.
  • NtNTP2 mutants root development is monitored in seedlings growing on agar plates and in young plantlets in hydroponic growth. The results are shown in Figure 8. In both cases NtNTP2-T single homozygous mutants and NtNTP2-S and -T double mutant plants display an increase in root development, expressed as number of lateral roots (A) or maximum length of aquatic roots (B). NtNTP2 activity impairment therefore results in an increase in root development.
  • Ntntp2-S W211stop/Ntntp2-T W212stop BC2S2 TN90 and K326 mutant plants and their wild type outsegregant controls are grown in a greenhouse in hydroponic solution carrying normal 50% of the standard nitrogen fertilization (N50%). At 4 to 6 weeks after transplant, the number of primary roots that sprout from the stele per plant is recorded, together with the average diameter.
  • the diameter of the primary lateral roots for plants grown in N50% is measured with a thickness gage (Mitutoyo ABSOLUTE, Mitutoyo Europe GmbH, BorsigstraBe 8-10 D-41469 Neuss).
  • the mutant plants generated 20% thinner primary roots compared to the outsegregant wild types, as indicated in Figure 10.
  • NtNTP2 activity results in a change in root development, generating more and thinner roots compared to wild type plants grown in the same conditions, as summarised in Figure 11. This may increase the ability of the plants to uptake nutrients from the soil.
  • RNAi RNA interference
  • Nicotiana tabacum TN90 cells are transformed with Agrobacterium tumefaciens carrying the binary vector for RNAi of NtNtp2 and plants are regenerated on kanamycin.
  • TN90 T2 seedlings, transgenics for the RNAi construct (RNAi-T2) and for the empty vector (control plants, CT-T2) are selected on kanamycin and transplanted to grow in a greenhouse in hydroponic solution in 50% nitrogen fertilization compared to standard practices (N50%).
  • N50% nitrogen fertilization compared to standard practices
  • the transgenic plants for the RNAi construct display a statistically validated higher number of primary lateral roots compared to their respective outsegregant controls (+39.7% with a p value of 0.00083 for TN90 and +47.7% with a p value of 0.000587 for K326), as shown in Figure 14.
  • the diameter of the primary lateral roots for RNAi and control plants grown in N50% is measured with a thickness gage (Mitutoyo ABSOLUTE, Mitutoyo Europe GmbH, BorsigstraBe 8-10 D-41469 Neuss).
  • the RNAi plants generated thinner primary roots compared to the outsegregant wild types, as indicated in Figure 15.
  • Impairing NtNTP2 protein activity does not determine a decrease in nitrate levels in cured lamina nor midrib in AA37 tobacco plants grown in Burley conditions, nor a major change in amount of the other chemical compounds measured.
  • a phenotype of Ntntp2 double stop mutation or Ntntp2-T homozygous mutation in tobacco is the increase in cured leaf biomass in both standard and nitrogen starvation conditions when compared to out-segregant wild type plants.
  • a further phenotype of Ntntp2 double stop mutation or Ntntp2-T homozygous mutation in tobacco is an increase in plant NUE response, when expressed as kilograms of cured leaf biomass produced per kilogram of nitrogen input per hectare. This makes NtNtp2 genes good targets for NUE solutions.
  • a phenotype of RNAi engineered NtNtp2-T tobacco plants in which NtNTP2 expression and/or activity is impaired is a change in root development, generating more and thinner roots compared to wild type plants grown in the same conditions. This may increase the ability of the plants to uptake nutrients from soil.
  • SEQ ID NO: 8 NtNtp2-S truncated polypeptide encoded by SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6; stop codon at position W211 of SEQ ID NO: 7
  • SEQ ID NO: 12 NtNtp2-T polypeptide sequence encoded by SEQ ID NO: 11

Abstract

There is disclosed a mutant, non-naturally occurring or transgenic plant or part of the plant having reduced or inhibited expression or activity of NtNTP2-T or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or (v) a NtNTP2-T polypeptide having at least 77% sequence identity to SEQ ID NO:12; wherein the expression or activity of the NtNTP2-T or the expression or activity of the NtNTP2-T and the NtNTP2-S is reduced or inhibited as compared to a control plant.

Description

INCREASING LEAF BIOMASS AND NITROGEN USE EFFICIENCY BY REGULATING NTP2
FIELD OF THE INVENTION
The present invention generally relates to a mutant, non-naturally occurring or transgenic plant leaf or part of the plant leaf having modulated expression or activity of NTP2. In particular, the present invention relates to a mutant, non-naturally occurring or transgenic plant or part of the plant (such as leaf) having reduced or inhibited expression or activity of NTP2 which confers an increase in biomass and Nitrogen Use Efficiency (NUE) response, without decreasing nitrate levels in the plant or part of the plant.
BACKGROUND OF THE INVENTION
Modern industrial agriculture seeks to constantly improve crop yield against costs. Nitrogen fertilizers enable farmers to achieve these improved yields. Depending on the crop, fertilizer use is accountable for a significant part of the cost of production and has associated risks of a negative impact on the environment as 50-70% of the applied nitrogen is lost from the plantsoil system and causes pollution. Improving nitrogen utilization efficiency (NUE) is important to reduce the cost of crop production as well as damage to the environment.
Reducing tobacco nitrate levels in tobacco plants reduces tobacco-specific nitrosamine (TSNAs) accumulation in cured leaves and cigarette smoke (Lu et al. (2016) Plant Biotechnology Journal, 14: 1500-1510 and WO 2016046288). TSNAs are a class of compounds that are predominantly produced during the curing of tobacco leaves, though additional formation can occur in the subsequent processing and storage of leaf, and possibly via pyrosynthesis during combustion. Two of the TSNAs found in the cured leaf, N- nitrosonornicotine (NNN) and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), are classified as Group I carcinogens (the highest designation) by the International Agency for Research on Cancer. Due to the volume of evidence implicating these compounds with various tobacco-associated cancers, the World Health organization and other experts in the field have recommended that mandates be implemented to ensure that future tobacco products have reduced levels of these toxicants. TSNAs have been shown to be strong carcinogens in numerous animal studies (Hecht (1998) Chem Res Toxicol, Jun;11 (6):559- 603; Ma etal. (2018) Carcinogenesis, Feb 9;39(2):232-241 ; Kovi et al. (2018) Toxicol Pathol., Feb;46(2):184-192; Carlson et al. (2018) Chem Res Toxicol., May 21 ;31 (5):358-370).
Whilst reducing nitrate levels can have a positive impact on reducing TSNA levels in tobacco, growing tobacco in such conditions can have a negative effect on plant biomass and quality and is therefore not a viable option, especially in relation to commercial tobacco production. There is a general need in the art to develop plants - such as tobacco plants - that have increased yield with lower fertilization input and therefore improved NUE. SUMMARY OF THE INVENTION
Two genes (NtNTP2-S and NtNTP2-T) from Nicotiana tabacum are disclosed herein which belong to the nitrate transporter family 1 NRT1.4 (PTR2 family of peptide transporters) according to homology searches against Arabidopsis thaliana AtNTP2 At2g26690. Reducing or inhibiting (for example, switching off) the expression or activity of the endogenous NtNTP2- T or both NtNTP2-S and NtNTP2-T in Nicotiana tabacum using various different methods is found to confer an advantageous phenotype that improves an agronomic characteristic as compared to a control plant grown in the same conditions. In particular: (i) nitrate levels are not decreased; (ii) biomass (for example, leaf biomass) is increased in both standard and nitrogen starvation conditions; and (iii) NUE response, intended as biomass per unit of nitrogen fertilization applied, is increased. As used herein, ‘unit’ in the context of a unit of nitrogen fertilization applied means ‘kg per hectare’, as discussed herein These results make NtNTP2-T or both NtNTP2-S and NtNTP2-T an excellent target for developing plants which in lower fertilisation input have increased yield compared to control plants grown in the same conditions. It is surprising that NtNTP2-T alone but not NtNTP2-S alone can confer this advantageous phenotype.
It is also shown herein that reducing or inhibiting the function of NtNTP2 using, for example, mutation or RNAi, surprisingly results in a change (for example, an increase) in root development by generating more and thinner roots compared to wild type plants grown in the same conditions. Without being bound by any particular theory, this may increase the ability of the plants to uptake nutrients from the soil.
The advantageous phenotype of the present invention is also surprising in view of the results reported by Chiu et al. (2004) Plant Cell Physiol., 45(9), 1139-1148, which discloses results for Arabidopsis thaliana homozygous AtNTP2 insertional mutants. The Arabidopsis thaliana homozygous AtNTP2 insertional mutants developed (i) nitrate levels that were 50-64% lower; (ii) increased plant leaf width due to cell expansion; and (iii) no mention of NUE improvement. Accordingly, despite the 77% identity between NtNTP2 and AtNTP2, it is surprising and unexpected that NtNtp2 is an excellent target for developing plants which have increased yield and higher NUE response (ratio biomass per unit of nitrogen applied) compared to wild type plants grown in the same conditions, whereas this is not demonstrated for AtNTP2 in the mentioned publication.
This advantageous phenotype is further surprising in view of the results reported in US2014/0201863 in which loss of function, via a transgenic approach, of Arabidopsis NRT1.7 in nitrogen starvation conditions resulted in growth retardation and a 30% decrease in rosette diameter.
In one aspect, there is disclosed a mutant, non-naturally occurring or transgenic plant or part of the plant having reduced or inhibited expression or activity of NtNTP2-T, or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or (v) a NtNTP2-T polypeptide having at least 77 % % sequence identity to SEQ ID NO: 12; wherein the expression or activity of the NtNTP2- T or the expression or activity of the NtNTP2-T and the NtNTP2-S is reduced or inhibited as compared to a control plant.
Suitably, the plant or the part of the plant: (i) does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response, indicated as biomass per units of nitrogen applied, as compared to the control plant grown in the same conditions. The plant or the part of the plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
In one embodiment, the plant or the part of the plant (for example, leaf): (i) does not have decreased nitrate levels as compared to the control plant grown in the same conditions; and (ii) has at least a 5% biomass increase as compared to the control plant grown in the same conditions; and (iii) has at least a 5% increase in NUE response as compared to the control plant grown in the same conditions. The plant or the part of the plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
In another embodiment, the plant or the part of the plant (for example, leaf): (i) does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and (ii) has at least a 5% biomass increase as compared to the control plant grown in the same fertilization conditions; and (iii) has at least a 5% increase in NUE response as compared to the control plant grown in the same fertilization conditions. The plant or the part of the plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
The mutant, non-naturally occurring or transgenic plant or part of the plant can be modified in various ways with the aim of reducing or inhibiting the expression or activity of NtNTP2-T, or reducing or inhibiting the expression or activity of NtNTP2-T and NtNTP2-S. Suitably, the mutant, non-naturally occurring or transgenic plant or part of the plant in which the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited comprises: (i) one or more sequence-specific polynucleotides that can interfere with the transcription of NtNTP2-T or NtNTP2-T and NtNTP2-S~, (ii) one or more sequence-specific polypeptides that can interfere with the stability of NtNTP2-T or NtNTP2-T and NtNTP2-S; (iii) one or more sequence-specific polynucleotides that can interfere with the enzymatic activity of NtNTP2-T or NtNTP2-T and NtNTP2-S or the binding activity of NtNTP2-T or NtNTP2-T and NtNTP2-S with respect to substrates or regulatory proteins; (iv) gene edited NtNTP2-T or NtNTP2-T and NtNTP2-S' or (v) at least one genetic alteration in the NtNTP2-T polynucleotide sequence or at least one genetic alteration in the NtNTP2-T polynucleotide and the NtNTP2-S polynucleotide sequence or at least one genetic alteration in the NtNTP2-T polypeptide sequence or at least one genetic alteration in the NtNTP2-T polypeptide sequence and the NtNTP2-S polypeptide sequence, suitably at least one genetic alteration that causes the encoded polypeptide(s) to terminate or end translation earlier than in the control plant.
Suitably, the NtNTP2-T or NtNTP2-T and NtNTP2-S is gene edited using the bacterial CRISPR/Cas system.
Suitably, the at least one genetic alteration is at least one mutation.
Suitably, the mutant, non-naturally occurring or transgenic plant or part of the plant comprises at least one nonsense mutation in the NtNTP2-T polynucleotide or the NtNTP2-T polypeptide or at least one nonsense mutation in the NtNTP2-T polynucleotide or NtNTP2-T polypeptide and at least one nonsense mutation in the NtNTP2-S polynucleotide or the NtNTP2-S polypeptide.
Suitably, the mutant, non-naturally occurring or transgenic plant or part of the plant comprises a single nucleotide polymorphism in NtNTP2-S at nucleotide position 632 or 633 or 632 and 633 of SEQ ID NO: 3, suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 632 or 633 of SEQ I D NO: 3 or a ‘g ’ to ‘a’ mutation at nucleotide positions 632 and 633 of SEQ ID NO: 3.
Suitably, the mutated NtNTP2-S polynucleotide sequence comprises, consists or consists essentially of SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6.
Suitably, the mutant, non-naturally occurring or transgenic plant or part of the plant comprises a single nucleotide polymorphism in NtNTP2-T at nucleotide position 636 of SEQ ID NO: 11 , suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 635 or 636 of SEQ ID NO: 11 or a ‘g’ to ‘a’ mutation at nucleotide positions 635 and 636 of SEQ ID NO: 11.
Suitably, the mutated NtNTP2-T polynucleotide sequence comprises, consists or consists essentially of SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15.
Suitably, the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide each have at least one nonsense mutation at position W212 and W211 , respectively. Suitably, the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide comprise(s), consist(s) or consist(s) essentially of either SEQ ID NO: 16 or SEQ ID NO: 8 and SEQ ID NO: 16, respectively; optionally, wherein the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and the mutated NtNTP2-S polypeptide are truncated.
Suitably, the plant part is selected from: (i) green leaf or part thereof; or (ii) dried leaf or part thereof, suitably, wherein the dried leaf or part thereof is air dried, suitably, sun dried or fire dried, flue cured; or (iii) cured leaf or part thereof, suitably, wherein the cured leaf is air cured, more suitably, sun cured or fire cured, flue cured.
In a further aspect, there is disclosed a method of preparing a plant or a part of the plant comprising: (a) providing a plant comprising: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2- S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or (v) a NtNTP2-T polypeptide having at least 77 % sequence identity to SEQ ID NO: 12; (b) reducing the expression or activity of the NtNTP2-T or the combination of the NtNTP2-T and NtNTP2-S in the plant; and (c) obtaining a plant or part of the plant which: (i) does not have decreased nitrate levels as compared to a control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response as compared to the control plant grown in the same fertilization conditions. The plant or the part of the plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions. In a further aspect, there is disclosed a mutant, non-naturally occurring or transgenic plant or a part thereof obtained or obtainable by the method of the present invention.
In a further aspect, there is disclosed a mutant, non-naturally occurring or transgenic plant or a part thereof in which there is no significant difference in nitrate levels as compared to a control plant grown in the same fertilization conditions, wherein biomass yield is higher as compared to the control plant grown in the same fertilization conditions, and wherein the NUE of the plant is higher as compared to the control plant grown in the same fertilization conditions. The mutant, non-naturally occurring or transgenic plant or part thereof can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
Suitably, this mutant, non-naturally occurring or transgenic plant or part of the plant has reduced or inhibited expression or activity of NtNTP2-T, or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77% sequence identity to SEQ ID NO: 7; or (v) a NtNTP2-T polypeptide having at least 77 % sequence identity to SEQ ID NO: 12; wherein the expression or activity of the NtNTP2-T or the expression or activity of the NtNTP2-T and the NtNTP2-S is reduced or inhibited as compared to a control plant.
In a further aspect, there is disclosed a tobacco product or a smoking article comprising the mutant, non-naturally occurring or transgenic plant or part of the plant according to the present invention.
In a further aspect, there is provided a method of improving an agronomic characteristic of a plant, the method comprising reducing or inhibiting the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or (v) a NtNTP2-T polypeptide having at least 77 % % sequence identity to SEQ ID NO: 12; wherein the expression or activity of the NtNTP2-T or the expression or activity of the NtNTP2-T and the NtNTP2-S is reduced or inhibited as compared to a control plant.
Suitably, the agronomic characteristic is: (i) nitrate levels are not decreased; (ii) biomass (for example, leaf biomass) is increased in both standard and nitrogen starvation conditions; and (iii) NUE response, intended as biomass per unit of nitrogen fertilization applied, is increased.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a photograph showing the morphology of AA37 ntp2-S W211stop and ntp2-T W212stop double mutant lines in field at harvest time. Representative pictures of two topped plants in field at harvest time. Double wild type indicates an example of an outsegregant wild type plant; Double mutant indicates an example of ntp2-S W211stop/ntp2-T W212stop double mutant plant.
Figure 2 is a graph of nitrate levels in AA37 ntp2 double stop mutants cured leaf material. Indicated are the average nitrate levels measured in cured lamina (A) and midribs (B) of mid position leaves from plants grown in Burley regime, sstt indicates the ntp2 double mutant homozygous genotype, SSTT indicates the ntp2 double mutant out-segregant wild type genotype. Error bars indicate standard errors. N=8 plots of 10 plants each for the out- segregant wild type and n=7 plots of 10 plants each for the double mutant.
Figure 3 is a graph showing the morphology of AA37 ntp2-S W211stop and ntp2-T W212stop double mutant lines in the field. Representative pictures plants growing in the field 3 months after transplant. Wild type indicates an example of out-segregant wild type 10 plant plot; Double mutant indicates an example of 10 plant plots for ntp2-S W211stop/ntp2-T W212stop double mutant; AA37 indicates the control AA37 non mutated plots. Upper panel (Burley regime) indicates the field part fertilized with 254 units of nitrogen; lower panel (Virginia regime) indicates the field part fertilized with 55 units of nitrogen.
Figure 4 is a graph showing the results of total harvest leaf cured biomass in AA37 ntp2 double stop mutant lines grown in different nitrogen regimes. Indicated is the cured biomass (expressed as grams per plant) of total leaf harvest from plants grown in standard conditions (Burley regime) or nitrogen starvation (Virginia regime) during a first year trial, sstt indicates the ntp2 double mutant homozygous genotype, SSTT indicates the ntp2 double mutant out- segregant wild type genotype. Error bars indicate confidence intervals at 95%. N=19 plots of 8 plants each (the two edge plants were discarded to minimize positional effects).
Figure 5 is a graph showing segregation of the mutant phenotype. Indicated is the cured biomass (expressed as grams per plant) of total leaf harvest from plants grown in standard conditions during a second year trial. SSTT indicates the outsegregant wild type plants, sstt indicates the ntp2 double mutant homozygous genotype, ssTT and SStt indicate the homozygoyus single mutants of the -S and -T forms respectively. Error bars indicate standard errors. N=8 to 10 plots of 8 plants each (the two edge plants were discarded to minimize positional effects).
Figure 6 is a graph showing the NUE index, intended as biomass per unit of nitrogen fertilization applied, of AA37 ntp2 double stop mutant lines grown in different nitrogen regimes. Indicated is the NUE index (expressed as kilograms of cured leaf biomass produced per kilogram of N input per hectare, assuming a number of 12 thousand plants per hectare) of total leaf harvest from plants grown in standard conditions (Burley regime) or Flue cured nitrogen fertilization (Virginia regime) during growing season 2019. sstt indicates the ntp2 double mutant homozygous genotype, SSTT indicates the ntp2 double mutant out-segregant wild type genotype. Error bars indicate confidence intervals at 95%. N=19 plots of 8 plants each (the two edge plants were discarded to minimize positional effects).
Figure 7 is a graph showing the expression level of NtNTP2 genes in tobacco leaves from different varieties before and shortly after harvesting. Microarray expression profiles of NtNTP2 during plant growth (green mature), at harvest time (harvest) and during early curing (7h curing and 15h curing) of Swiss-Burley (Burley) and Swiss-Flue-cured (Virginia) are shown. Expression levels are indicated as fold changes in a Iog2 scale normalized on each total transcript level.
Figure 8 is two graphs showing how NtNTP2 mutation effects root development. Figure 8(A) shows the number of lateral roots in seedlings grown for 13 days on agar plates. Figure 8(B) shows the maximum length of aquatic root in 6 week old young plants grown in hydroponic conditions. SSTT, sstt, ssTT and SStt indicate respectively the outsegregant wild type, the homozygous double -S/-T mutant, and the -S and -T single mutants. Letters on top of the columns in Figure 8(B) indicates statistical groups in a one-way ANOVA test. Error bars indicate confidence intervals at 95%. P value is indicated.
Figure 9 is two graphs of average root number per plant of Ntntp2-S W211stop/Ntntp2-T W212stop BC2S2 TN90 (A) and K326 (B) mutant plants and their wild type outsegregant controls. SSTT indicates wild type plants, sstt indicates the double mutant Ntntp2-S W211stop/Ntntp2-T W212stop genetic background. TN90 and K326 BC2S2 plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions. Between 4 to 6 weeks after transplant, the primary roots sprouting from the stele were counted for a minimum of 18 plants per genotype for the TN90 variety and a minimum of 25 plants for K326. The statistical validity of the data was tested in t-Student test and p values are reported. Error bars reported the confidence interval at 95%. Figure 10 is a graph showing average root diameter for TN90 BC2S2 plants. SSTT indicates wild type plants, sstt indicates the double mutant Ntntp2-S W211stop/Ntntp2-T W212stop genetic background. TN90 BC2S2 plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions. Between 4 to 6 weeks after transplant, the diameter of the primary roots sprouting from the stele is measured for a minimum of 655 roots per genotype. The statistical validity of the data was tested in t-Student test and the p value is reported in figure. Error bars indicate the confidence interval at 95%.
Figure 11 is a graph showing total root diameter distribution in BC2S2 TN90 plants grown in 50% N fertilization. TN90 BC2S2 plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions. Between 4 to 6 weeks after transplant, the primary roots sprouting from the stele are counted and their diameter is measured with a thickness gage instrument. The distribution of the root diameter measured for 15 plants per genotype plotted against the number of roots measured is reported. SSTT indicates wild type plants, sstt indicates the double mutant Ntntp2-S W211stop/Ntntp2-T W212stop genetic background.
Figure 12 is a diagram showing a synthetized RNAi loop with 35S CaMV terminator, having the DNA sequence shown in SEQ ID NO: 45.
Figure 13 is a diagram showing the RNAi loop from Figure 12 cloned via Hindi I l-Avrl I into a binary vector carrying an MMV promoter and translator enhancer. Figure 14 is a graph showing average root number per plant in TN90 transgenic plants grown in 50% N fertilization. TN90 transgenic plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions. Between 4 to 6 weeks after transplant, the primary roots sprouting from the stele are counted for a minimum of 24 plants per genotype. CT-T2 indicates control plants transformed with an empty binary vector, RNAi-T2 indicates the plants transformed with an RNAi vector. The statistical validity of the data is tested in t-Student test and p value is reported together with the percentage of increase of the RNAi lines compared to the control. Error bars report the confidence interval at 95%.
Figure 15 is a graph of total root diameter distribution in TN90 transgenic plants grown in 50% N fertilization. TN90 transgenic plants are grown in hydroponic solution at N fertilization at 50% compared to standard agricultural practices in greenhouse conditions. Between 4 to 6 weeks after transplant, the primary roots sprouting from the stele are counted and their diameter is measured. The distribution of the root diameter measured for 7 plants per construct plotted against the number of roots measured is reported. CT-T2 indicates control plants transformed with an empty binary vector, RNAi-T2 indicates the plants transformed with an RNAi vector.
SOME ADVANTAGES
Plants that have increased yield with lower fertilization input and therefore an improved NUE, intended as biomass per unit of nitrogen fertilization applied, can be obtained.
The phenotype can be achieved via a non-transgenic approach to develop non-genetically modified (non-GM) plants. This is highly desirable due to the difficulties of growing and commercialising GM plants in various countries, including Europe. Mutant plants featuring one or more single nucleotide polymorphisms are not considered to be GM plants. In the EU for example, there are no special regulations for plants derived from mutation breeding. Thus, in certain embodiments, it is preferred that the plant contains only one or more single nucleotide polymorphisms (that is, one or more mutations) to result in a non-genetically modified plant.
It may be possible to grow plants at a nitrogen rate that is lower than what is normally required but with no major biomass loss. Thus, less nitrogen fertilizer can be used, which can achieve a reduction in plant nitrate levels and therefore in TSNAs, but not yield. This is highly advantageous in commercial plant production, for example, commercial tobacco plant production.
A further advantage is that the present invention can be applied to green leaves or dried leaves. When applied to tobacco this means that green tobacco or dried tobacco can be obtained in greater yield. This can be used in various tobacco applications that can include: (i) smoking products; (ii) biofuel production; (iii) producing recombinant products; and (iv) extraction of bioactive compounds. The present invention can also be applied to cured leaves.
DETAILED DESCRIPTION
Section headings as used in this disclosure are for organisation purposes and are not intended to be limiting.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures.
The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.
The term “and/or” means (a) or (b) or both (a) and (b).
The present disclosure contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of’ the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
As used throughout the specification and the claims, the following terms have the following meanings:
“Coding sequence” or “polynucleotide encoding” means the nucleotides (RNA or DNA molecule) that comprise a polynucleotide which encodes a polypeptide. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the polynucleotide is administered. The coding sequence may be codon optimized.
“Complement” or “complementary” can mean Watson-Crick (for example, A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs. “Complementarity” refers to a property shared between two polynucleotides, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary. "Construct" refers to a double-stranded, recombinant polynucleotide fragment comprising one or more polynucleotides. The construct comprises a "template strand" base-paired with a complementary "sense or coding strand." A given construct can be inserted into a vector in two possible orientations, either in the same (or sense) orientation or in the reverse (or antisense) orientation with respect to the orientation of a promoter positioned within a vector - such as an expression vector.
The term "control" in the context of a control plant or control plant cells means a plant or plant cells in which the expression, function or activity of one or more NtNTP2 genes or NtNTP2 polypeptides has not been modified (for example, increased or reduced or inhibited) and so it can provide a comparison with a plant in which the expression, function or activity of the same one or more NtNTP2 genes or NtNTP2 polypeptides has been modified. As used herein, a “control plant” is a plant that is substantially equivalent to a test plant or modified plant in all parameters with the exception of the test parameters. For example, when referring to a plant into which a polynucleotide has been introduced or a polynucleotide has been modified, a control plant is an equivalent plant into which no such polynucleotide has been introduced or no such polynucleotide has been modified. A control plant can be an equivalent plant into which a control polynucleotide has been introduced. In such instances, the control polynucleotide is one that is expected to result in little or no phenotypic effect on the plant. The control plant may comprise an empty vector. The control plant may correspond to a wildtype (WT) plant. The control plant may be a null segregant wherein the T 1 segregant no longer possesses the transgene. For comparison purposed, the control plant and the plant which is being compared to the control plant will be grown under the same conditions.
“Donor DNA” or “donor template” refers to a double-stranded DNA fragment or molecule that includes at least a portion of the gene of interest. The donor DNA may encode a fully- functional polypeptide or a partially-functional polypeptide.
“Endogenous gene or polypeptide” refers to a gene or polypeptide that originates from the genome of an organism and has not undergone a change, such as a loss, gain, or exchange of genetic material. An endogenous gene undergoes normal gene transmission and gene expression. An endogenous polypeptide undergoes normal expression.
"Enhancer sequences" refer to the sequences that can increase gene expression. These sequences can be located upstream, within introns or downstream of the transcribed region. The transcribed region is comprised of the exons and the intervening introns, from the promoter to the transcription termination region. The enhancement of gene expression can be through various mechanisms including increasing transcriptional efficiency, stabilization of mature mRNA and translational enhancement.
"Expression" refers to the production of a functional product. For example, expression of a polynucleotide fragment may refer to transcription of the polynucleotide fragment (for example, transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature polypeptide. "Overexpression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in a null segregating (or non- transgenic) organism from the same experiment.
“Functional” and “full-functional” describes a polypeptide that has biological function or activity. A “functional gene” refers to a gene transcribed to mRNA, which is translated to a functional or active polypeptide.
“Genetic construct" refers to DNA or RNA molecules that comprise a polynucleotide that encodes a polypeptide. The coding sequence can include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression.
“Genome editing" refers to changing an endogenous gene that encodes an endogenous polypeptide, such that polypeptide expression of a truncated endogenous polypeptide or an endogenous polypeptide having an amino acid substitution is obtained. Genome editing can include replacing the region of the endogenous gene to be targeted or replacing the entire endogenous gene with a copy of the gene that has a truncation or an amino acid substitution with a repair mechanism - such as HDR. Genome editing may also include generating an amino acid substitution in the endogenous gene by generating a double stranded break in the endogenous gene that is then repaired using NHEJ. NHEJ may add or delete at least one base pair during repair which may generate an amino acid substitution. Genome editing may also include deleting a gene segment by the simultaneous action of two nucleases on the same DNA strand in order to create a truncation between the two nuclease target sites and repairing the DNA break by NHEJ.
"Heterologous" with respect to a sequence means a sequence 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.
“Homology-directed repair” or “HDR” refers to a mechanism in cells to repair double strand DNA lesions when a homologous piece of DNA is present in the nucleus, mostly in G2 and S phase of the cell cycle. HDR uses a donor DNA or donor template to guide repair and may be used to create specific sequence changes to the genome, including the targeted addition of whole genes. If a donor template is provided along with the site-specific nuclease, then the cellular machinery will repair the break by homologous recombination, which is enhanced several orders of magnitude in the presence of DNA cleavage. When the homologous DNA piece is absent, NHEJ may take place instead.
The terms "homology” or “similarity" refer to the degree of sequence similarity between two polypeptides or between two polynucleotide molecules compared by sequence alignment. The degree of homology between two discrete polynucleotides being compared is a function of the number of identical, or matching, nucleotides at comparable positions.
"Identical" or "identity" in the context of two or more polynucleotides or polypeptides means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. Percentage identity can be determined over the full length of a sequence. When comparing DNA and RNA, thymine (T) and uracil (II) may be considered equivalent. Identity may be determined manually or by using a computer sequence algorithm such as ClustalW, ClustalX, BLAST, FASTA or Smith-Waterman. The popular multiple alignment program ClustalW (Nucleic Acids Research (1994) 22, 4673-4680; Nucleic Acids Research (1997), 24, 4876-4882) is a suitable way for generating multiple alignments of polypeptides or polynucleotides. Suitable parameters for ClustalW maybe as follows: For polynucleotide alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For polypeptide alignments: Gap Open Penalty = 10. o, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1 , and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment. Suitably, calculation of percentage identities is then calculated from such an alignment as (N/T), where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
The term "increase" or "increased" refers to an increase of from about 5% to about 99%, or an increase of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, at least about 150%, or at least about 200% or more or more of a quantity or a function or an activity, such as but not limited to polypeptide function or activity, transcriptional function or activity, and/or polypeptide expression. The term “increased,” or the phrase “an increased amount” can refer to a quantity or a function or an activity in a modified plant or a product generated from the modified plant that is more than what would be found in a plant or a product from the same variety of plant processed in the same manner, which has not been modified. Thus, in some contexts, a wild- type plant of the same variety that has been processed in the same manner is used as a control by which to measure whether an increase in quantity is obtained.
The term "reduce" or " reduced" as used herein, refers to a reduction of from about 5% to about 99%, or a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100% or, or at least about 150%, or at least about 200% more of a quantity or a function - such as polypeptide function, transcriptional function, or gene expression. The term “reduced,” or the phrase “a reduced amount” can refer to a quantity or a function in a modified plant or a product generated from the modified plant that is less than what would be found in a plant or a product from the same plant processed in the same manner, which has not been modified. Thus, in some contexts, a wild-type plant of the same species or variety that has been processed in the same manner is used as a control by which to measure whether a reduction in quantity is obtained.
The term "inhibit" or "inhibited" refers to a reduction of from about 98% to about 100%, or a reduction of at least about 98%, at least about 99%, but particularly about 100%, of a quantity or a function or an activity, such as but not limited to polypeptide function or activity, transcriptional function or activity, and/or polypeptide expression.
The term “introduced” means providing a polynucleotide (for example, a construct) or polypeptide into a cell. Introduced includes reference to the incorporation of a polynucleotide into a eukaryotic cell where the polynucleotide may be incorporated into the genome of the cell, and includes reference to the transient provision of a polynucleotide or polypeptide to the cell. Introduced includes reference to stable or transient transformation methods, as well as sexually crossing. Thus, "introduced" in the context of inserting a polynucleotide (for example, a recombinant construct/expression construct) into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a polynucleotide into a eukaryotic cell where the polynucleotide may be incorporated into the genome of the cell (for example, chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (for example, transfected mRNA).
The terms "isolated" or "purified" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. In particular, an isolated polynucleotide is separated from open reading frames that flank the desired gene and encode polypeptides other than the desired polypeptide. The term "purified" as used herein denotes that a polynucleotide or polypeptide gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the polynucleotide or polypeptide is at least 85% pure, more suitably at least 95% pure, and most suitably at least 99% pure. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional polynucleotide purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
The term “level” refers to an amount and is used interchangeably with “content”.
"Modulate" or “modulating” refers to causing or facilitating a qualitative or quantitative change, alteration, or modification in a process, pathway, function or activity of interest. Without limitation, such a change, alteration, or modification may be an increase or a reduction in the relative process, pathway, function or activity of interest. For example, NtNTP2 gene expression or NtNTP2 polypeptide expression or NtNTP2 polypeptide function or activity can be modulated. Typically, the relative change, alteration, or modification will be determined by comparison to a control.
“Non-homologous end joining (NHEJ) pathway” as used herein refers to a pathway that repairs double-strand breaks in DNA by directly ligating the break ends without the need for a homologous template. The template-independent re-ligation of DNA ends by NHEJ is a stochastic, error-prone repair process that introduces random micro-insertions and microdeletions (indels) at the DNA breakpoint. This method may be used to intentionally disrupt, delete, or alter the reading frame of targeted gene sequences. NHEJ typically uses short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the end of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately, yet imprecise repair leading to loss of nucleotides may also occur, but is much more common when the overhangs are not compatible.
The term 'non-naturally occurring' describes an entity - such as a polynucleotide, a genetic mutation, a polypeptide, a plant, a plant cell and plant material - that is not formed by nature or that does not exist in nature. Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by methods described herein or that are known in the art. Such non-naturally occurring entities or artificial entities may be made, synthesized, initiated, modified, intervened, or manipulated by man. Thus, by way of example, a non-naturally occurring plant, a non-naturally occurring plant cell or non- naturally occurring plant material may be made using traditional plant breeding techniques - such as backcrossing - or by genetic manipulation technologies - such as antisense RNA, interfering RNA, meganuclease and the like. By way of further example, a non-naturally occurring plant, a non-naturally occurring plant cell or non-naturally occurring plant material may be made by introgression of or by transferring one or more genetic mutations (for example one or more polymorphisms) from a first plant or plant cell into a second plant or plant cell (which may itself be naturally occurring), such that the resulting plant, plant cell or plant material or the progeny thereof comprises a genetic constitution (for example, a genome, a chromosome or a segment thereof) that is not formed by nature or that does not exist in nature. The resulting plant, plant cell or plant material is thus artificial or non-naturally occurring. Accordingly, an artificial or non-naturally occurring plant or plant cell may be made by modifying a genetic sequence in a first naturally occurring plant or plant cell, even if the resulting genetic sequence occurs naturally in a second plant or plant cell that comprises a different genetic background from the first plant or plant cell. In certain embodiments, a mutation is not a naturally occurring mutation that exists naturally in a polynucleotide or a polypeptide. Differences in genetic background can be detected by phenotypic differences or by molecular biology techniques known in the art - such as polynucleotide sequencing, presence or absence of genetic markers (for example, microsatellite RNA markers).
“Oligonucleotide” or “polynucleotide” means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a polynucleotide also encompasses the complementary strand of a depicted single strand. Many variants of a polynucleotide may be used for the same purpose as a given polynucleotide. A polynucleotide also encompasses substantially identical polynucleotides and complements thereof. A single strand provides a probe that may hybridize to a given sequence under stringent hybridization conditions. Thus, a polynucleotide also encompasses a probe that hybridizes under stringent hybridization conditions. Polynucleotides may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The polynucleotide may be DNA, both genomic and cDNA, RNA, or a hybrid, where the polynucleotide may contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Polynucleotides may be obtained by chemical synthesis methods or by recombinant methods. The specificity of singlestranded DNA to hybridize complementary fragments is determined by the "stringency" of the reaction conditions (Sambrook et al., Molecular Cloning and Laboratory Manual, Second Ed., Cold Spring Harbor (1989)). Hybridization stringency increases as the propensity to form DNA duplexes reduces. In polynucleotide hybridization reactions, the stringency can be chosen to favor specific hybridizations (high stringency), which can be used to identify, for example, full- length clones from a library. Less-specific hybridizations (low stringency) can be used to identify related, but not exact (homologous, but not identical), DNA molecules or segments. DNA duplexes are stabilised by: (1) the number of complementary base pairs; (2) the type of base pairs; (3) salt concentration (ionic strength) of the reaction mixture; (4) the temperature of the reaction; and (5) the presence of certain organic solvents, such as formamide, which reduces DNA duplex stability. In general, the longer the probe, the higher the temperature required for proper annealing. A common approach is to vary the temperature; higher relative temperatures result in more stringent reaction conditions. To hybridize under "stringent conditions" describes hybridization protocols in which polynucleotides at least 60% homologous to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and polynucleotide concentration) at which 50% of the probes complementary to the given sequence hybridize to the given sequence at equilibrium. Since the given sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
“NtNTP2” is used herein to refer to: (i) a NtNTP2-S polynucleotide or a NtNTP2-T polynucleotide or a combination of NtNTP2-S and NtNTP2-T polynucleotides; or (ii) a NtNTP2- S polypeptide or a NtNTP2-T polypeptide or a combination of NtNTP2-S and NtNTP2-T polypeptides.
"Stringent hybridisation conditions" are conditions that enable a probe, primer, or oligonucleotide to hybridize only to its specific sequence. Stringent conditions are sequencedependent and will differ. Stringent conditions typically comprise: (1) low ionic strength and high temperature washes, for example 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1 % sodium dodecyl sulfate, at 50°C; (2) a denaturing agent during hybridization, for example, 50% (v/v) formamide, 0.1 % bovine serum albumin, 0.1% Ficoll, 0.1 % polyvinylpyrrolidone, 50 mM sodium phosphate buffer (750 mM sodium chloride, 75 mM sodium citrate; pH 6.5), at 42°C; or (3) 50% formamide. Washes typically also comprise 5xSSC (0.75 M NaCI, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5xDenhardt's solution, sonicated salmon sperm DNA (50 pg/mL), 0.1% SDS, and 10% dextran sulfate at 42°C, with a wash at 42°C in 0.2xSSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of O.IxSSC containing EDTA at 55°C. Suitably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
"Moderately stringent conditions" use washing solutions and hybridization conditions that are less stringent, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide. One example comprises hybridization in 6xSSC, 5xDenhardt's solution, 0.5% SDS and 100 pg/mL denatured salmon sperm DNA at 55°C, followed by one or more washes in 1xSSC, 0.1% SDS at 37°C. The temperature, ionic strength, etc., can be adjusted to accommodate experimental factors such as probe length. Other moderate stringency conditions have been described (see Ausubel et al., Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., Hoboken, N.J. (1993); Kriegler, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, N.Y. (1990); Perbal, A Practical Guide to Molecular Cloning, 2nd edition, John Wiley & Sons, New York, N.Y. (1988)). "Low stringent conditions" use washing solutions and hybridization conditions that are less stringent than those for moderate stringency, such that a polynucleotide will hybridize to the entire, fragments, derivatives, or analogs of the polynucleotide. A non-limiting example of low stringency hybridization conditions includes hybridization in 35% formamide, 5xSSC, 50 mM Tris HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/mL denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2xSSC, 25 mM Tris HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency, such as those for cross-species hybridizations, are well-described (see Ausubel et al., 1993; Kriegler, 1990).
“Operably linked” means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function. "Operably linked" refers to the association of polynucleotide fragments in a single fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a polynucleotide fragment when it is capable of regulating the transcription of that polynucleotide fragment.
The term "plant" refers to any plant at any stage of its life cycle or development, and its progenies. The term includes reference to whole plants, plant organs, plant tissues - such as leaf, plant propagules, plant seeds, plant cells and progeny of same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Suitable species, cultivars, hybrids and varieties of tobacco plant are described herein. "Polynucleotide", "polynucleotide sequence" or "polynucleotide fragment" are used interchangeably herein and refer to a polymer of RNA or DNA that is single- or doublestranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide. A polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) thereof. Moreover, a polynucleotide can be single-stranded or double-stranded, a mixture of single-stranded and double-stranded regions, a hybrid molecule comprising DNA and RNA, or a hybrid molecule with a mixture of single-stranded and double-stranded regions or a fragment(s) thereof. In addition, the polynucleotide can be composed of triple-stranded regions comprising DNA, RNA, or both or a fragment(s) thereof. A polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid (PNA). Generally, polynucleotides can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or a combination of the foregoing. Although the polynucleotides described herein are shown as DNA sequences, the polynucleotides include their corresponding RNA sequences, and their complementary (for example, completely complementary) DNA or RNA sequences, including the reverse complements thereof. The polynucleotides of the present disclosure are set forth in the accompanying sequence listing.
"Polypeptide” or "polypeptide sequence" refer to a polymer of amino acids in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring polymers of amino acids. The terms are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. The polypeptides of the present disclosure are set forth in the accompanying sequence listing. “Promoter” means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a polynucleotide in a cell. The term refers to a polynucleotide element/sequence, typically positioned upstream and operably-linked to a double-stranded polynucleotide fragment. Promoters can be derived entirely from regions proximate to a native gene of interest, or can be composed of different elements derived from different native promoters or synthetic polynucleotide segments. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
"Tissue-specific promoter" and "tissue-preferred promoter" as used interchangeably herein refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell. A "developmentally regulated promoter" refers to a promoter whose function is determined by developmental events. A “constitutive promoter” refers to a promoter that causes a gene to be expressed in most cell types at most times. An “inducible promoter” selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Examples of inducible or regulated promoters include promoters regulated by light, heat, stress, flooding or drought, pathogens, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
“Recombinant" as used herein refers to an artificial combination of two otherwise separated segments of sequence - such as by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques. The term also includes reference to a cell or vector, that has been modified by the introduction of a heterologous polynucleotide or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (for example, spontaneous mutation, natural transformation or transduction or transposition) such as those occurring without deliberate human intervention.
"Recombinant construct" refers to a combination of polynucleotides that are not normally found together in nature. Accordingly, a recombinant construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature. The recombinant construct can be a recombinant DNA construct. "Regulatory sequences" and "regulatory elements" as used interchangeably herein refer to polynucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include promoters, translation leader sequences, introns, and polyadenylation recognition sequences. The terms "regulatory sequence" and "regulatory element" are used interchangeably herein.
“Site-specific nuclease” refers to an enzyme capable of specifically recognizing and cleaving DNA sequences. The site-specific nuclease may be engineered. Examples of engineered sitespecific nucleases include zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), CRISPR/Cas9-based systems, and meganucleases.
The term “tobacco” is used in a collective sense to refer to tobacco crops (for example, a plurality of tobacco plants grown in the field and not hydroponically grown tobacco), tobacco plants and parts thereof, including but not limited to, root, stem, leaf, flower, and seed prepared and/or obtained, as described herein. It is understood that “tobacco” includes Nicotiana tabacum plants and parts thereof. The term “tobacco products” refers to consumer tobacco products, including but not limited to, smoking materials (for example, cigarettes, cigars, and pipe tobacco), snuff, chewing tobacco, gum, and lozenges, as well as components, materials and ingredients for manufacture of consumer tobacco products. Suitably, these tobacco products are manufactured from tobacco leaves and stems harvested from tobacco and cut, dried, cured, and/or fermented according to conventional techniques in tobacco preparation. The tobacco in the tobacco products may be combined with a binder, as described herein.
"Transcription terminator", "termination sequences", or "terminator" refers to DNA sequences located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
"Transgenic" refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous polynucleotide, such as a recombinant construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event. The term does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events - such as random cross-fertilization, nonrecombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
"Transgenic plant" refers to a plant which comprises within its genome one or more heterologous polynucleotides, that is, a plant that contains recombinant genetic material not normally found therein and which has been introduced into the plant in question (or into progenitors of the plant) by human manipulation. For example, the heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide can be integrated into the genome alone or as part of a recombinant construct. The commercial development of genetically improved germplasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking approach. In this approach, multiple genes conferring different characteristics of interest can be introduced into a plant. Gene stacking can be accomplished by many means including but not limited to co-transformation, retransformation, and crossing lines with different transgenes. Thus, a plant that is grown from a plant cell into which recombinant DNA is introduced by transformation is a transgenic plant, as are all offspring of that plant that contain the introduced transgene (whether produced sexually or asexually). It is understood that the term transgenic plant encompasses the entire plant or tree and parts of the plant or tree, for instance grains, seeds, flowers, leaves, roots, fruit, pollen, stems and the like. Each heterologous polynucleotide may confer a different trait to the transgenic plant.
“Transcription activator-like effector” or “TALE” refers to a polypeptide structure that recognizes and binds to a particular DNA sequence. The “TALE DNA-binding domain” refers to a DNA-binding domain that includes an array of tandem 33-35 amino acid repeats, also known as RVD modules, each of which specifically recognizes a single base pair of DNA. RVD modules may be arranged in any order to assemble an array that recognizes a defined sequence. A binding specificity of a TALE DNA-binding domain is determined by the RVD array followed by a single truncated repeat of 20 amino acids. A TALE DNA-binding domain may have 12 to 27 RVD modules, each of which contains an RVD and recognizes a single base pair of DNA. Specific RVDs have been identified that recognize each of the four possible DNA nucleotides (A, T, C, and G). Because the TALE DNA-binding domains are modular, repeats that recognize the four different DNA nucleotides may be linked together to recognize any particular DNA sequence. These targeted DNA-binding domains may then be combined with catalytic domains to create functional enzymes, including artificial transcription factors, methyltransferases, integrases, nucleases, and recombinases.
“Transcription activator-like effector nucleases” or “TALENs” as used interchangeably herein refers to engineered fusion polypeptides of the catalytic domain of a nuclease, such as endonuclease Fokl, and a designed TALE DNA-binding domain that may be targeted to a custom DNA sequence.
A “TALEN monomer” refers to an engineered fusion polypeptide with a catalytic nuclease domain and a designed TALE DNA-binding domain. Two TALEN monomers may be designed to target and cleave a TALEN target region.
“Transgene” refers to a gene or genetic material containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or polypeptide in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code. The introduction of a transgene has the potential to change the phenotype of an organism.
“Variant” or “mutant” with respect to a polynucleotide refers to a polynucleotide that differs from the wild-type polynucleotide (such as SEQ ID NO: 3 or SEQ ID NO: 11) by one or more nucleic acid deletions, additions, substitutions or side-chain modifications. Exemplary polynucleotide variants or mutants are shown in SEQ ID NOs: 4, 5, 6, 13, 14 and 15. “Variant” or “mutant” with respect to a polypeptide means a polypeptide that differs in sequence by the insertion, deletion, or conservative substitution of one or more amino acids. The variant or mutant may retain all or some or no biological function or activity as compared to the polypeptide that does not contain the insertion, deletion, or conservative substitution of the one or more amino acids. A conservative substitution of an amino acid, that is, replacing an amino acid with a different amino acid of similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. Exemplary polypeptide variants or mutants are shown in SEQ ID NOs: 8 and 16.
The term "variety" in the context of a plant refers to a population of plants that share constant characteristics which separate them from other plants of the same species. While possessing one or more distinctive traits, a variety is further characterized by a very small overall variation between individuals within that variety. A variety is often sold commercially.
"Vector" refers to a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the transport of polynucleotides, polynucleotide constructs and polynucleotide conjugates and the like. A vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. Suitable vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other vectors of any origin. An "expression vector" as used herein is a polynucleotide vehicle that comprises a combination of polynucleotide components for enabling the expression of polynucleotide(s), polynucleotide constructs and polynucleotide conjugates and the like. Suitable expression vectors include episomes capable of extra- chromosomal replication such as circular, double-stranded nucleotide plasmids; linearized double-stranded nucleotide plasmids; and other functionally equivalent expression vectors of any origin. An expression vector comprises at least a promoter positioned upstream and operably-linked to a polynucleotide, polynucleotide constructs or polynucleotide conjugate, as defined below.
“Zinc finger” refers to a polypeptide structure that recognizes and binds to DNA sequences. The zinc finger domain is the most common DNA-binding motif in the human proteome. A single zinc finger contains approximately 30 amino acids and the domain typically functions by binding 3 consecutive base pairs of DNA via interactions of a single amino acid side chain per base pair.
“Zinc finger nuclease” or “ZFN” refers to a chimeric polypeptide molecule comprising at least one zinc finger DNA binding domain effectively linked to at least one nuclease or part of a nuclease capable of cleaving DNA when fully assembled.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and polypeptide and polynucleotide chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
In one embodiment, there is provided an isolated polynucleotide comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to any of the sequences described herein, including any of polynucleotides shown in the sequence listing. Suitably, the isolated polynucleotide comprises, consists or consists essentially of a sequence having at least 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or 100% sequence identity thereto.
Suitably, the polynucleotide(s) described herein encode an active polypeptide that has at least about 45%, 50%, 60%, 70%, 80%, 90% 95%, 96%, 97%, 98%, 99%, 100% or more of the function or activity of the polypeptide(s) shown in the sequence listing.
In another embodiment, there is provided an isolated polynucleotide comprising, consisting or consisting essentially of a polynucleotide having at least 70% sequence identity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO: 15 (provided that the recited mutation is maintained). Suitably, the isolated polynucleotide comprises, consists or consist essentially of a sequence having at least about 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO: 15 (provided that the recited mutation is maintained).
In another embodiment, there is provided polynucleotides comprising, consisting or consisting essentially of polynucleotides with substantial homology (that is, sequence similarity) or substantial identity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO:15 (provided that the recited mutation is maintained).
In another embodiment, there is provided fragments of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO: 15 (provided that the recited mutation is maintained) with substantial homology (that is, sequence similarity) or substantial identity thereto that have at least about70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the corresponding fragments of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO: 15 (provided that the recited mutation is maintained). In another embodiment, there is provided polynucleotides comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4 (provided that the recited mutation is maintained), SEQ ID NO:5 (provided that the recited mutation is maintained), SEQ ID NO:6 (provided that the recited mutation is maintained), SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13 (provided that the recited mutation is maintained), SEQ ID NO:14 (provided that the recited mutation is maintained) or SEQ ID NO: 15 (provided that the recited mutation is maintained) that encode a polypeptide that functions as a NtNTP2.
In another embodiment, there is provided a polymer of polynucleotides which comprises, consists or consists essentially of a polynucleotide designated herein as SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO: 13, SEQ ID NO:14 or SEQ ID NO:15.
Suitably, the NtNTP2 polynucleotide(s) described herein can encode a functional NtNTP2(s). In the alternative, the NtNTP2 polynucleotide(s) described herein can contain at least one mutation, suitably at least one mutation encoding a stop codon that causes the encoded polypeptide to terminate or end its translation earlier than expected, which will result in a nonfunctional fragment of NtNTP2(s). Such exemplary poly nucleotides are disclosed herein.
A polynucleotide as described herein can include a polymer of nucleotides, which may be unmodified or modified deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Accordingly, a polynucleotide can be, without limitation, a genomic DNA, complementary DNA (cDNA), mRNA, or antisense RNA or a fragment(s) thereof. Moreover, a polynucleotide can be singlestranded or double-stranded DNA, DNA that is a mixture of single-stranded and doublestranded regions, a hybrid molecule comprising DNA and RNA, or a hybrid molecule with a mixture of single-stranded and double-stranded regions or a fragment(s) thereof. In addition, the polynucleotide can be composed of triple-stranded regions comprising DNA, RNA, or both or a fragment(s) thereof. A polynucleotide can contain one or more modified bases, such as phosphothioates, and can be a peptide nucleic acid. Generally, polynucleotides can be assembled from isolated or cloned fragments of cDNA, genomic DNA, oligonucleotides, or individual nucleotides, or a combination of the foregoing. Although the polynucleotides described herein are shown as DNA sequences, they include their corresponding RNA sequences, and their complementary (for example, completely complementary) DNA or RNA sequences, including the reverse complements thereof.
A polynucleotide as described herein will generally contain phosphodiester bonds, although in some cases, polynucleotide analogues are included that may have alternate backbones, comprising, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O- methylphophoroamidite linkages; and peptide polynucleotide backbones and linkages. Other analogue polynucleotides include those with positive backbones; non-ionic backbones, and non-ribose backbones. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring polynucleotides and analogues can be made; alternatively, mixtures of different polynucleotide analogues, and mixtures of naturally occurring polynucleotides and analogues may be made. A variety of polynucleotide analogues are known, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages and peptide polynucleotide backbones and linkages. Other analogue polynucleotides include those with positive backbones, non-ionic backbones and non-ribose backbones. Polynucleotides containing one or more carbocyclic sugars are also included.
Other analogues include peptide polynucleotides which are peptide polynucleotide analogues. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring polynucleotides. This may result in advantages. First, the peptide polynucleotide backbone may exhibit improved hybridization kinetics. Peptide polynucleotides have larger changes in the melting temperature for mismatched versus perfectly matched base pairs. DNA and RNA typically exhibit a 2-4 °C drop in melting temperature for an internal mismatch. With the non-ionic peptide polynucleotide backbone, the drop is closer to 7-9 °C. Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. In addition, peptide polynucleotides may not be degraded or degraded to a lesser extent by cellular enzymes, and thus may be more stable.
Among the uses of the disclosed polynucleotides, and fragments thereof, is the use of fragments as probes in hybridisation assays or primers for use in amplification assays. Such fragments generally comprise at least about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least about 10, 15, 20, 30, 40, 50 or 60 or more contiguous nucleotides of a DNA sequence. Thus, in one aspect, there is also provided a method for detecting a polynucleotide comprising the use of the probes or primers or both. Exemplary primers are described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are described by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Using knowledge of the genetic code in combination with the polypeptide sequences described herein, sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, for example, in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for genetic libraries. Such libraries include cDNA libraries, genomic libraries, and even electronic express sequence tag or DNA libraries. Homologous sequences identified by this method would then be used as probes to identify homologues of the sequences identified herein.
Also of potential use are polynucleotides and oligonucleotides (for example, primers or probes) that hybridize under reduced stringency conditions, typically moderately stringent conditions, and commonly highly stringent conditions to the polynucleotide(s), as described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and can be readily determined by those having ordinary skill in the art based on, for example, the length or base composition of the polynucleotide.
One way of achieving moderately and high stringent conditions are defined herein. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, for example, Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y). When hybridizing a polynucleotide to a polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10 °C less than the melting temperature of the hybrid, where melting temperature is determined according to the following equations. For hybrids less than 18 base pairs in length, melting temperature (°C)=2(number of A+T bases)+4(number of G+C bases). For hybrids above 18 base pairs in length, melting temperature (°C)=81.5+16.6(log10 [Na+])+0.41(% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1x Standard Sodium Citrate=0.165M). Typically, each such hybridizing polynucleotide has a length that is at least 25% (commonly at least 50%, 60%, or 70%, and most commonly at least 80%) of the length of a polynucleotide to which it hybridizes, and has at least 60% sequence identity (for example, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) with a polynucleotide to which it hybridizes.
As will be understood by the person skilled in the art, a linear DNA has two possible orientations: the 5'-to-3' direction and the 3'-to-5' direction. For example, if a first sequence is positioned in the 5'-to-3' direction, and if a second sequence is positioned in the 5'-to-3' direction within the same polynucleotide molecule/strand, then the first sequence and the second sequence are orientated in the same direction, or have the same orientation. Typically, a promoter sequence and a gene of interest under the regulation of the given promoter are positioned in the same orientation. However, with respect to the first sequence positioned in the 5'-to-3' direction, if a second sequence is positioned in the 3'-to-5' direction within the same polynucleotide molecule/strand, then the first sequence and the second sequence are orientated in anti-sense direction, or have anti-sense orientation. Two sequences having antisense orientations with respect to each other can be alternatively described as having the same orientation, if the first sequence (5'-to-3' direction) and the reverse complementary sequence of the first sequence (first sequence positioned in the 5'-to-3') are positioned within the same polynucleotide molecule/strand. The sequences set forth herein are shown in the 5'- to-3' direction.
Fragments of the polynucleotides described herein are also disclosed and may range from at least about 25 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300 nucleotides or about 1400 nucleotides and up to the full-length polynucleotide.
In another aspect, there is provided an isolated polypeptide comprising, consisting or consisting essentially of a polypeptide having at least 77 % sequence identity to any of the polypeptide described herein, including any of the polypeptides shown in the sequence listing. Suitably, the isolated polypeptide comprises, consists or consists essentially of a sequence having at least 77%, 78%, 79%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity thereto. In one embodiment, there is provided a polypeptide encoded by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:16.
In another embodiment, there is provided an isolated polypeptide comprising, consisting or consisting essentially of a sequence having at least 77%, 78%, 79%, 80%, 85%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12 or SEQ ID NO:16.
The polypeptide can include sequences comprising a sufficient or substantial degree of identity or similarity to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 12 or SEQ ID NO:16to function as a NtNTP2. The fragments of the polypeptide(s) typically retain some or all of the NtNTP2 function or activity of the full-length sequence.
As discussed herein, the polypeptides can include mutations - such as mutations encoding a stop codon as in SEQ ID NOs:8 and 16. SEQ ID NO: 8 has a mutation at amino acid position 211 which mutates amino acid W211 of NtNtp2-S into a stop codon. SEQ ID NO: 16 has a mutation at amino acid position 212 which mutates amino acid W212 of NtNtp2-T into a stop codon.
Whilst exemplary mutations are disclosed herein, it will be understood by the skilled person that the advantageous phenotype can be obtained by other mutations in the NtNtp2 polynucleotide sequence(s) described herein that cause a truncation of the expressed polypeptide. Likewise, other genetic techniques as described herein can be used to obtain the advantageous phenotype by modulating (for example, reducing or inhibiting) the expression or activity of NtNtp2.
Mutants can be produced by introducing any type of alterations (for example, insertions, deletions, or substitutions of amino acids; changes in glycosylation states; changes that affect refolding or isomerizations, three-dimensional structures, or self-association states), which can be deliberately engineered or isolated naturally provided that they still have some or all of their function or activity. Suitably, this function or activity is modulated, more suitably, reduced or lost.
A deletion refers to removal of one or more amino acids from a polypeptide. An insertion refers to one or more amino acid residues being introduced into a predetermined site in a polypeptide. Insertions may comprise intra-sequence insertions of single or multiple amino acids. A substitution refers to the replacement of amino acids of the polypeptide with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break a-helical structures or p-sheet structures). Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide and may range from about 1 to about 10 amino acids. The amino acid substitutions are suitably conservative amino acid substitutions as described below. Amino acid substitutions, deletions and/or insertions can be made using peptide synthetic techniques - such as solid phase peptide synthesis or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion mutations of a polypeptide are well known in the art. The mutant may have alterations which produce a silent change and result in a functionally equivalent polypeptide. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and the amphipathic nature of the residues as long as the secondary binding of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and suitably in the same line in the third column may be substituted for each other:
Figure imgf000032_0001
The polypeptide may be a mature polypeptide or an immature polypeptide or a polypeptide derived from an immature polypeptide. Polypeptides may be in linear form or cyclized using known methods. Polypeptides typically comprise at least 10, at least 20, at least 30, or at least 40 contiguous amino acids.
Fragments of the disclosed polypeptides are also disclosed. Fragments of a polypeptide may range from at least about 25 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, and up to the full-length polypeptide described herein. The encoded polypeptide fragment may retain all or a portion or none of the biological activity of the full-length polypeptide. Recombinant constructs can be used to transform plants or plant cells to modulate NtNTP2 polypeptide expression, function or activity. A recombinant NtNTP2 polynucleotide construct can comprise a NtNTP2 polynucleotide encoding one or more NtNTP2 polynucleotides as described herein, operably linked to a regulatory region suitable for expressing the NtNTP2 polypeptide. Thus, a NtNTP2 polynucleotide can comprise a coding sequence that encodes the NtNTP2 polypeptide as described herein. Plants or plant cells in which NtNTP2 polypeptide expression, function or activity are modulated can include mutant, non-naturally occurring, transgenic, man-made or genetically engineered plants or plant cells. Suitably, the transgenic plant or plant cell comprises a genome that has been altered by the stable integration of recombinant DNA. Recombinant DNA includes DNA which has been genetically engineered and constructed outside of a cell and includes DNA containing naturally occurring DNA or cDNA or synthetic DNA. A transgenic plant can include a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant. Suitably, the transgenic modification alters the expression or function or activity of the NtNTP2 polynucleotide or the NtNTP2 polypeptide described herein as compared to a control plant.
The NtNTP2 polypeptide encoded by a recombinant NtNTP2 polynucleotide can be a native NtNTP2 polypeptide or it can be heterologous to the cell. In some cases, the recombinant construct contains a NtNTP2 polynucleotide that modulates expression, operably linked to a regulatory region. Examples of suitable regulatory regions are described herein.
Vectors containing recombinant polynucleotide constructs, including recombinant NtNTP2 polynucleotide constructs are also provided. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, or bacteriophage artificial chromosomes. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available.
The vectors can include, for example, origins of replication, scaffold attachment regions or markers. A marker gene can confer a selectable phenotype on a plant cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (for example, kanamycin, G418, bleomycin, or hygromycin), or an herbicide (for example, glyphosate, chlorsulfuron or phosphinothricin). In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (for example, purification or localization) of the expressed polypeptide. Tag sequences, such as luciferase, betaglucuronidase, green fluorescent polypeptide, glutathione S-transferase, polyhistidine, c-myc or hemagglutinin sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.
A plant or plant cell can be transformed by having the recombinant NtNTP2 polynucleotide integrated into its genome to become stably transformed. The plant or plant cell described herein can be stably transformed. Stably transformed cells typically retain the introduced NtNTP2 polynucleotide with each cell division. A plant or plant cell can be transiently transformed such that the recombinant NtNTP2 polynucleotide is not integrated into its genome. Transiently transformed cells typically lose all or some portion of the introduced recombinant NtNTP2 polynucleotide with each cell division such that the introduced recombinant NtNTP2 polynucleotide cannot be detected in daughter cells after a sufficient number of cell divisions.
A number of methods are available in the art for transforming a plant cell including biolistics, gene gun techniques, Agrobacterium-mediated transformation, viral vector-mediated transformation, freeze-thaw method, microparticle bombardment, direct DNA uptake, sonication, microinjection, plant virus-mediated transfer, and electroporation. The Agrobacterium system for integration of foreign DNA into plant chromosomes has been extensively studied, modified, and exploited for plant genetic engineering. Naked recombinant DNA molecules comprising DNA sequences corresponding to the subject purified polypeptide operably linked, in the sense or antisense orientation, to regulatory sequences are joined to appropriate T-DNA sequences by conventional methods. These are introduced into protoplasts by polyethylene glycol techniques or by electroporation techniques, both of which are standard. Alternatively, such vectors comprising recombinant DNA molecules encoding the subject purified polypeptide are introduced into live Agrobacterium cells, which then transfer the DNA into the plant cells. Transformation by naked DNA without accompanying T- DNA vector sequences can be accomplished via fusion of protoplasts with DNA-containing liposomes or via electroporation. Naked DNA unaccompanied by T-DNA vector sequences can also be used to transform cells via inert, high velocity microprojectiles.
If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
The choice of regulatory regions to be included in a recombinant construct depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue- preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. Transcription of a NtNTP2 polynucleotide can be modulated in a similar manner. Some suitable regulatory regions initiate transcription only, or predominantly, in certain cell types. Methods for identifying and characterizing regulatory regions in plant genomic DNA are known in the art.
Suitable promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (for example, root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present during different developmental stages, or present in response to different environmental conditions. Suitable promoters include constitutive promoters that can be activated in most cell types without requiring specific inducers. Examples of suitable promoters for controlling RNAi polypeptide production include the cauliflower mosaic virus 35S (CaMV/35S), SSU, OCS, Iib4, usp, STLS1 , B33, nos or ubiquitin- or phaseolin-promoters. Persons skilled in the art are capable of generating multiple variations of recombinant promoters.
Tissue-specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues. Tissue-specific expression can be advantageous, for example, when the expression of polynucleotides in certain tissues is preferred. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, for example, roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue. Reproductive tissue-specific promoters may be, for example, anther-specific, ovulespecific, embryo-specific, endosperm-specific, integument-specific, seed and seed coatspecific, pollen-specific, petal-specific, sepal-specific, or combinations thereof.
Suitable leaf-specific promoters include pyruvate, orthophosphate dikinase (PPDK) promoter from C4 plant (maize), cab-m1Ca+2 promoter from maize, the Arabidopsis thaliana myb- related gene promoter (Atmyb5), the ribulose biphosphate carboxylase (RBCS) promoters (for example, the tomato RBCS 1 , RBCS2 and RBCS3A genes expressed in leaves and light- grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruits or ribulose bisphosphate carboxylase promoter expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels).
Suitable senescence-specific promoters include a tomato promoter active during fruit ripening, senescence and abscission of leaves, a maize promoter of gene encoding a cysteine protease, the promoter of 82E4 and the promoter of SAG genes. Suitable anther-specific promoters can be used. Suitable root- preferred promoters known to persons skilled in the art may be selected. Suitable seed-preferred promoters include both seed-specific promoters (those promoters active during seed development such as promoters of seed storage polypeptides) and seed-germinating promoters (those promoters active during seed germination). Such seed-preferred promoters include Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1 -phosphate synthase); mZE40-2, also known as Zm-40; nuclc; and celA (cellulose synthase). Gama-zein is an endosperm-specific promoter. Glob-1 is an embryo-specific promoter. For dicots, seed-specific promoters include bean beta-phaseolin, napin, p-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include a maize 15 kDa zein promoter, a 22 kDa zein promoter, a 27 kDa zein promoter, a g-zein promoter, a 27 kDa gamma-zein promoter (such as gzw64A promoter, see Genbank Accession number S78780), a waxy promoter, a shrunken 1 promoter, a shrunken 2 promoter, a globulin 1 promoter (see Genbank Accession number L22344), an Itp2 promoter, cim1 promoter, maize end1 and end2 promoters, nuc1 promoter, Zm40 promoter, eep1 and eep2; led , thioredoxin H promoter; mlip15 promoter, PCNA2 promoter; and the shrunken-2 promoter.
Examples of inducible promoters include promoters responsive to pathogen attack, anaerobic conditions, elevated temperature, light, drought, cold temperature, or high salt concentration. Pathogen-inducible promoters include those from pathogenesis-related polypeptides (PR polypeptides), which are induced following infection by a pathogen (for example, PR polypeptides, SAR polypeptides, beta-1 , 3-glucanase, chitinase).
In addition to plant promoters, other suitable promoters may be derived from bacterial origin for example, the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from Ti plasmids, or may be derived from viral promoters (for example, 35S and 19S RNA promoters of cauliflower mosaic virus (CaMV), constitutive promoters of tobacco mosaic virus, cauliflower mosaic virus (CaMV) 19S and 35S promoters, or figwort mosaic virus 35S promoter).
Suitable methods of introducing polynucleotides into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al., Biotechniques 4:320-334 (1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602-5606 (1986)), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,981 ,840 and 5,563,055), direct gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)), and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al., in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin) (1995); and McCabe et al., Biotechnology 6:923-926 (1988)).
A plant or plant cell comprising a mutation in one or more NtNTP2 polynucleotides or NtNTP2 polypeptides described herein is disclosed, wherein said mutation results in modulated function or activity of NtNTP2 (including total loss of function or activity). Suitably, the mutation reduces or inhibits the expression or activity of NtNTP2. Aside from the specific mutations described herein, the mutant plants or plant cells can have one or more further mutations either in the same NtNTP2 polynucleotides or NtNTP2 polypeptides as described herein or in one or more other polynucleotides or polypeptides within the genome. Aside from mutation, other techniques can be utilised by the skilled person to modulate the function or activity of NtNTP2, as described in detail herein.
There is also provided a method for modulating the level of a NtNTP2 polypeptide in a plant or in plant material said method comprising introducing into the genome of said plant one or more mutations that modulate expression of at least one NtNTP2 gene, wherein said at least one NtNTP2 gene is selected from the sequences according to the present disclosure.
There is also provided a method for identifying a plant that does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and has increased biomass as compared to the control plant grown in the same fertilization conditions; and has increased NUE response as compared to the control plant grown in the same fertilization conditions, said method comprising screening a polynucleotide sample from a plant of interest for the presence of one or more mutations in the NtNTP2 sequences described herein. The plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
There is also disclosed a plant or plant cell that is heterozygous or homozygous for one or more mutations in one or more NtNTP2 genes according to the present disclosure, wherein said mutation results in modulated expression of the NtNTP2 gene(s) or function or activity of the NtNTP2 polypeptide encoded thereby.
A number of approaches can be used to combine mutations in one plant including sexual crossing. A plant having one or more favourable heterozygous or homozygous mutations in a NtNTP2 gene that modulates expression of the NtNTP2 gene or the function or activity of the NtNTP2 polypeptide encoded thereby can be crossed with a plant having one or more favourable heterozygous or homozygous mutations in one or more other genes that modulate expression thereof or the function or activity of the polypeptide encoded thereby. In one embodiment, crosses are made to introduce one or more favourable heterozygous or homozygous mutations within the NtNTP2 gene(s) within the same plant.
The function or activity of one or more NtNTP2 polypeptides of the present disclosure in a plant is increased or reduced if the function or activity is lower or higher than the function or activity of the same polypeptide(s) in a plant that has not been modified to modulate the function or activity of that polypeptide and which has been cultured, harvested and optionally cured using the same protocols.
In some embodiments, the mutation(s) is introduced into a plant or plant cell using a mutagenesis approach, and the introduced mutation is identified or selected using methods known to those of skill in the art - such as Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. Mutations that impact NtNTP2 gene expression or that interfere with the function of the encoded NtNTP2 polypeptide can be determined using methods that are well known in the art. Insertional mutations in NtNTP2 gene exons usually result in null-mutants. Mutations in conserved residues can be particularly effective in reducing or inhibiting the metabolic function of the encoded NtNTP2 polypeptide. It will be appreciated, for example, that a mutation in one or more of the highly conserved regions would likely alter NtNTP2 polypeptide function, while a mutation outside of those highly conserved regions would likely have little to no effect on NtNTP2 polypeptide function. In addition, a mutation in a single nucleotide can create a stop codon, which would result in a truncated NtNTP2 polypeptide and, depending on the extent of truncation, reduction or complete loss of function. Suitably, the mutation(s) results in reduced expression of NtNTP2 and reduced biological function of NtNTP2, more suitably, the mutation(s) results in complete loss of expression of NtNTP2 and complete loss of biological function of NtNTP2.
Methods for obtaining mutant NtNTP2 polynucleotides and mutant NtNTP2 polypeptides are also disclosed. Any plant of interest, including a plant cell or plant material or plant leaf can be genetically modified by various methods known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically-induced mutagenesis, irradiation-induced mutagenesis, mutagenesis utilizing modified bases, mutagenesis utilizing gapped duplex DNA, double-strand break mutagenesis, mutagenesis utilizing repair-deficient host strains, mutagenesis by total gene synthesis, DNA shuffling and other equivalent methods.
Mutant NtNTP2 polypeptides can be used to create mutant, non-naturally occurring or transgenic plants (for example, mutant, non-naturally occurring, transgenic, man-made or genetically engineered plants) or plant cells comprising one or more mutant polypeptides. Suitably, mutant NtNTP2 polypeptides can retain all, some or no function of the unmutated polypeptide. Thus, the function of the mutant NtNTP2 polypeptide may be higher, lower or about the same as the unmutated polypeptide. In certain embodiments, it is preferred that the mutant NtNTP2 polypeptide has reduced function or does not retain any function.
Mutations in the NtNTP2 polynucleotides and NtNTP2 polypeptides can include man-made mutations or synthetic mutations or genetically engineered mutations. Mutations in the NtNTP2 polynucleotides and NtNTP2 polypeptides described herein can be mutations that are obtained or obtainable via a process which includes an in vitro or an in vivo manipulation step. Mutations in the NtNTP2 polynucleotides and NtNTP2 polypeptides described herein can be mutations that are obtained or obtainable via a process which includes intervention by man. Methods that introduce a mutation randomly in a polynucleotide can include chemical mutagenesis and radiation mutagenesis. Chemical mutagenesis involves the use of exogenously added chemicals - such as mutagenic, teratogenic, or carcinogenic organic compounds - to induce mutations. Mutagens that create primarily point mutations and short deletions, insertions, missense mutations, simple sequence repeats, transversions, and/or transitions, including chemical mutagens or radiation, may be used to create the mutations. Mutagens include ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosurea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, and the like), 2-methoxy-6-chloro-9[3-(ethyl- 2-chloro-ethyl)aminopropylamino]acridine dihydrochloride and formaldehyde.
Spontaneous mutations in the locus that may not have been directly caused by the mutagen are also contemplated provided that they result in the desired phenotype. Suitable mutagenic agents can also include, for example, ionising radiation - such as X-rays, gamma rays, fast neutron irradiation and UV radiation. The dosage of the mutagenic chemical or radiation is determined experimentally for each type of plant tissue such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility. Any method of plant polynucleotide preparation known to those of skill in the art may be used to prepare the plant polynucleotide for mutation screening.
The mutation process may include one or more plant crossing steps.
After mutation, screening can be performed to identify mutations that create premature stop codons or otherwise non-functional genes. After mutation, screening can be performed to identify mutations that create functional genes that are capable of being expressed at increased or reduced levels. Screening of mutants can be carried out by sequencing, or by the use of one or more probes or primers specific to the NtNTP2 gene or NtNTP2 polypeptide. Specific mutations in NtNTP2 polynucleotides can also be created that can result in modulated NtNTP2 gene expression, modulated stability of mRNA, or modulated stability of polypeptide. Such plants are referred to herein as "non-naturally occurring" or "mutant" plants. Typically, the mutant or non-naturally occurring plants will include at least a portion of foreign or synthetic or man-made nucleotide (for example, DNA or RNA) that was not present in the plant before it was manipulated. The foreign nucleotide may be a single nucleotide, two or more nucleotides, two or more contiguous nucleotides or two or more non-contiguous nucleotides - such as at least 10, 20, 30, 40, 50,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400 or 1500 or more contiguous or non-contiguous nucleotides.
Other than mutagenesis, compositions that can modulate the expression or function or activity of one or more of the NtNTP2 polynucleotides or NtNTP2 polypeptides include sequencespecific polynucleotides that can interfere with the transcription of one or more endogenous gene(s); sequence-specific polynucleotides that can interfere with the translation of RNA transcripts (for example, double-stranded RNAs, siRNAs, ribozymes); sequence-specific polypeptides that can interfere with the stability of one or more polypeptides; sequencespecific polynucleotides that can interfere with the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides with respect to substrates or regulatory polypeptides; antibodies that exhibit specificity for one or more polypeptides; small molecule compounds that can interfere with the stability of one or more polypeptides or the enzymatic function of one or more polypeptides or the binding function of one or more polypeptides; zinc finger polypeptides that bind one or more polynucleotides; and meganucleases that have function towards one or more polynucleotides. Gene editing technologies, genetic editing technologies and genome editing technologies are well known in the art.
Zinc finger polypeptides can be used to modulate the expression or function or activity of one or more of the NtNTP2 polynucleotides described herein. In various embodiments, a genomic DNA sequence comprising a part of or all of the coding sequence of the NtNTP2 polynucleotide is modified by zinc finger nuclease-mediated mutagenesis. The genomic DNA sequence is searched for a unique site for zinc finger polypeptide binding. Alternatively, the genomic DNA sequence is searched for two unique sites for zinc finger polypeptide binding wherein both sites are on opposite strands and close together, for example, 1 , 2, 3, 4, 5, 6 or more base pairs apart. Accordingly, zinc finger polypeptides that bind to NtNTP2 polynucleotides are provided. A zinc finger polypeptide may be engineered to recognize a selected target site in a gene. A zinc finger polypeptide can comprise any combination of motifs derived from natural zinc finger DNA-binding domains and non-natural zinc finger DNA- binding domains by truncation or expansion or a process of site-directed mutagenesis coupled to a selection method such as, but not limited to, phage display selection, bacterial two-hybrid selection or bacterial one-hybrid selection. The term “non-natural zinc finger DNA-binding domain” refers to a zinc finger DNA-binding domain that binds a three-base pair sequence within the polynucleotide target and that does not occur in the cell or organism comprising the polynucleotide which is to be modified. Methods for the design of zinc finger polypeptides which bind specific polynucleotides which are unique to a target gene are known in the art.
In other embodiments, a zinc finger polypeptide may be selected to bind to a regulatory sequence of a NtNTP2 polynucleotide. More specifically, the regulatory sequence may comprise a transcription initiation site, a start codon, a region of an exon, a boundary of an exon-intron, a terminator, or a stop codon. Accordingly, the disclosure provides a mutant, non- naturally occurring or transgenic plant or plant cells, produced by zinc finger nuclease- mediated mutagenesis in the vicinity of or within one or more NtNTP2 polynucleotides described herein, and methods for making such a plant or plant cell by zinc finger nuclease- mediated mutagenesis. Methods for delivering zinc finger polypeptide and zinc finger nuclease to a plant are similar to those described below for delivery of meganuclease.
In another aspect, methods for producing mutant, non-naturally occurring or transgenic or otherwise genetically-modified plants using meganucleases, such as l-Crel, are described. Naturally occurring meganucleases as well as recombinant meganucleases can be used to specifically cause a double-stranded break at a single site or at relatively few sites in the genomic DNA of a plant to allow for the disruption of one or more NtNTP2 polynucleotides described herein. The meganuclease may be an engineered meganuclease with altered DNA- recognition properties. Meganuclease polypeptides can be delivered into plant cells by a variety of different mechanisms known in the art.
The disclosure encompasses the use of meganucleases to inactivate a NtNTP2 polynucleotide(s) described herein (or any combination thereof as described herein) in a plant cell or plant. Particularly, the disclosure provides a method for inactivating a NtNTP2 polynucleotide in a plant using a meganuclease comprising: a) providing a plant cell comprising a NtNTP2 polynucleotide as described herein; (b) introducing a meganuclease or a construct encoding a meganuclease into said plant cell; and (c) allowing the meganuclease to substantially inactivate the NtNTP2 polynucleotide(s)
Meganucleases can be used to cleave meganuclease recognition sites within the coding regions of a NtNTP2 polynucleotide. Such cleavage frequently results in the deletion of DNA at the meganuclease recognition site following mutagenic DNA repair by non-homologous end joining. Such mutations in the gene coding sequence are typically sufficient to inactivate the gene. This method to modify a plant cell involves, first, the delivery of a meganuclease expression cassette to a plant cell using a suitable transformation method. For highest efficiency, it is desirable to link the meganuclease expression cassette to a selectable marker and select for successfully transformed cells in the presence of a selection agent. This approach will result in the integration of the meganuclease expression cassette into the genome, however, which may not be desirable if the plant is likely to require regulatory approval. In such cases, the meganuclease expression cassette (and linked selectable marker gene) may be segregated away in subsequent plant generations using conventional breeding techniques.
Following delivery of the meganuclease expression cassette, plant cells are grown, initially, under conditions that are typical for the particular transformation procedure that was used. This may mean growing transformed cells on media at temperatures below 26°C, frequently in the dark. Such standard conditions can be used for a period of time, suitably 1-4 days, to allow the plant cell to recover from the transformation process. At any point following this initial recovery period, growth temperature may be raised to stimulate the function of the engineered meganuclease to cleave and mutate the meganuclease recognition site.
One method of gene editing involves the use of transcription activator-like effector nucleases (TALENs) which induce double-strand breaks which cells can respond to with repair mechanisms. NHEJ reconnects DNA from either side of a double-strand break where there is very little or no sequence overlap for annealing. This repair mechanism induces errors in the genome via insertion or deletion, or chromosomal rearrangement. Any such errors may render the gene products coded at that location non-functional. For certain applications, it may be desirable to precisely remove the NtNTP2 polynucleotide from the genome of the plant. Such applications are possible using a pair of engineered meganucleases, each of which cleaves a meganuclease recognition site on either side of the intended deletion. TALENs that are able to recognize and bind to a gene and introduce a double-strand break into the genome can also be used. Thus, in another aspect, methods for producing mutant, non-naturally occurring or transgenic or otherwise genetically-modified plants as described herein using TAL Effector Nucleases are contemplated.
Another method of gene editing involves the use of the bacterial CRISPR/Cas system. Bacteria and archaea exhibit chromosomal elements called clustered regularly interspaced short palindromic repeats (CRISPR) that are part of an adaptive immune system that protects against invading viral and plasmid DNA. In Type II CRISPR systems, CRISPR RNAs (crRNAs) function with trans-activating crRNA (tracrRNA) and CRISPR-associated (Cas) polypeptides to introduce double-stranded breaks in target DNA. Target cleavage by Cas9 requires basepairing between the crRNA and tracrRNA as well as base pairing between the crRNA and the target DNA. Target recognition is facilitated by the presence of a short motif called a protospacer-adjacent motif (PAM) that conforms to the sequence NGG. This system can be harnessed for genome editing. Cas9 is normally programmed by a dual RNA consisting of the crRNA and tracrRNA. However, the core components of these RNAs can be combined into a single hybrid ‘guide RNA’ for Cas9 targeting. The use of a noncoding RNA guide to target DNA for site-specific cleavage promises to be significantly more straightforward than existing technologies - such as TALENs. Using the CRISPR/Cas strategy, retargeting the nuclease complex only requires introduction of a new RNA sequence and there is no need to reengineer the specificity of polypeptide transcription factors. CRISPR/Cas technology was implemented in plants in the method of international application WO 2015/189693 A1 , which discloses a viral-mediated genome editing platform that is broadly applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and deliver guide RNA into Nicotiana benthamiana plants overexpressing Cas9 endonuclease. In the context of the present disclosure, a guide RNA may be derived from any of the sequences disclosed herein and the teaching of WO 2015/189693 A1 applied to edit the genome of a plant cell and obtain a desired mutant plant. The fast pace of the development of the technology has generated a great variety of protocols with broad applicability in plantae, which have been well catalogued in a number of recent scientific review articles (for example, Schiml et al. Plant Methods 2016 12:8; and Khatodia et al. Front Plant Sci. 2016; 7: 506). A review of CRISPR/Cas systems with a particular focus on its application in plants is given by Bortesi and Fischer (Biotechnology Advances (2015) 33, 1 , 41-52). Bortesi and Fischer also make comparisons between the CRISPR/Cas technology, zinc finger nucleases, and TALENs. More recent developments in the use of CRISPR/Cas for manipulating plant genomes are discussed by Liu et al. (2017) Acta Pharmaceutica Sinica B 7, 3, 292-302 and Curr. Op. in Plant Biol. (2017) 36, 1-8. CRISPR/Cas9 plasmids for use in plants are listed in “addgene”, the nonprofit plasmid repository (addgene.org), and CRISPR/Cas plasmids are commercially available.
Antisense technology is another well-known method that can be used to modulate the expression or activity of a NtNTP2 polypeptide. A polynucleotide of the gene to be repressed is cloned and operably linked to a regulatory region and a transcription termination sequence so that the antisense strand of RNA is transcribed. The recombinant construct is then transformed into a plant cell and the antisense strand of RNA is produced. The polynucleotide need not be the entire sequence of the gene to be repressed, but typically will be substantially complementary to at least a portion of the sense strand of the gene to be repressed.
A polynucleotide may be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA. Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. Heterologous polynucleotides can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide. Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site-specific recognition sequences can be used. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target RNA contains a 5'-UG-3' polynucleotide. The construction and production of hammerhead ribozymes is known in the art. Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo.
In one embodiment, the sequence-specific polynucleotide that can interfere with the translation of RNA transcript(s) is interfering RNA. RNA interference or RNA silencing is an evolutionarily conserved process by which specific mRNAs can be targeted for enzymatic degradation. A double-stranded RNA (double-stranded RNA) is introduced or produced by a cell (for example, double-stranded RNA virus, or interfering RNA polynucleotides) to initiate the interfering RNA pathway. The double-stranded RNA can be converted into multiple small interfering RNA (siRNA) duplexes of 21-24 bp length by RNases III, which are double-stranded RNA-specific endonucleases. The siRNAs can be subsequently recognized by RNA-induced silencing complexes that promote the unwinding of siRNA through an ATP-dependent process. The unwound antisense strand of the siRNA guides the activated RNA-induced silencing complexes to the targeted mRNA comprising a sequence complementary to the siRNA anti-sense strand. The targeted mRNA and the anti-sense strand can form an A-form helix, and the major groove of the A-form helix can be recognized by the activated RNA- induced silencing complexes. The target mRNA can be cleaved by activated RNA-induced silencing complexes at a single site defined by the binding site of the 5'-end of the siRNA strand. The activated RNA-induced silencing complexes can be recycled to catalyze another cleavage event.
Interfering RNA expression vectors may comprise interfering RNA constructs encoding interfering RNA polynucleotides that exhibit RNA interference by reducing the expression level of mRNAs, pre-mRNAs, or related RNA variants. The expression vectors may comprise a promoter positioned upstream and operably-linked to an Interfering RNA construct, as further described herein. Interfering RNA expression vectors may comprise a suitable minimal core promoter, a Interfering RNA construct of interest, an upstream (5') regulatory region, a downstream (3') regulatory region, including transcription termination and polyadenylation signals, and other sequences known to persons skilled in the art, such as various selection markers.
The double-stranded RNA molecules may include siRNA molecules assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a polynucleotide based or non- polynucleotide-based linker(s), as well as circular single-stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating interfering RNA.
The use of small hairpin RNA molecules is also contemplated. They comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a singlestranded RNA molecule and its reverse complement, such that they may anneal to form a double-stranded RNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3' end or the 5' end of either or both strands). The spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double-stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end or the 5' end of either or both strands). The spacer sequence is typically an unrelated polynucleotide that is situated between two complementary polynucleotides regions which, when annealed into a doublestranded polynucleotide, comprise a small hairpin RNA. The spacer sequence generally comprises between about 3 and about 100 nucleotides.
Any RNA polynucleotide of interest can be produced by selecting a suitable sequence composition, loop size, and stem length for producing the hairpin duplex. A suitable range for designing stem lengths of a hairpin duplex, includes stem lengths of at least about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides - such as about 14-30 nucleotides, about 30-50 nucleotides, about 50-100 nucleotides, about 100-150 nucleotides, about 150-200 nucleotides, about 200-300 nucleotides, about 300-400 nucleotides, about 400-500 nucleotides, about 500-600 nucleotides, and about 600-700 nucleotides. A suitable range for designing loop lengths of a hairpin duplex, includes loop lengths of about 4-25 nucleotides, about 25-50 nucleotides, or longer if the stem length of the hair duplex is substantial. In certain embodiments, a double-stranded RNA or ssRNA molecule is between about 15 and about 40 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule between about 15 and about 35 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule between about 17 and about 30 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule between about 19 and about 25 nucleotides in length. In another embodiment, the siRNA molecule is a double-stranded RNA or ssRNA molecule between about 21 to about 23 nucleotides in length. In certain embodiments, hairpin structures with duplexed regions longer than 21 nucleotides may promote effective siRNA- directed silencing, regardless of loop sequence and length. Exemplary sequences for RNA interference are described herein.
The target mRNA sequence is typically between about 14 to about 50 nucleotides in length. The target mRNA can, therefore, be scanned for regions between about 14 and about 50 nucleotides in length that suitably meet one or more of the following criteria: an A+T/G+C ratio of between about 2:1 and about 1 :2; an AA dinucleotide or a CA dinucleotide at the 5' end; a sequence of at least 10 consecutive nucleotides unique to the target mRNA (that is, the sequence is not present in other mRNA sequences from the same plant); and no "runs" of more than three consecutive guanine (G) nucleotides or more than three consecutive cytosine (C) nucleotides. These criteria can be assessed using various techniques known in the art, for example, computer programs such as BLAST can be used to search publicly available databases to determine whether the selected sequence is unique to the target mRNA. Alternatively, a sequence can be selected (and a siRNA sequence designed) using computer software available commercially (for example, OligoEngine, Target Finder and the siRNA Design Tool which are commercially available).
In one embodiment, target mRNA sequences are selected that are between about 14 and about 30 nucleotides in length that meet one or more of the above criteria. In another embodiment, sequences are selected that are between about 16 and about 30 nucleotides in length that meet one or more of the above criteria. In a further embodiment, sequences are selected that are between about 19 and about 30 nucleotides in length that meet one or more of the above criteria. In another embodiment, sequences are selected that are between about 19 and about 25 nucleotides in length that meet one or more of the above criteria.
In an exemplary embodiment, the siRNA molecules comprise a specific antisense sequence that is complementary to at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more contiguous nucleotides of any one of the polynucleotides described herein. The specific antisense sequence comprised by the siRNA molecule can be identical or substantially identical to the complement. In one embodiment, the specific antisense sequence comprised by the siRNA molecule is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the complement of the target mRNA sequence. Methods of determining sequence identity are known in the art and can be determined, for example, by using the BLASTN program of the University of Wisconsin Computer Group (GCG) software or provided on the NCBI website.
One method for inducing double stranded RNA-silencing in plants is transformation with a gene construct producing hairpin RNA (see Nature (2000) 407, 319-320). Such constructs comprise inverted regions of the target gene sequence, separated by an appropriate spacer. The insertion of a functional plant intron region as a spacer fragment additionally increases the efficiency of the gene silencing induction, due to generation of an intron spliced hairpin RNA (Plant J. (2001), 27, 581-590). Suitably, the stem length is about 50 nucleotides to about 1 kilobases in length. Methods for producing intron spliced hairpin RNA are well described in the art (see for example, Bioscience, Biotechnology, and Biochemistry (2008) 72, 2, 615-617). Interfering RNA molecules having a duplex or double-stranded structure, for example doublestranded RNA or small hairpin RNA, can have blunt ends, or can have 3' or 5' overhangs. As used herein, "overhang" refers to the unpaired nucleotide or nucleotides that protrude from a duplex structure when a 3'-terminus of one RNA strand extends beyond the 5'-terminus of the other strand (3' overhang), or vice versa (5' overhang). The nucleotides comprising the overhang can be ribonucleotides, deoxyribonucleotides or modified versions thereof. In one embodiment, at least one strand of the interfering RNA molecule has a 3' overhang from about 1 to about 6 nucleotides in length. In other embodiments, the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides and from about 2 to about 4 nucleotides in length.
When the interfering RNA molecule comprises a 3' overhang at one end of the molecule, the other end can be blunt-ended or have also an overhang (5' or 3'). When the interfering RNA molecule comprises an overhang at both ends of the molecule, the length of the overhangs may be the same or different. In one embodiment, the interfering RNA molecule comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule. In a further embodiment, the interfering RNA molecule is a double-stranded RNA having a 3' overhang of 2 nucleotides at both ends of the molecule. In yet another embodiment, the nucleotides comprising the overhang of the interfering RNA are TT dinucleotides or ULI dinucleotides.
The interfering RNA molecules can comprise one or more 5' or 3'-cap structures. The term "cap structure" refers to a chemical modification incorporated at either terminus of an oligonucleotide, which protects the molecule from exonuclease degradation, and may also facilitate delivery or localisation within a cell.
Another modification applicable to interfering RNA molecules is the chemical linkage to the interfering RNA molecule of one or more moieties or conjugates which enhance the function, cellular distribution, cellular uptake, bioavailability or stability of the interfering RNA molecule. The polynucleotides may be synthesized or modified by methods well established in the art. Chemical modifications include 2' modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and typically two, chemical linkages.
The nucleotides at one or both of the two single strands may be modified to modulate the activation of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for reducing or inhibiting the activation of cellular enzymes are known in the art including, but not limited to, 2'-amino modifications, 2'-fluoro modifications, 2'-alkyl modifications, uncharged backbone modifications, morpholino modifications, 2'-O-methyl modifications, and phosphoramidate.
Ligands may be conjugated to an interfering RNA molecule, for example, to enhance its cellular absorption. In certain embodiments, a hydrophobic ligand is conjugated to the molecule to facilitate direct permeation of the cellular membrane. In certain instances, conjugation of a cationic ligand to oligonucleotides often results in improved resistance to nucleases.
"Targeted Induced Local Lesions In Genomes" (TILLING) is another mutagenesis technology that can be used to generate and/or identify NtNTP2 polynucleotides encoding NtNTP2 polypeptides with modified expression, function or activity. TILLING also allows selection of plants carrying such mutants. TILLING combines high-density mutagenesis with high- throughput screening methods. Methods for TILLING are well known in the art (see McCallum et al., (2000) Nat Biotechnol 18: 455-457 and Stemple (2004) Nat Rev Genet 5(2): 145-50). Various embodiments are directed to expression vectors comprising one or more of the NtNTP2 polynucleotides or interfering RNA constructs that comprise one or more NtNTP2 polynucleotides described herein.
Various embodiments are directed to expression vectors comprising one or more of the NtNTP2 polynucleotides or one or more interfering RNA constructs described herein. Various embodiments are directed to expression vectors comprising one or more NtNTP2 polynucleotides or one or more interfering RNA constructs encoding one or more interfering RNA polynucleotides described herein that are capable of self-annealing to form a hairpin structure, in which the construct comprises (a) one or more of the NtNTP2 polynucleotides described herein; (b) a second sequence encoding a spacer element that forms a loop of the hairpin structure; and (c) a third sequence comprising a reverse complementary sequence of the first sequence, positioned in the same orientation as the first sequence, wherein the second sequence is positioned between the first sequence and the third sequence, and the second sequence is operably-linked to the first sequence and to the third sequence.
The disclosed sequences can be utilised for constructing various NtNTP2 polynucleotides that do not form hairpin structures. For example, a double-stranded RNA can be formed by (1) transcribing a first strand of the DNA by operably-linking to a first promoter, and (2) transcribing the reverse complementary sequence of the first strand of the DNA fragment by operably- linking to a second promoter. Each strand of the polynucleotide can be transcribed from the same expression vector, or from different expression vectors. The RNA duplex having RNA interference can be enzymatically converted to siRNAs to modulate RNA levels.
Thus, various embodiments are directed to expression vectors comprising one or more NtNTP2 polynucleotides or interfering RNA constructs described herein encoding interfering RNA polynucleotides capable of self-annealing, in which the construct comprises (a) one or more of the NtNTP2 polynucleotides described herein; and (b) a second sequence comprising a complementary (for example, reverse complementary) sequence of the first sequence, positioned in the same orientation as the first sequence.
Various compositions and methods are provided for modulating the endogenous expression levels of one or more of the NtNTP2 polypeptides described herein (or any combination thereof as described herein) by promoting co-suppression of gene expression.
Various compositions and methods are provided for modulating the endogenous gene expression level by modulating the translation of mRNA. A host plant cell can be transformed with an expression vector comprising: a promoter operably-linked to a NtNTP2 polynucleotide, positioned in anti-sense orientation with respect to the promoter to enable the expression of RNA polynucleotides having a sequence complementary to a portion of mRNA.
Various expression vectors for modulating the translation of mRNA may comprise: a promoter operably-linked to a NtNTP2 polynucleotide in which the sequence is positioned in anti-sense orientation with respect to the promoter. The lengths of anti-sense RNA polynucleotides can vary, and may be from about 15-20 nucleotides, about 20-30 nucleotides, about 30-50 nucleotides, about 50-75 nucleotides, about 75-100 nucleotides, about 100-150 nucleotides, about 150-200 nucleotides, and about 200-300 nucleotides. Alternatively, genes can be targeted for inactivation by introducing transposons (for example, IS elements) into the genomes of plants of interest. These mobile genetic elements can be introduced by sexual cross-fertilization and insertion mutants can be screened for loss in polypeptide function. The disrupted gene in a parent plant can be introduced into other plants by crossing the parent plant with plant not subjected to transposon-induced mutagenesis by, for example, sexual cross-fertilization. Any standard breeding techniques known to persons skilled in the art can be utilized. In one embodiment, one or more genes can be inactivated by the insertion of one or more transposons. Mutations can result in homozygous disruption of one or more genes, in heterozygous disruption of one or more genes, or a combination of both homozygous and heterozygous disruptions if more than one gene is disrupted. Suitable transposable elements include retrotransposons, retroposons, and SI NE-like elements. Such methods are known to persons skilled in the art.
Alternatively, genes can be targeted for inactivation by introducing ribozymes derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. These RNAs can replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples of suitable RNAs include those derived from avocado sunblotch viroid and satellite RNAs derived from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus, and subterranean clover mottle virus. Various target RNA-specific ribozymes are known to persons skilled in the art.
The mutant or non-naturally occurring plants or plant cells can have any combination of one or more mutations in one or more genes which results in modulated expression or function or activity of those genes or their products. For example, the mutant or non-naturally occurring plants or plant cells may have a single mutation in a single gene; multiple mutations in a single gene; a single mutation in two or more or three or more or four or more genes; or multiple mutations in two or more or three or more or four or more genes. Examples of such mutations are described herein. By way of further example, the mutant or non-naturally occurring plants or plant cells may have one or more mutations in a specific portion of the NtNTP2 gene(s) - such as in a region of the gene that encodes an active site of the NtNTP2 polypeptide or a portion thereof. By way of further example, the mutant or non-naturally occurring plants or plant cells may have one or more mutations in a region outside of one or more NtNTP2 gene(s) - such as in a region upstream or downstream of the gene it regulates provided that they modulate the function or expression of the NtNTP2 gene(s). Upstream elements can include promoters, enhancers or transcription factors. Some elements - such as enhancers - can be positioned upstream or downstream of the gene it regulates. The element(s) need not be located near to the gene that it regulates since some elements have been found located several hundred thousand base pairs upstream or downstream of the gene that it regulates. The mutant or non-naturally occurring plants or plant cells may have one or more mutations located within the first 100 nucleotides of the gene(s), within the first 200 nucleotides of the gene(s), within the first 300 nucleotides of the gene(s), within the first 400 nucleotides of the gene(s), within the first 500 nucleotides of the gene(s), within the first 600 nucleotides of the gene(s), within the first 700 nucleotides of the gene(s), within the first 800 nucleotides of the gene(s), within the first 900 nucleotides of the gene(s), within the first 1000 nucleotides of the gene(s), within the first 1100 nucleotides of the gene(s), within the first 1200 nucleotides of the gene(s), within the first 1300 nucleotides of the gene(s), within the first 1400 nucleotides of the gene(s) or within the first 1500 nucleotides of the gene(s). The mutant or non-naturally occurring plants or plant cells may have one or more mutations located within the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth set of 100 nucleotides of the gene(s) or combinations thereof. Mutant or non-naturally occurring plants or plant cells (for example, mutant, non-naturally occurring or transgenic plants or plant cells and the like, as described herein) comprising the mutant NtNTP2 polypeptides are disclosed.
In one embodiment, seeds from plants are mutagenised and then grown into first generation mutant plants. The first generation plants are then allowed to self-pollinate and seeds from the first generation plant are grown into second generation plants, which are then screened for mutations in their loci. Though the mutagenized plant material can be screened for mutations, an advantage of screening the second generation plants is that all somatic mutations correspond to germline mutations. One of skill in the art would understand that a variety of plant materials, including but not limited to, seeds, pollen, plant tissue or plant cells, may be mutagenised in order to create the mutant plants. However, the type of plant material mutagenised may affect when the plant polynucleotide is screened for mutations. For example, when pollen is subjected to mutagenesis prior to pollination of a non-mutagenized plant the seeds resulting from that pollination are grown into first generation plants. Every cell of the first generation plants will contain mutations created in the pollen; thus these first generation plants may then be screened for mutations instead of waiting until the second generation.
Prepared NtNTP2 polynucleotide from individual plants, plant cells, or plant material can optionally be pooled in order to expedite screening for mutations in the population of plants originating from the mutagenized plant tissue, cells or material. One or more subsequent generations of plants, plant cells or plant material can be screened. The size of the optionally pooled group is dependent upon the sensitivity of the screening method used.
After the samples are optionally pooled, they can be subjected to NtNTP2 polynucleotidespecific amplification techniques, such as PCR. Any one or more primers or probes specific to the NtNTP2 gene or the sequences immediately adjacent to the NtNTP2 gene may be utilized to amplify the sequences within the optionally pooled sample. Suitably, the one or more primers or probes are designed to amplify the regions of the locus where useful mutations are most likely to arise. Most suitably, the primer is designed to detect mutations within regions of the NtNTP2 polynucleotide. Additionally, it is preferable for the primer(s) and probe(s) to avoid known polymorphic sites in order to ease screening for point mutations. To facilitate detection of amplification products, the one or more primers or probes may be labelled using any conventional labelling method. Primer(s) or probe(s) can be designed based upon the sequences described herein using methods that are well understood in the art.
To facilitate detection of amplification products, the primer(s) or probe(s) may be labelled using any conventional labelling method. These can be designed based upon the sequences described herein using methods that are well understood in the art.
Polymorphisms may be identified by means known in the art and some have been described in the literature.
In some embodiments, a plant may be regenerated or grown from the plant, plant tissue or plant cell. Any suitable methods for regenerating or growing a plant from a plant cell or plant tissue may be used, such as, without limitation, tissue culture or regeneration from protoplasts. Suitably, plants may be regenerated by growing transformed plant cells on callus induction media, shoot induction media and/or root induction media. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. Thus as used herein, "transformed seeds" refers to seeds that contain the nucleotide construct stably integrated into the plant genome.
Accordingly, in a further aspect there is provided a method of preparing a mutant plant. The method involves providing at least one cell of a plant comprising a NtNTP2 gene encoding a functional NtNTP2 polypeptide. Next, the at least one cell of the plant is treated under conditions effective to modulate (reduce) the function of the NtNTP2 polynucleotide(s) described herein. The at least one mutant plant cell is then propagated into a mutant plant, where the mutant plant has a modulated (reduced or inhibited) level of NtNTP2 polypeptide(s) as compared to that of a control plant. In one embodiment of this method of making a mutant plant, the treating step involves subjecting the at least one cell to a chemical mutagenising agent as described above and under conditions effective to yield at least one mutant plant cell. In another embodiment of this method, the treating step involves subjecting the at least one cell to a radiation source under conditions effective to yield at least one mutant plant cell. The term "mutant plant" includes mutant plants in which the genotype is modified as compared to a control plant, suitably by means other than genetic engineering or genetic modification.
In certain embodiments, the mutant plant, mutant plant cell or mutant plant material may comprise one or more mutations that have occurred naturally in another plant, plant cell or plant material and confer a desired trait. This mutation can be incorporated (for example, introgressed) into another plant, plant cell or plant material (for example, a plant, plant cell or plant material with a different genetic background to the plant from which the mutation was derived) to confer the trait thereto. Thus, by way of example, a mutation that occurred naturally in a first plant may be introduced into a second plant - such as a second plant with a different genetic background to the first plant. The skilled person is therefore able to search for and identify a plant carrying naturally in its genome one or more mutant alleles of the genes described herein which confer a desired trait. The mutant allele(s) that occurs naturally can be transferred to the second plant by various methods including breeding, backcrossing and introgression to produce a lines, varieties or hybrids that have one or more mutations in the genes described herein. The same technique can also be applied to the introgression of one or more non-naturally occurring mutation(s) from a first plant into a second plant. Plants showing a desired trait may be screened out of a pool of mutant plants. Suitably, the selection is carried out utilising the knowledge of the NtNTP2 polynucleotide as described herein. Consequently, it is possible to screen for a genetic trait as compared to a control. Such a screening approach may involve the application of conventional amplification and/or hybridization techniques as discussed herein. Thus, a further aspect of the present disclosure relates to a method for identifying a mutant plant comprising the steps of: (a) providing a sample comprising NtNTP2 polynucleotide from a plant; and (b) determining the sequence of the NtNTP2 polynucleotide, wherein a difference in the sequence of the NtNTP2 polynucleotide as compared to the NtNTP2 polynucleotide of a control plant is indicative that said plant is a mutant plant. In another aspect there is provided a method for identifying a mutant plant which: (i) does not have decreased nitrate levels as compared to a control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response as compared to the control plant grown in the same fertilization conditions comprising: (a) providing a sample from a plant to be screened; (b) determining if said sample comprises one or more mutations in one or more of the NtNTP2 polynucleotides described herein; and (c) determining the nitrate levels, the biomass and the NUE response as compared to the control plant grown in the same fertilization conditions.
In another aspect there is provided a method for preparing a mutant plant which: (i) does not have decreased nitrate levels as compared to a control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response as compared to the control plant grown in the same fertilization conditions comprising: (a) providing a sample from a first plant;
(b) determining if said sample comprises one or more mutations in one or more the NtNTP2 polynucleotides described herein that result in: (i) decreased nitrate levels as compared to a control plant grown in the same fertilization conditions; and (ii) increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) increased NUE response as compared to the control plant grown in the same fertilization conditions; and
(c) transferring the one or more mutations into a second plant. The mutant plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions.
The mutation(s) can be transferred into the second plant using various methods that are known in the art - such as by genetic engineering, genetic manipulation, introgression, plant breeding, backcrossing and the like. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background to the first plant.
In another aspect there is provided a method for preparing a mutant plant which: (i) has decreased nitrate levels as compared to a control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response as compared to the control plant grown in the same fertilization conditions comprising: (a) providing a sample from a first plant; (b) determining if said sample comprises one or more mutations in one or more of the NtNTP2 polynucleotides described herein that results in decreased nitrate levels, increased biomass and increased NUE response as compared to the control plant grown in the same fertilization conditions; and (c) introgressing the one or more mutations from the first plant into a second plant. The mutant plant can also have an increase in root development as compared to the control plant grown in the same fertilization conditions. In one embodiment, the step of introgressing comprises plant breeding, optionally including backcrossing and the like. In one embodiment, the first plant is a naturally occurring plant. In one embodiment, the second plant has a different genetic background to the first plant. In one embodiment, the first plant is not a cultivar or an elite cultivar. In one embodiment, the second plant is a cultivar or an elite cultivar.
A further aspect relates to a mutant plant (including a cultivar or elite cultivar mutant plant) obtained or obtainable by the methods described herein. In certain embodiments, the “mutant plants” may have one or more mutations localised only to a specific region of the plant - such as within the sequence of the one or more NtNTP2 polynucleotide(s) described herein. According to this embodiment, the remaining genomic sequence of the mutant plant will be the same or substantially the same as the plant prior to the mutagenesis. In certain embodiments, the mutant plants may have one or more mutations localised in more than one genomic region of the plant - such as within the sequence of one or more of the NtNTP2 polynucleotides described herein and in one or more further regions of the genome. According to this embodiment, the remaining genomic sequence of the mutant plant will not be the same or will not be substantially the same as the plant prior to the mutagenesis. In certain embodiments, the mutant plants may not have one or more mutations in one or more, two or more, three or more, four or more or five or more exons of the polynucleotide(s) described herein; or may not have one or more mutations in one or more, two or more, three or more, four or more or five or more introns of the polynucleotide(s) described herein; or may not have one or more mutations in a promoter of the polynucleotide(s) described herein; or may not have one or more mutations in the 3’ untranslated region of the polynucleotide(s) described herein; or may not have one or more mutations in the 5’ untranslated region of the polynucleotide(s) described herein; or may not have one or more mutations in the coding region of the polynucleotide(s) described herein; or may not have one or more mutations in the non-coding region of the polynucleotide(s) described herein; or any combination of two or more, three or more, four or more, five or more; or six or more thereof parts thereof.
In a further aspect there is provided a method of identifying a plant, a plant cell or plant material comprising a mutation in a gene encoding a NtNTP2 polynucleotide as described herein comprising: (a) subjecting a plant, a plant cell or plant material to mutagenesis; (b) obtaining a sample from said plant, plant cell or plant material or descendants thereof; and (c) determining the polynucleotide sequence of the NtNTP2 gene or a variant or a fragment thereof, wherein a difference in said sequence is indicative of one or more mutations therein. This method also allows the selection of plants having mutation(s) that occur(s) in genomic regions that affect the expression of the NtNTP2 gene in a plant cell, such as a transcription initiation site, a start codon, a region of an intron, a boundary of an exon-intron, a terminator, or a stop codon.
The mutant, non-naturally occurring or transgenic plant or part thereof according to the present disclosure has an advantageous phenotype in which there is no significant difference in nitrate levels as compared to a control plant grown in the same fertilization conditions, leaf biomass yield is higher as compared to the control plant grown in the same fertilization conditions, and the NUE of the plant is higher as compared to the control plant grown in the same fertilization conditions. A morphological difference observed between the mutant, non-naturally occurring or transgenic plants as compared to the control is an increase in root development as compared to the control plant grown in the same fertilization conditions.
No significant difference in nitrate levels has been observed (in cured lamina and midrib from mid-stalk position leaves) between the -S and -T mutant, non-naturally occurring or transgenic plants as compared to a control. Thus, reducing NtNTP2 activity of both the -S and -T forms is not expected to lead to evident effects on nitrate levels.
The mutant, non-naturally occurring or transgenic plant or part thereof according to the present disclosure generates more biomass - (for example, leaf biomass) as compared to a control. Suitably, leaf biomass is green leaves or dried leaves or cured leaves, more suitably, green leaves. The increase in biomass can be at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29% or at least about 30% or at least about 31%, or at least about 34% or at least about 35%, or at least about 36%, or at least about 37% or more as compared to the control grown in the same conditions. The amount of biomass that is generated can be higher in a nitrogen starvation regime as compared to a standard fertilization regime. For example, in a standard fertilization regimeor in a nitrogen starvation regime, the increase in biomass can be at least about 5% or more as compared to the control grown in the same conditions. In one embodiment, the increase in biomass in nitrogen starvation conditions is at least about 28% as compared to a control grown in the same conditions. Accordingly, NtNTP2 loss of activity results in an improved yield in different nitrogen regimes.
The NUE index (i.e. the units of biomass produced (expressed as kilograms per hectare, assuming a number of 12 thousand plants per hectare) per unit of nitrogen fertilization input (expressed as kilograms of nitrogen per hectare)) determined for the mutant, non-naturally occurring or transgenic plant or part thereof according to the present disclosure can be increased when grown in nitrogen starving conditions by at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21 %, or at least about 22% or more as compared to the control. In a standard fertilization regime, the NUE index can be increased by at least about 5%, at least about 9%, at least about 10%, at least about 13%, or at least about 14%, at least about 20%, a least about 25% or at least about 28% or more as compared to the control grown in the same conditions. In one embodiment, the increase in NUE index in nitrogen starvation conditions is at least about 28% as compared to the control grown in the same conditions. Accordingly, NtNTP2 loss of activity results in an improved NUE in different nitrogen regimes. Thus, impairment in NTP2 protein activity can increase the plant’s ability to adjust to nitrogen starvation, thereby increasing the NUE of the plant, intended as biomass per unit of nitrogen fertilization applied.
Accordingly, the mutant, non-naturally occurring or transgenic plant or part thereof according to the present disclosure has an advantageous phenotype in which there is no significant difference in nitrate levels as compared to a control plant grown in the same fertilization conditions, biomass yield is increased by at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29% or at least about 30% or at least about 31%, or at least about 34% or at least about 35%, or at least about 36%, or at least about 37% or more as compared to the control and the NUE of the plant is increased by at least about 5%, at least about 9%, at least about 10%, at least about 13%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, or at least about 22% or more as compared to the control. The mutant, non-naturally occurring or transgenic plant or part thereof can have an increase in root development as compared to the control plant grown in the same fertilization conditions.
In one embodiment, in standard fertilisation conditions the plant or the part of the plant (for example, leaf): (i) does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and (ii) has at least a 5% biomass increase as compared to the control plant grown in the same fertilization conditions; and (iii) has at least a 5% increase in NUE response as compared to the control plant grown in the same fertilization conditions. The plant or part thereof can have an increase in root development as compared to the control plant grown in the same fertilization conditions.
In another embodiment, in nitrogen starvation conditions the plant or the part of the plant (for example, leaf): (i) does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and (ii) has at least a 5% biomass increase as compared to the control plant grown in the same fertilization conditions; and (iii) has at least a 5% increase in NUE response as compared to the control plant grown in the same fertilization conditions. The plant or part thereof can have an increase in root development as compared to the control plant grown in the same fertilization conditions.
In embodiments, the phenotype differs according to the fertilization input. Thus, in one embodiment, in standard fertilisation conditions, the plant or the part of the plant (for example, leaf): (i) does not have decreased nitrate levels as compared to the control plant; and (ii) has at least a 5 % biomass increase as compared to the control plant; and (iii) has at least a 5% increase in NUE response as compared to the control plant. In another embodiment, in nitrogen starvation conditions, the plant or the part of the plant (for example, leaf): (i) does not have decreased nitrate levels as compared to the control plant; and (ii) has at least a 5% biomass increase as compared to the control plant; and (iii) has at least a 5% increase in NUE response as compared to the control plant. The plant or part thereof can have an increase in root development as compared to the control plant grown in the same fertilization conditions. The plants of the present disclosure therefore have increased yield with lower fertilization input and therefore an improved NUE. The lower fertilization input can achieve a reduction in TSNAs.
According to the present disclosure, ‘standard conditions’ are 254 nitrogen units (one unit expressed as kilograms per hectare) and ‘nitrogen starving conditions’ are 55 nitrogen units (one unit expressed as kilograms per hectare).
Plants according to the present disclosure include, but are not limited to, monocotyledonous and dicotyledonous plants and plant cell systems, including species from one of the following families: Acanthaceae, Alliaceae, Alstroemeriaceae, Amaryllidaceae, Apocynaceae, Arecaceae, Asteraceae, Berberidaceae, Bixaceae, Brassicaceae, Bromeliaceae, Cannabaceae, Caryophyllaceae, Cephalotaxaceae, Chenopodiaceae, Colchicaceae, Cucurbitaceae, Dioscoreaceae, Ephedraceae, Erythroxylaceae, Euphorbiaceae, Fabaceae, Lamiaceae, Linaceae, Lycopodiaceae, Malvaceae, Melanthiaceae, Musaceae, Myrtaceae, Nyssaceae, Papaveraceae, Pinaceae, Plantaginaceae, Poaceae, Rosaceae, Rubiaceae, Salicaceae, Sapindaceae, Solanaceae, Taxaceae, Theaceae, or itaceae.
Suitable species may include members of the genera Abelmoschus, Abies, Acer, Agrostis,
Allium, Alstroemeria, Ananas, Andrographis, Andropogon, Artemisia, Arundo, Atropa, Berberis, Beta, Bixa, Brassica, Calendula, Camellia, Camptotheca, Cannabis, Capsicum, Carthamus, Catharanthus, Cephalotaxus, Chrysanthemum, Cinchona, Citrullus, Coffea, Colchicum, Coleus, Cucumis, Cucurbita, Cynodon, Datura, Dianthus, Digitalis, Dioscorea, Elaeis, Ephedra, Erianthus, Erythroxylum, Eucalyptus, Festuca, Fragaria, Galanthus, Glycine, Gossypium, Helianthus, Hevea, Hordeum, Hyoscyamus, Jatropha, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Lycopodium, Manihot, Medicago, Mentha, Miscanthus, Musa, Nicotiana, Oryza, Panicum, Papaver, Parthenium, Pennisetum, Petunia, Phalaris, Phleum, Pinus, Poa, Poinsettia, Populus, Rauwolfia, Ricinus, Rosa, Saccharum, Salix, Sanguinaria, Scopolia, Secale, Solanum, Sorghum, Spartina, Spinacea, Tanacetum, Taxus, Theobroma, Triticosecale, Triticum, llniola, Veratrum, Vinca, Vitis, and Zea.
Suitable species may include Panicum spp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Andropogon gerardii (big bluestem), Pennisetum purpureum (elephant grass), Phalaris arundinacea (reed canarygrass), Cynodon dactylon (bermudagrass), Festuca arundinacea (tall fescue), Spartina pectinata (prairie cord-grass), Medicago sativa (alfalfa), Arundo donax (giant reed), Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale (tritic wheat times rye), bamboo, Helianthus annuus (sunflower), Carthamus tinctorius (safflower), Jatropha curcas (jatropha), Ricinus communis (castor), Elaeis guineensis (palm), Linum usitatissimum (flax), Brassica juncea, Beta vulgaris (sugarbeet), Manihot esculenta (cassaya), Lycopersicon esculentum (tomato), Lactuca sativa (lettuce), Musyclise alca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, Brussels sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea ycliseca (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant), Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Lupinus albus (lupin), llniola paniculata (oats), bentgrass (Agrostis spp.), Populus tremuloides (aspen), Pinus spp. (pine), Abies spp. (fir), Acer spp. (maple), Hordeum vulgare (barley), Poa pratensis (bluegrass), Lolium spp. (ryegrass) and Phleum pratense (timothy), Panicum virgatum (switchgrass), Sorghu56yclise56or (sorghum, sudangrass), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), or Pennisetum glaucum (pearl millet).
Suitable species may include species of the genus Nicotiana, including N. rustica and N. tabacum (for example, LA B21 , LN KY171 , Tl 1406, Basma, Galpao, Perique, Beinhart 1000- 1 , and Petico). Other Nicotiana species include N. acaulis, N. acuminata, N. africana, N. alata, N. ameghinoi, N. amplexicaulis, N. arentsii, N. attenuata, N. azambujae, N. benavidesii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. Cleveland ii, N. cordi folia, N. corymbosa, N. debneyi, N. excelsior, N. forgetiana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. linearis, N. longiflora, N. maritime, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N. occidentalis subsp. hesperis, N. otophora, N. paniculata, N. pauciflora, N. petunioides, N. plumbaginifolia, N. quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata subsp. ingulba, N. rotundifolia, N. setchellii, N. simulans, N. solanifolia, N. spegazzinii, N. stocktonii, N. suaveolens, N. sylvestris, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides, and N. x sanderae. In one embodiment, the plant is N. tabacum.
The use of tobacco cultivars and elite tobacco cultivars is also contemplated herein. The transgenic, non-naturally occurring or mutant plant may therefore be a tobacco variety or elite tobacco cultivar that comprises one or more transgenes, or one or more genetic mutations or a combination thereof. The genetic mutation(s) (for example, one or more polymorphisms) can be mutations that do not exist naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar) or can be genetic mutation(s) that do occur naturally provided that the mutation does not occur naturally in the individual tobacco variety or tobacco cultivar (for example, elite tobacco cultivar). Particularly useful N. tabacum varieties include Burley type, dark type, flue-cured type, and Oriental type tobaccos. Non-limiting examples of varieties or cultivars are: BD 64, CC 101 , CC 200, CC 27, CC 301 , CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, Coker 176, Coker 319, Coker 371 Gold, Coker 48, CD 263, DF911 , DT 538 LC Galpao tobacco, GL 26H, GL 350, GL 600, GL 737, GL 939, GL 973, HB 04P, HB 04P LC, HB3307PLC, Hybrid 403LC, Hybrid 404LC, Hybrid 501 LC, K 149, K 326, K 346, K 358, K394, K 399, K 730, KDH 959, KT 200, KT204LC, KY10, KY14, KY 160, KY 17, KY 171 , KY 907, KY907LC, KY14xL8 LC, Little Crittenden, McNair 373, McNair 944, msKY 14xL8, Narrow Leaf Madole, Narrow Leaf Madole LC, NBH 98, N-126, N-777LC, N-7371 LC, NC 100, NC 102, NC 2000, NC 291 , NC 297, NC 299, NC 3, NC 4, NC 5, NC 6, NC7, NC 606, NC 71 , NC 72, NC 810, NC BH 129, NC 2002, Neal Smith Madole, OXFORD 207, PD 7302 LC, PD 7309 LC, PD 7312 LC, ’Perique' tobacco, PVH03, PVH09, PVH19, PVH50, PVH51 , R 610, R 630, R 7-11 , R 7-12, RG 17, RG 81 , RG H51 , RGH 4, RGH 51 , RS 1410, Speight 168, Speight 172, Speight 179, Speight 210, Speight 220, Speight 225, Speight 227, Speight 234, Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3, Tl 1406, Tl 1269, TN 86, TN86LC, TN 90, TN 97, TN97LC, TN D94, TN D950, TR (Tom Rosson) Madole, VA 309, VA359, AA 37-1 , B13P, Xanthi (Mitchell- Mor), Bel-W3, 79-615, Samsun Holmes NN, KTRDC number 2 Hybrid 49, Burley 21 , KY8959, KY9, MD 609, PG01 , PG04, PO1 , PO2, PO3, RG11 , RG 8, VA509, AS44, Banket A1 , Basma Drama B84/31 , Basma I Zichna ZP4/B, Basma Xanthi BX 2A, Batek, Besuki Jember, C104, Coker 347, Criollo Misionero, Delcrest, Djebel 81 , DVH 405, Galpao Comum, HB04P, Hicks Broadleaf, Kabakulak Elassona, Kutsage E1 , LA BU 21 , NC 2326, NC 297, PVH 2110, Red Russian, Samsun, Saplak, Simmaba, Talgar 28, Wislica, Yayaldag, Prilep HC-72, Prilep P23, Prilep PB 156/1 , Prilep P12-2/1 , Yaka JK-48, Yaka JB 125/3, TI-1068, KDH-960, TI-1070, TW136, Basma, TKF 4028, L8, TKF 2002, GR141 , Basma xanthi, GR149, GR153, Petit Havana. Low converter subvarieties of the above, even if not specifically identified herein, are also contemplated.
Embodiments are also directed to compositions and methods for producing mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that have been modified to modulate the expression or function of a NtNTP2 polynucleotide(s) described herein (or any combination thereof as described herein). Advantageously, the mutant plants, non-naturally occurring plants, hybrid plants, or transgenic plants that are obtained may be similar or substantially the same in overall appearance to control plants. Various phenotypic characteristics such as degree of maturity, number of leaves per plant, stalk height, leaf insertion angle, leaf size (width and length), internode distance, and lamina-midrib ratio can be assessed by field observations.
One aspect relates to a seed of a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant described herein. A further aspect relates to pollen or an ovule of a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant that is described herein. In addition, there is provided a mutant plant, a non-naturally occurring plant, a hybrid plant or a transgenic plant as described herein which further comprises a polynucleotide conferring male sterility. Also provided is a tissue culture of regenerable cells of the mutant plant, non-naturally occurring plant, hybrid plant, or transgenic plant or a part thereof as described herein, which culture regenerates plants capable of expressing all the morphological and physiological characteristics of the parent. The regenerable cells include cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers and a part thereof, ovules, shoots, stems, stalks, pith and capsules or callus or protoplasts derived therefrom.
The mutant, non-naturally occurring or transgenic plant leaf or part of the plant leaf obtained according to this disclosure can be similar or substantially the same in visual appearance to the corresponding control plant leaf or part of the plant leaf. In one embodiment, the leaf number is substantially the same as the control. In another embodiment, the chlorophyll content is substantially the same as the control plant grown in the same fertilization conditions. In other embodiments, the size or form or number or colouration of the leaf is substantially the same as the control plant grown in the same fertilization conditions.
Polynucleotides and recombinant constructs described herein can be used to modulate the expression or function or activity of the NtNTP2 polynucleotides or NtNTP2 polypeptides described herein.
A plant carrying a mutant allele of one or more NtNTP2 polynucleotides described herein (or any combination thereof as described herein) can be used in a plant breeding program to create useful lines, varieties and hybrids containing leaf of the desired genotype and phenotype. In particular, the mutant allele is introgressed into the commercially important varieties described above. Thus, methods for breeding plants are provided, that comprise crossing a mutant plant, a non-naturally occurring plant or a transgenic plant as described herein with a plant comprising a different genetic identity. The method may further comprise crossing the progeny plant with another plant, and optionally repeating the crossing until a progeny with the desirable genotype and phenotype is obtained. One purpose served by such breeding methods is to introduce a desirable genetic trait into other varieties, breeding lines, hybrids or cultivars, particularly those that are of commercial interest. Another purpose is to facilitate stacking of genetic modifications of different genes in a single plant variety, lines, hybrids or cultivars. Intraspecific as well as interspecific matings are contemplated. The progeny plants that arise from such crosses, also referred to as breeding lines, are examples of non-naturally occurring plants of the disclosure.
In one embodiment, a method is provided for producing a non-naturally occurring plant comprising: (a) crossing a mutant or transgenic plant with a second plant to yield progeny tobacco seed; (b) growing the progeny tobacco seed, under plant growth conditions, to yield the non-naturally occurring plant. The method may further comprise: (c) crossing the previous generation of non-naturally occurring plant with itself or another plant to yield progeny tobacco seed; (d) growing the progeny tobacco seed of step (c) under plant growth conditions, to yield additional non-naturally occurring plants; and repeating the crossing and growing steps of (c) and (d) multiple times to generate further generations of non-naturally occurring plants. The method may optionally comprises prior to step (a), a step of providing a parent plant which comprises a genetic identity that is characterized and that is not identical to the mutant or transgenic plant. In some embodiments, depending on the breeding program, the crossing and growing steps are repeated from 0 to 2 times, from 0 to 3 times, from 0 to 4 times, 0 to 5 times, from 0 to 6 times, from 0 to 7 times, from 0 to 8 times, from 0 to 9 times or from 0 to 10 times, in order to generate generations of non-naturally occurring plants. Backcrossing is an example of such a method wherein a progeny is crossed with one of its parents or another plant genetically similar to its parent, in order to obtain a progeny plant in the next generation that has a genetic identity which is closer to that of one of the parents. Techniques for plant breeding, particularly plant breeding, are well known and can be used in the methods of the disclosure. The disclosure further provides non-naturally occurring plants produced by these methods. Certain embodiments exclude the step of selecting a plant. Suitably, leaf or a part of the leaf is harvested from the produced plant(s).
In some embodiments of the methods described herein, lines resulting from breeding and screening for variant NtNTP2 genes are evaluated in the field using standard field procedures. Control genotypes including the original unmutagenised parent are included and entries are arranged in the field in a randomized complete block design or other appropriate field design. For tobacco, standard agronomic practices are used, for example, the tobacco is harvested, weighed, and sampled for chemical and other common testing before and during curing. Statistical analyses of the data are performed to confirm the similarity of the selected lines to the parental line. Cytogenetic analyses of the selected plants are optionally performed to confirm the chromosome complement and chromosome pairing relationships.
DNA fingerprinting, single nucleotide polymorphism, microsatellite markers, or similar technologies may be used in a marker-assisted selection (MAS) breeding program to transfer or breed mutant alleles of a NtNTP2 gene into other tobaccos, as described herein. For example, a breeder can create segregating populations from hybridizations of a genotype containing a mutant NtNTP2 allele of interest with an agronomically desirable genotype. Plants in the F2 or backcross generations can be screened using a marker developed from a genomic sequence or a fragment thereof, using one of the techniques listed herein. Plants identified as possessing the mutant NtNTP2 allele can be backcrossed or self-pollinated to create a second population to be screened. Depending on the expected inheritance pattern, it may be necessary to self-pollinate the selected plants before each cycle of backcrossing to aid identification of the desired individual plants. Backcrossing or other breeding procedure can be repeated until the desired phenotype of the recurrent parent is recovered.
According to the disclosure, in a breeding program, successful crosses yield F1 plants that are fertile. Selected F1 plants can be crossed with one of the parents, and the first backcross generation plants are self-pollinated to produce a population that is again screened for variant NtNTP2 gene expression (for example, the null version of the gene). The process of backcrossing, self-pollination, and screening is repeated, for example, at least 4 times until the final screening produces a plant that is fertile and reasonably similar to the recurrent parent. This plant, if desired, is self-pollinated and the progeny are subsequently screened again to confirm that the plant exhibits variant NtNTP2 gene expression. In some embodiments, a plant population in the F2 generation is screened for variant NtNTP2 gene expression, for example, a plant is identified that fails to express a NtNTP2 polypeptide due to the absence of the NtNTP2 gene according to standard methods, for example, by using a PCR method with primers based upon the polynucleotide sequence information for the NtNTP2 polynucleotide(s) described herein (or any combination thereof as described herein). Hybrid tobacco varieties can be produced by preventing self-pollination of female parent plants (that is, seed parents) of a first variety, permitting pollen from male parent plants of a second variety to fertilize the female parent plants, and allowing F1 hybrid seeds to form on the female plants. Self-pollination of female plants can be prevented by emasculating the flowers at an early stage of flower development. Alternatively, pollen formation can be prevented on the female parent plants using a form of male sterility. For example, male sterility can be produced by cytoplasmic male sterility (CMS), or transgenic male sterility wherein a transgene inhibits microsporogenesis and/or pollen formation, or self-incompatibility. Female parent plants containing CMS are particularly useful. In embodiments in which the female parent plants are CMS, pollen is harvested from male fertile plants and applied manually to the stigmas of CMS female parent plants, and the resulting F1 seed is harvested.
Varieties and lines described herein can be used to form single-cross tobacco F1 hybrids. In such embodiments, the plants of the parent varieties can be grown as substantially homogeneous adjoining populations to facilitate natural cross-pollination from the male parent plants to the female parent plants. The F1 seed formed on the female parent plants is selectively harvested by conventional means. One also can grow the two parent plant varieties in bulk and harvest a blend of F1 hybrid seed formed on the female parent and seed formed upon the male parent as the result of self-pollination. Alternatively, three-way crosses can be carried out wherein a single-cross F1 hybrid is used as a female parent and is crossed with a different male parent. As another alternative, double-cross hybrids can be created wherein the F1 progeny of two different single-crosses are themselves crossed. A population of mutant, non-naturally occurring or transgenic plants can be screened or selected for those members of the population that have a desired trait or phenotype. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression or function of the NtNTP2 polypeptide(s) encoded thereby. Physical and biochemical methods can be used to identify expression or activity levels. These include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, S1 RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme function of polypeptides and polynucleotides; and polypeptide gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining and enzyme assays also can be used to detect the presence or expression, function or activity of NtNTP2 polypeptides or NtNTP2 polynucleotides.
Mutant, non-naturally occurring or transgenic plant cells and plants are described herein comprising one or more recombinant NtNTP2 polynucleotides, one or more NtNTP2 polynucleotide constructs, one or more double-stranded RNAs, one or more conjugates or one or more vectors/expression vectors.
One or more of the following further genetic modifications can be present in the mutant, non- naturally occurring or transgenic plant leaf or part of the plant leaf.
One or more genes that are involved in the conversion of nitrogenous metabolic intermediates can be modified resulting in lower levels of at least one tobacco-specific nitrosamine (TSNA). Non-limiting examples of such genes include those encoding nicotine demethylase - such as CYP82E4, CYP82E5 and CYP82E10 as described in W02006/091194, W02008/070274, W02009/064771 and WO2011/088180 - and nitrate reductase, as described in WO2016046288.
One or more genes that are involved in heavy metal uptake or heavy metal transport can be modified resulting in lower heavy metal content. Non-limiting examples include genes in the family of multidrug resistance associated polypeptides, the family of cation diffusion facilitators (CDF), the family of Zrt- 1 rt-like polypeptides (ZIP), the family of cation exchangers (CAX), the family of copper transporters (COPT), the family of heavy-metal ATPases (for example, HMAs, as described in W02009/074325 and WO2017/129739), the family of homologs of natural resistance-associated macrophage polypeptides (NRAMP), and other members of the family of ATP-binding cassette (ABC) transporters (for example, MRPs), as described in WO20 12/028309, which participate in transport of heavy metals - such as cadmium.
Other exemplary modifications can result in plants with modulated expression or function of isopropylmalate synthase which results in a change in sucrose ester composition which can be used to alter favour profile (see WO2013029799). Other exemplary modifications can result in plants with modulated expression or function of threonine synthase in which levels of methional can be modulated (see WO2013029800).
Other exemplary modifications can result in plants with modulated expression or function of one or more of neoxanthin synthase, lycopene beta cyclase and 9-cis-epoxycarotenoid dioxygenase to modulate beta-damascenone content to alter flavour profile (see WO20 13064499).
Other exemplary modifications can result in plants with modulated expression or function of members of the CLC family of chloride channels to modulate nitrate levels therein (see WO2014096283 and WO2015197727).
Other exemplary modifications can result in plants with modulated expression or function of one or more asparagine synthetases to modulate levels of asparagine in leaf and modulated levels of acrylamide in aerosol produced upon heating or combusting the leaf (see WO20 17042162).
Examples of other modifications include modulating herbicide tolerance, for example, glyphosate is an active ingredient of many broad spectrum herbicides. Glyphosate resistant transgenic plants have been developed by transferring the aroA gene (a glyphosate EPSP synthetase from Salmonella typhimurium and E.coli). Sulphonylurea resistant plants have been produced by transforming the mutant ALS (acetolactate synthetase) gene from Arabidopsis. OB polypeptide of photosystem II from mutant Amaranthus hybridus has been transferred in to plants to produce atrazine resistant transgenic plants; and bromoxynil resistant transgenic plants have been produced by incorporating the bxn gene from the bacterium Klebsiella pneumoniae.
Another exemplary modification results in plants that are resistant to insects. Bacillus thuringiensis (Bt) toxins can provide an effective way of delaying the emergence of Bt-resistant pests, as recently illustrated in broccoli where pyramided crylAc and cry1C Bt genes controlled diamondback moths resistant to either single polypeptide and significantly delayed the evolution of resistant insects.
Another exemplary modification results in plants that are resistant to diseases caused by pathogens (for example, viruses, bacteria, fungi). Plants expressing the Xa21 gene (resistance to bacterial blight) with plants expressing both a Bt fusion gene and a chitinase gene (resistance to yellow stem borer and tolerance to sheath) have been engineered.
Another exemplary modification results in altered reproductive capability, such as male sterility.
Another exemplary modification results in plants that are tolerant to abiotic stress (for example, drought, temperature, salinity), and tolerant transgenic plants have been produced by transferring acyl glycerol phosphate enzyme from Arabidopsis; genes coding mannitol dehydrogenase and sorbitol dehydrogenase which are involved in synthesis of mannitol and sorbitol improve drought resistance.
Another exemplary modification results in plants in which the activity of one or more endogenous glycosyltransferases - such as N-acetylglucosaminyltransferase, (3(1 ,2)- xylosyltransferase and a(1 ,3)-fucosyl- transferase is modulated (see WO/2011/117249).
Another exemplary modification results in plants in which the activity of one or more nicotine N-demethylases is modulated such that the levels of nornicotine and metabolites of nornicotine - that are formed during curing can be modulated (see WO2015169927).
Other exemplary modifications can result in plants with improved storage polypeptides and oils, plants with enhanced photosynthetic efficiency, plants with prolonged shelf life, plants with enhanced carbohydrate content, and plants resistant to fungi. Transgenic plants in which the expression of S-adenosyl-L-methionine (SAM) and/or cystathionine gamma-synthase (CGS) has been modulated are also contemplated.
One or more genes that are involved in the nicotine synthesis pathway can be modified resulting in plants or parts of plants that when cured, produce modulated levels of nicotine. The nicotine synthesis genes can be selected from the group consisting of: A622, BBLa, BBLb, JRE5L1 , JRE5L2, MATE1 , MATE 2, MPO1 , MPO2, MYC2a, MYC2b, NBB1 , nic1 , nic2, NUP1 , NLIP2, PMT 1 , PMT2, PMT3, PMT4 and QPT or a combination of one or more thereof.
One or more genes that are involved in controlling the amount of one or more alkaloids can be modified resulting in plants or parts of plants that produce modulated levels of alkaloid. Alkaloid level controlling genes can be selected from the group consisting of; BBLa, BBLb, JRE5L1 , JRE5L2, MATE1 , MATE 2, MYC2a, MYC2b, nic1 , nic2, NUP1 and NUP2 or a combination of two or more thereof.
Leaf material - such as lamina and midrib - can be incorporated into or used in making various consumable products including but not limited to aerosol forming materials, aerosol forming devices, smoking articles, smokable articles, smokeless products, medicinal or cosmetic products, intravenous preparations, tablets, powders, and tobacco products. Examples of aerosol forming materials include tobacco compositions, tobaccos, tobacco extract, cut tobacco, cut filler, cured tobacco, expanded tobacco, homogenized tobacco, reconstituted tobacco, and pipe tobaccos. Smoking articles and smokable articles are types of aerosol forming devices. Examples of smoking articles or smokable articles include cigarettes, cigarillos, and cigars. Examples of smokeless products comprise chewing tobaccos, and snuffs. In certain aerosol forming devices, rather than combustion, a tobacco composition or another aerosol forming material is heated by one or more electrical heating elements to produce an aerosol. In another type of heated aerosol forming device, an aerosol is produced by the transfer of heat from a combustible fuel element or heat source to a physically separate aerosol forming material, which may be located within, around or downstream of the heat source. Smokeless tobacco products and various tobacco-containing aerosol forming materials may contain tobacco in any form, including as dried particles, shreds, granules, powders, or a slurry, deposited on, mixed in, surrounded by, or otherwise combined with other ingredients in any format, such as flakes, films, tabs, foams, or beads. As used herein, the term ‘smoke’ is used to describe a type of aerosol that is produced by smoking articles, such as cigarettes, or by combusting an aerosol forming material.
In one example, leaf material - such as lamina and midrib - can be processed according to the methods described in US20190142058A1 in which a cast sheet of homogenized tobacco material is prepared by pulping cellulose fibres with water; grinding a blend of tobacco of one or more tobacco types to tobacco particles; combining the pulped cellulose fibres with the tobacco particles and with a binder to form a slurry; homogenizing the slurry; casting the slurry to form a cast sheet of homogenized tobacco material from the slurry; discarding undesired portions of the cast sheet; and introducing the discarded undesired portions of the cast sheet into the slurry. Accordingly, leaf material - such as lamina and midrib - can be combined with a binder - such as natural pectins, such as fruit, citrus or tobacco pectins; guar gums, such as hydroxyethyl guar and hydroxypropyl guar; locust bean gums, such as hydroxyethyl and hydroxypropyl locust bean gum; alginate; starches, such as modified or derivitized starches; celluloses, such as methyl, ethyl, ethyl hydroxymethyl and carboxymethyl cellulose; tamarind gum; dextran; pullalon; konjac flour; xanthan gum and the like. Thus, tobacco material can comprise the leaf material as described herein and a binder. In one embodiment, there is also provided cured plant leaf material. Processes of curing green tobacco leaves are known by those having skills in the art and include without limitation air-curing, fire-curing, flue-curing and sun-curing as described herein.
In another embodiment, there is described tobacco products including tobacco-containing aerosol forming materials comprising plant leaf material, suitably cured leaf. The tobacco products described herein can be a blended tobacco product which may further comprise unmodified tobacco.
The mutant, non-naturally occurring or transgenic plant leaf or part of the plant leaf may have other uses in, for example, agriculture. For example, mutant, non-naturally occurring or transgenic plant leaf or part of the plant leaf described herein can be used to make animal feed and human food products.
The disclosure also provides methods for producing seeds comprising: cultivating the mutant plant, non-naturally occurring plant, or transgenic plant described herein, and collecting seeds from the cultivated plants. Seeds from plants described herein can be conditioned and bagged in packaging material by means known in the art to form an article of manufacture. Packaging material such as paper and cloth are well known in the art. A package of seed can have a label, for example, a tag or label secured to the packaging material, a label printed on the package that describes the nature of the seeds therein.
Compositions, methods and kits for genotyping plants for identification, selection, or breeding can comprise a means of detecting the presence of a NtNTP2 polynucleotide (or any combination thereof as described herein) in a sample of polynucleotide. Accordingly, a composition is described comprising one or more primers for specifically amplifying at least a portion of one or more of the NtNTP2 polynucleotides and optionally one or more probes and optionally one or more reagents for conducting the amplification or detection.
Accordingly, NtNTP2 gene specific oligonucleotide primers or probes comprising about 10 or more contiguous polynucleotides corresponding to the NtNTP2 polynucleotide(s) described herein are disclosed. Said primers or probes may comprise or consist of about 15, 20, 25, 30, 40, 45 or 50 more contiguous polynucleotides that hybridise (for example, specifically hybridise) to the polynucleotide(s) described herein. In some embodiments, the primers or probes may comprise or consist of about 10 to 50 contiguous nucleotides, about 10 to 40 contiguous nucleotides, about 10 to 30 contiguous nucleotides or about 15 to 30 contiguous nucleotides that may be used in sequence-dependent methods of NtNTP2 gene identification (for example, Southern hybridization) or isolation (for example, in situ hybridization of bacterial colonies or bacteriophage plaques) or NtNTP2 gene detection (for example, as one or more amplification primers in amplification or detection). The one or more specific primers or probes can be designed and used to amplify or detect a part or all of the polynucleotide(s). By way of specific example, two primers may be used in a PCR protocol to amplify a NtNTP2 polynucleotide fragment. The PCR may also be performed using one primer that is derived from a NtNTP2 polynucleotide sequence and a second primer that hybridises to the sequence upstream or downstream of the polynucleotide sequence - such as a promoter sequence, the 3' end of the mRNA precursor or a sequence derived from a vector. Examples of thermal and isothermal techniques useful for in vitro amplification of polynucleotides are well known in the art. The sample may be or may be derived from a plant, a plant cell or plant material or a tobacco product made or derived from the plant, the plant cell or the plant material as described herein.
In a further aspect, there is also provided a method of detecting a NtNTP2 polynucleotide(s) described herein (or any combination thereof as described herein) in a sample comprising the step of: (a) providing a sample comprising, or suspected of comprising, a NtNTP2 polynucleotide; (b) contacting said sample with one or more primers or one or more probes for specifically detecting at least a portion of the NtNTP2 polynucleotide(s); and (c) detecting the presence of an amplification product, wherein the presence of an amplification product is indicative of the presence of the NtNTP2 polynucleotide(s) in the sample. In a further aspect, there is also provided the use of one or more primers or probes for specifically detecting at least a portion of the NtNTP2 polynucleotide(s). Kits for detecting at least a portion of the NtNTP2 polynucleotide(s) are also provided which comprise one or more primers or probes for specifically detecting at least a portion of the NtNTP2 polynucleotide(s). The kit may comprise reagents for NtNTP2 polynucleotide amplification - such as PCR - or reagents for probe hybridization-detection technology - such as Southern Blots, Northern Blots, in-situ hybridization, or microarray. The kit may comprise reagents for antibody bindingdetection technology such as Western Blots, ELISAs, SELDI mass spectrometry or test strips. The kit may comprise reagents for DNA sequencing. The kit may comprise reagents and instructions for using the kit.
In some embodiments, a kit may comprise instructions for one or more of the methods described. The kits described may be useful for genetic identity determination, phylogenetic studies, genotyping, haplotyping, pedigree analysis or plant breeding particularly with codominant scoring.
The present disclosure also provides a method of genotyping a plant, a plant cell or plant material comprising a NtNTP2 polynucleotide as described herein. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. The specific method of genotyping may employ any number of molecular marker analytic techniques including amplification fragment length polymorphisms (AFLPs). AFLPs are the product of allelic differences between amplification fragments caused by polynucleotide variability. Thus, the present disclosure further provides a means to follow segregation of one or more NtNTP2 genes or polynucleotides as well as chromosomal sequences genetically linked to these NtNTP2 genes or polynucleotides using such techniques as AFLP analysis.
The present disclosure also provides a method of improving an agronomic characteristic of a plant by reducing or inhibiting the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of: (i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or (ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or (iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or (iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or (v) a NtNTP2-T polypeptide having at least 77 % % sequence identity to SEQ ID NO: 12; wherein the expression or activity of the NtNTP2-T or the expression or activity of the NtNTP2-T and the NtNTP2-S is reduced or inhibited as compared to a control plant. Suitably, the agronomic characteristic is: (i) nitrate levels are not decreased; (ii) biomass (for example, leaf biomass) is increased in both standard and nitrogen starvation conditions; and (iii) NUE response, intended as biomass per unit of nitrogen fertilization applied, is increased. The expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S can be reduced or inhibited using any of the methods that are described herein. By way of example, one or more sequence-specific polynucleotides that can interfere with the transcription of NtNTP2-T or NtNTP2-T and NtNTP2-S can be used. By way of further example, one or more sequencespecific polypeptides that can interfere with the stability of NtNTP2-T or NtNTP2-T and NtNTP2-S can be used. By way of example, one or more sequence-specific polynucleotides that can interfere with the enzymatic activity of NtNTP2-T or NtNTP2-T and NtNTP2-S or the binding activity of NtNTP2-T or NtNTP2-T and NtNTP2-S with respect to substrates or regulatory proteins. By way of further example, gene edited NtNTP2-T or NtNTP2-T and NtNTP2-S can be used. Suitably, the NtNTP2-T or NtNTP2-T and NtNTP2-S are gene edited using the bacterial CRISPR/Cas system. By way of further example, at least one genetic alteration in the NtNTP2-T polynucleotide sequence or at least one genetic alteration in the NtNTP2-T polynucleotide and the NtNTP2-S polynucleotide sequence or at least one genetic alteration in the NtNTP2-T polypeptide sequence or at least one genetic alteration in the NtNTP2-T polypeptide sequence and the NtNTP2-S polypeptide sequence can be used. Suitably, the at least one genetic alteration can be at least one genetic alteration that causes the encoded polypeptide(s) to terminate or end translation earlier than in the control plant. By way of further example, the at least one genetic alteration can reduce or inhibit the expression or activity of NtNTP2-T or of NtNTP2-T and NtNTP2-S. The at least one genetic alteration can comprise at least one nonsense mutation in the NtNTP2-T polynucleotide or the NtNTP2- T polypeptide or at least one nonsense mutation in the NtNTP2-T polynucleotide or NtNTP2- T polypeptide and at least one nonsense mutation in the NtNTP2-S polynucleotide or the NtNTP2-S polypeptide. For example, the mutation can be a single nucleotide polymorphism in NtNTP2-S at nucleotide position 632 or 633 or 632 and 633 of SEQ ID NO: 3, suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 632 or 633 of SEQ ID NO: 3 or a ‘g’ to ‘a’ mutation at nucleotide positions 632 and 633 of SEQ ID NO: 3. The mutated NtNTP2-S polynucleotide sequence can comprise, consist or consist essentially of SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6. The mutation can be a single nucleotide polymorphism in NtNTP2-T at nucleotide position 636 of SEQ ID NO: 11 , suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 635 or 636 of SEQ ID NO: 11 or a ‘g’ to ‘a’ mutation at nucleotide positions 635 and 636 of SEQ ID NO: 11. The mutated NtNTP2-T polynucleotide sequence can comprise, consist or consist essentially of SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15. The mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide can each have at least one nonsense mutation at position W212 or position W212 and W211 , respectively. The mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide can comprise, consist or consist essentially of either SEQ ID NO: 16 or SEQ ID NO: 8 and SEQ ID NO: 16, respectively. Sequences deposited in databases are described herein and can change over time. Suitably the current version of sequence database(s) are relied upon. Alternatively, the release in force at the date of filing is relied upon. As the skilled person knows, the accession numbers may be version/dated accession numbers. The citeable accession numbers for the current database entry are the same as herein, but omitting the decimal point and any subsequent digits. GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences (National Center for Biotechnology Information, U.S. National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA; Nucleic Acids Research, 2013 Jan;41 (D1):D36-42) and accession numbers provided relate to this unless otherwise apparent. Suitably the current release is relied upon. More suitably the release available at the effective filing date is relied upon. Most suitably, the GenBank database release referred to is NCBI-GenBank Release 241 : 15 December 2020. Suitably the current version of sequence database(s) are relied upon. Alternatively, the release in force at the date of filing is relied upon. For the avoidance of doubt, the UniProt consortium European Bioinformatics Institute (EBI), SIB Swiss Institute of Bioinformatics and Protein Information Resource (PIR)’s UniProt Knowledgebase (UniProtKB) Release 2021_01 published 10 February 2021 is relied upon. UniProt (Universal Protein Resource) is a comprehensive catalogue of information on proteins (“UniProt: the universal protein knowledgebase” Nucleic Acids Res. 45: D158-D169 (2017)).
The invention is further described in the Examples below, which are provided to describe the invention in further detail. These examples, which set forth a preferred mode presently contemplated for carrying out the invention, are intended to illustrate and not to limit the invention.
EXAMPLES
Example 1 - EMS mutant population screening for ntp2 mutations and ntp2-S W211stop/ntp2- T W212stop double mutant breeding
To find specific mutations in an EMS-generated AA37 mutant population, primers are designed covering part of the NtNtp2-Sar\d NtNtp2-T gene sequences. The resulting amplified fragments are sequenced. The primer pairs used to identify n/p2 stop mutants are reported in Table 1.
Point mutations leading to a stop codon and thus to a truncated non-functional version of the protein, are considered to be of interest. The list of stop mutants identified are reported in Table 2. The Table reports details of stop codon mutations identified during EMS screening. F seq and R seq columns indicate the SNP (single nucleotide polymorphism) mutation (in 5’ and 3’ to the mutation, respectively). The original wild type codons and corresponding amino acids (Codon ori and AS ori columns) and the corresponding mutated versions (Codon mut and AS mut columns) are also indicated.
A TaqMan assay is used to screen for the different ntp2 mutations and genotypes. Table 3 reports details of the primers and probes used for genotyping AA37 ntp2 W211stop and W212stop double mutant lines. The identity column indicates the gene and phenotype identified (wt indicates wild type genotype, mut indicates the mutant genotype; tAg and tgA indicate the two different mutations identified in the NtNtp2-S gene). F primer and R primer columns indicate the primer sequences of forward and reverse primers respectively. Probe column reports the sequences of the probes used (underlined bases indicate the discriminants between wt and different mutations).
The double mutant Ntntp2-S W211stop/Ntntp2-T W212stop in the AA37 background is generated by crossing single mutant plants Ntntp2-S W211stop (tgA mutation) and single mutant plants Ntntp2-T W212stop.
Example 2 - Field trials design and execution
Two season field trials are held. Seed bedding, sawing and seedling growth in a greenhouse is performed according to ID-160: Burley and Dark Tobacco Production Guide, 2021-2022 (uky.edu) for Burley and Dark tobaccos, 2022 Flue-Cured Tobacco Guide, NC State Extension Publications (ncsu.edu) for Virginia tobacco standards. 10 plants plots per genotype are transplanted in a randomly distributed field design, with 7 to 19 replicas per genotype, according to growing season. The first field trial is grown in Burley conditions, with a total input of 250 nitrogen units applied, being one unit expressed as kilograms per hectare. Plants are topped before flowering, and at harvest time, 5 mid stalk position leaves per plant are harvested and hanged to air dry for curing. Two identically randomly distributed fields are implemented in different nitrogen input conditions: one in Burley conditions (254 nitrogen units, being one unit expressed as kilograms per hectare) and one in nitrogen starving Virginia conditions (55 nitrogen units, i.e. 25% of nitrogen input compared to Burley conditions). Per each genotype, 19 replicas are grown per field. Plants are topped before flowering, and stalk cured after a few weeks.
Example 3 - Cured material analyses
After curing, the number of plants per plot and the weight of cured leaf material is recorded for biomass detection. Standard curing procedures are described in ID-160: Burley and Dark Tobacco Production Guide, 2021-2022 (uky.edu) for Burley and Dark tobaccos, 2022 Flue- Cured Tobacco Guide | NC State Extension Publications (ncsu.edu) for Virginia tobacco. All leaves are detached from the stalk and weighed, eliminating the material from edge plants to avoid positional effects. Representative lamina samples from 10 to 15 mid-stalk position leaves per plot are collected for nitrate level determination. Samples are lyophilized and reduced to powder. Nitrite- nitrate contents (in this report indicated as nitrate levels) are measured using the Cayman chemical Nitrate/Nitrite Colorimetric Assay Kit (Item No. 780001) according to supplier instructions and as described in FASEB Journal (1992) 6, 3051-3064; Anal. Biochem. (1982) 126, 131-138; and Methods (1995) 7, 48-54.
Example 4 - NtNtp2 gene and protein sequences
Two NtNtp2 genes are present in the tobacco genome, one on the chromosome 8 (-S form Ntab-TN90_AYMYSS948 in the publicly available TN90 genome) and the other on chromosome 21 (-T form Ntab-TN90_AYMYSS1317). Genomic, transcript, gene and protein sequences for the NtNtp2 genes are presented in the accompanying list of sequences.
The NtNTP2-T putative protein sequence is BLASTed in protein databases from different plant species (Solanum melongena, Arabidopsis thaliana, Nicotiana benthamiana and Solanum lycopersicum), and the highest homologies are reported in Table 4. Reported are the homologous proteins (predicted proteins) and the degree of identity expressed as percentage of identical residues. Table 5 reports the degree of identity with the respective predicted coding regions (CDS), expressed as percentage of identical residues.
Example 5 - Plant phenotype and nitrate levels in ntp2-S W211stop / ntp2-T W212stop double mutant
AA37 tobacco plants are grown in a field under Burley regime (254 nitrogen units, noted above) during two consecutive growing seasons. At harvest time, no morphological difference is evident between the double mutant and the wild type out-segregant genotypes by observing the plants in the field, as shown in Figure 1.
Nitrate levels in cured lamina and midrib from mid-stalk position leaves are measured as average per plot (n=7 to 8 plots of 10 plants each) and are reported in Figure 2. No significant difference is detected in the nitrate levels between out-segregant wt and double ntp2 mutant genotypes (p value=0.21361 for lamina data and p value=0.8485846 for midribs data in a two tailed Student test).
By contrast, in Arabidopsis, ntp2 insertional mutants display 50-64% less nitrate content in petiole and midrib compared to wild type, and a statistically significant 13% more nitrate in the lamina (Chiu et al. (2004) supra).
From these results there is no statistically evident difference in lamina nitrate content between homozygous mutants and wild type plants, nor in the midrib.
It is concluded that impairing NtNTP2 protein activity of both the -S and -T forms does not lead to evident effects on nitrate levels in AA37 tobacco cured leaf lamina nor midribs from plants grown in Burley conditions. Example 6 - Ntp2-S W211stop/ntp2-T W212stop mutant yield effect.
Two parallel fields are implemented in Burley and Virginia fertilization conditions (respectively 254 and 55 nitrogen units, i.e. 100% and 25% of nitrogen input). Double mutant and out- segregant wild type plants grown in Burley conditions did not display noticeable phenotypic differences (see Figure 3, upper panel). When grown in nitrogen starvation conditions (Virginia regime, 25% nitrogen units compared to the Burley conditions), ntp2 double mutant plants appear slightly bigger than the out-segregant wild type counterpart, with more developed leaves (see Figure 3, lower panel).
Plants are topped and stalk cured, and leaf cured biomass is recorded from all the commercially valuable leaf stages. Yield data reported in Figure 4 reports yield phenotype of the ntp2 double stop mutant in different nitrogen regimes. In the standard Burley fertilization regime, ntp2 double stop mutant plants generate 16.2% more leaf biomass than their out- segregant wild types (p value=0.00654 in two tailed Stuart test). In nitrogen starvation regime, the gain in biomass compared to the wild type is higher, reaching 28.3% (p value=0.003184). It is concluded that the characterizing phenotype for NtNTP2 loss of activity is an improved yield in the different nitrogen regimes tested.
In Chiu et al. (2004), supra, authors demonstrated that Arabidopsis homozygous Atntp2 insertional mutants develop wider and longer leaves compared to wild type plants, due to an increase in cell expansion. Interestingly, the present field results suggest that in tobacco AA37 plants a loss of function of NtNTP2 proteins not only give higher yield in standard fertilization regime, but also limit the biomass loss due to growth in nitrogen starvation. In fact, as shown in Figure 4, ntp2 double stop mutant plants have a yield difference between standard and nitrogen starvation conditions that is reduced compared to the out-segregant wild type genotype (30.8% compared to 37.3%), with a statistically significant higher cured biomass for the mutants compared to the wild type plants in all conditions (see Figure 4).
During a second experiment, a segregation test is performed to define contribution of the -S and -T mutations to the yield phenotype. Figure 5 reports total cured biomass data expressed in grams per plant of different segregant phenotypes of the Ntntp2 mutations. Ntntp2-S W211stop and Ntntp2-T W212stop double mutant plants (sstt) demonstrate a 9.4% increase in average total cure leaf biomass per plant compared to the out-segregant wild type (SSTT). The ntp2 loss of function biomass effect is not detected in the experiments conducted on the ntp2-S W211 stop single mutant (ssTT in Figure 5).
Therefore, a loss of function in the NTP2-S protein is not sufficient to gain this phenotype. Loss of function in the NTP2-T protein alone is sufficient for generating the yield improvement phenotype in AA37 tobacco plants (SStt), with a 13.9% gain in yield compared to the double outsegregant wild type (SSTT), with a p value=0.04545133. Example 7 - Ntp2-S W211stop/ntp2-T W212stop mutant NUE
From the cured biomass data reported in Figure 4, the difference in biomass between standard fertilization and nitrogen starving conditions (25% N units compared to standard conditions) is reduced for ntp2 double stop mutant plants compared to out-segregant wild types (30.8% compared to 37.3%, respectively). We calculated therefore the NUE index, i.e. the units of biomass produced (expressed as kilograms per hectare, assuming a number of 12 thousand plants per hectare) per unit of nitrogen fertilization input (expressed as kilograms of nitrogen per hectare), as indicated in Table 6. Per ntp2 genotype (Genotypes) are reported the total harvest cured biomass per hectare, considering 12000 plants per hectare, expressed in kilograms (Biomass), the nitrogen units available in the different fertilization regimes, expressed as kilograms of nitrogen per hectare (N units) and the NUE index calculated as Biomass per N unit applied.
As expected, both genotypes increase their NUE index, intended as biomass per unit of nitrogen fertilization applied, when grown in nitrogen starving conditions.
As indicated in Table 6 and Figure 6, in nitrogen starving conditions ntp2 double stop mutant plants increase their NUE index 16% more (3.97 vs 4.61) compared to the out-segregant wild type genotype and normal nitrogen conditions ntp2 double stop mutant plants increase their NUE index 28% more (11 .49 vs 14.75) compared to the out-segregant wild type genotype. Impairment in NTP2 protein activity in tobacco therefore increases the plant’s ability to adjust to nitrogen starvation, increasing the NUE of the plant.
Example 8 - Screening
To select potential candidate targets for increasing NUE in tobacco, microarray data is obtained for the identification of gene candidates differentially expressed in green and ripe leaves in different tobacco varieties. Expression dynamics during air curing of Swiss Burley material, notoriously exhibiting low NUE, is compared to the corresponding profiles in the other tobacco varieties more Nitrogen Use Efficient, such as Virginia type tobacco, along their maturity and respective early curing processes. Gene candidates are selected based on a literature search and their implication in nitrogen assimilation pathways and transport. Genes differentially expressed between Virginia and Burley tobaccos are focussed upon. The NtNtp2 gene is identified as highly expressed in Burley green mature leaves and at harvesting time; in Virginia tobacco, expression levels increase during curing to reach Burley levels as shown in Figure 7.
Example 9 - Root phenotype
To further understand the mechanism responsible for the NUE increase in NtNTP2 mutants, root development is monitored in seedlings growing on agar plates and in young plantlets in hydroponic growth. The results are shown in Figure 8. In both cases NtNTP2-T single homozygous mutants and NtNTP2-S and -T double mutant plants display an increase in root development, expressed as number of lateral roots (A) or maximum length of aquatic roots (B). NtNTP2 activity impairment therefore results in an increase in root development.
Example 10 - Mutant Ntntp2-S W211stop/Ntntp2-T W212stop root phenotype in greenhouse experiment
Ntntp2-S W211stop/Ntntp2-T W212stop BC2S2 TN90 and K326 mutant plants and their wild type outsegregant controls are grown in a greenhouse in hydroponic solution carrying normal 50% of the standard nitrogen fertilization (N50%). At 4 to 6 weeks after transplant, the number of primary roots that sprout from the stele per plant is recorded, together with the average diameter. Both TN90 (Burley tobacco type) and K326 (Virginia tobacco type) mutant plants display a statistically validated higher number of primary lateral roots compared to their respective outsegregant controls (+39.7% with a p value of 0.00083 for TN90 and +47.7% with a p value of 0.000587 for K326), as shown in Figure 9.
For TN90 plants, the diameter of the primary lateral roots for plants grown in N50% is measured with a thickness gage (Mitutoyo ABSOLUTE, Mitutoyo Europe GmbH, BorsigstraBe 8-10 D-41469 Neuss). The mutant plants generated 20% thinner primary roots compared to the outsegregant wild types, as indicated in Figure 10.
Surprisingly, impairment of NtNTP2 activity results in a change in root development, generating more and thinner roots compared to wild type plants grown in the same conditions, as summarised in Figure 11. This may increase the ability of the plants to uptake nutrients from the soil.
Example 11 - Transgenic construct for RNAi plants
A fragment of the coding region of NtNtp2-T gene is selected for designing an RNA interference (RNAi) construct. An RNAi loop is synthetized as shown in Figure 12. The DNA sequence is shown in SEQ ID NO: 45. The RNAi loop is cloned via Hind\\\-Avr\\ restriction sites into a binary vector (see WO2012/098111) carrying the MMV promoter and translator enhancer, to give the construct shown in Figure 12.
Nicotiana tabacum TN90 cells are transformed with Agrobacterium tumefaciens carrying the binary vector for RNAi of NtNtp2 and plants are regenerated on kanamycin. TN90 T2 seedlings, transgenics for the RNAi construct (RNAi-T2) and for the empty vector (control plants, CT-T2) are selected on kanamycin and transplanted to grow in a greenhouse in hydroponic solution in 50% nitrogen fertilization compared to standard practices (N50%). At 4 to 6 weeks after transplant, the number of primary roots that sprouted from the stele per plant is recorded, together with the average diameter. The transgenic plants for the RNAi construct display a statistically validated higher number of primary lateral roots compared to their respective outsegregant controls (+39.7% with a p value of 0.00083 for TN90 and +47.7% with a p value of 0.000587 for K326), as shown in Figure 14. The diameter of the primary lateral roots for RNAi and control plants grown in N50% is measured with a thickness gage (Mitutoyo ABSOLUTE, Mitutoyo Europe GmbH, BorsigstraBe 8-10 D-41469 Neuss). The RNAi plants generated thinner primary roots compared to the outsegregant wild types, as indicated in Figure 15.
Example 12 - Conclusions
Impairing NtNTP2 protein activity does not determine a decrease in nitrate levels in cured lamina nor midrib in AA37 tobacco plants grown in Burley conditions, nor a major change in amount of the other chemical compounds measured.
A phenotype of Ntntp2 double stop mutation or Ntntp2-T homozygous mutation in tobacco is the increase in cured leaf biomass in both standard and nitrogen starvation conditions when compared to out-segregant wild type plants.
A further phenotype of Ntntp2 double stop mutation or Ntntp2-T homozygous mutation in tobacco is an increase in plant NUE response, when expressed as kilograms of cured leaf biomass produced per kilogram of nitrogen input per hectare. This makes NtNtp2 genes good targets for NUE solutions.
A phenotype of RNAi engineered NtNtp2-T tobacco plants in which NtNTP2 expression and/or activity is impaired is a change in root development, generating more and thinner roots compared to wild type plants grown in the same conditions. This may increase the ability of the plants to uptake nutrients from soil.
Any publication cited or described herein provides relevant information disclosed prior to the filing date of the present application. Statements herein are not to be construed as an admission that the inventors are not entitled to antedate such disclosures. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in cellular, molecular and plant biology or related fields are intended to be within the scope of the following claims. TABLE 1
Primers used to screen the AA37 EMS mutagenized tobacco population searching for ntp2 mutations.
Figure imgf000077_0001
TABLE 2
Exemplary mutations leading to a stop codon and truncated NTP2-T and -S proteins.
Figure imgf000078_0001
TABLE 3
TaqMan primers and probes validated for breeding AA37 ntp2 W211stop and W212stop double mutant lines.
Figure imgf000079_0001
TABLE 4
BLAST results for NtNTP2 protein
Figure imgf000080_0001
TABLE 5
BLAST results for NtNtp2 CDS
Figure imgf000081_0001
TABLE 6
NUE index calculation for field results.
Figure imgf000082_0001
SEQUENCES
SEQ ID NO: 1 - NtNtp2-S genomic sequence aagagtgtgactacatacaagagaaagatcagtttcaaaaacattatagtcttttcttttcaatggggaaagaaa tggttagtagaattagttaatactactattattaaattatctacgtgttttaatattctcactttgtttcagatg agttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaaactggtggttgggtacct gctgctcttatcttaggtttgtactgtattatattgttccaagaaatattatacatttttttatatttggaaacc ctttggctttaatattttaagaccacaaattttaaaagactatatttatttgattaaattccgtgccgagtcaaa gtcacatatttaaatgaagagagcatttaattttcagatagtgtttttgaagtttttaactcaatctgtgtttcc tcatgaatagtgatgattgagaaacttggcatgcatttttgaattcattttcagatggttatgtttattatggta agtcatggaaataaggagttaagctgaattccaaaacatgcaggacatgcatgttgcttctgtaactaaacatta aaggaaatgacaagtgatgttaataattaaagatatactcctattaactaataatgaagaagtgattgatcttcc ttggtaaatttaacagtatgaaaattttacagggattgaaattgttgaaaggttgtcgacaatgggaatagcagt aaatctggtgacgtacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttcat gggcacatcattcctcctctgcctcctcggcggatttcttgctgactctttccttggcagatacaaaaccattgt aatctttgcccttatacagacactggtaattaaactaataataatcatgaggccgtcaatattaataatgctttc aactttatattaacaaaaaacataattattctactcagggaactgggatgttaacactggccgttagtctaccgc agctgcggccacctctttgccatcgtcatgatgtcaattgccaaccagcaaaaggttctcaaatgggaatcctat acatggccttgtacctcatagcattaggtactggtggcttaaaatcaagtgtctcaggatttggaacagaccagt ttgatgagaaagacgacaaggaaaaggcgcaaatggcgtatttcttcaacagattctttttcttcatcagcatag ggactttgacagcagttacagtgcttgtttacatccaagatgaagtcggacggagctgggcttacggtgtttgct ctgtatccatgctcattgccatcctaatcttccttctaggtactagaaggtaccgatacaagaaaagctccggaa gtcctattgtacacatttttcaagtcatttttgctgctataaggaaaaggaaaatggatcttccatatgatgttg gcatgctctatgagacaactaattcagaggctacaagaatccaacacacacaagaattcaggttagttttctttt tatatcaaaattatgttaggctaaactgaccttaaaatactcttaacatggtgtgatattgtttgctttgggcca agtccgcaaggttttttccaaaagggctcgtgccacacccgataaatacaatggttaactaagaatgtttactaa attttttgtagagtactggacaaggctgctatagtagctgagggagattttgaggaccaatatggatcatgctct gctccaaatccatggaagcttagcacagtgacaagagttgaggaagtgaaaatgatggcaaggctgctaccaatt tgggccaccaccataattttctggaccacctacgcacaaatgattactttttctgttgaacaagcttcaaccatg gaaagatcaattggcaatttccaaattccagcagggtctctcactgtcttctttgttgcggccatcttgattacc ttggctgtttatgaccgaattattatgccactttggaagaaatggaagggaaaaccaggtataattttcacacct tttaaatatttgaattgttaagtacggtaactaatgatgtactttccaaaaatttaaagaatctatgtttgaagt agcgtcaaaattaagaagtttgaccctcgaaatccgaactgcatcacataaattcggacggagggagtatatcta tgtggagaattaagctaaatgtgcttcatcacccctaaacatgcatgcatggttaattttgcaggttttagcagc ctacaaaaaatatccattggccttgtactttccataataggaatggcagcagctgccttagcggagaagaaaaga ttgacagttgcaaaatccgtggggcgccacagctcttcaactaatttgccaataagtgtattcttcttgatccca caattcttcttggtaggagcaggggaggccttcatatacactggccaactcgatttcttcataacacaatcgccg aaagggatgaagactatgagcactggccttttcttgacaaccctttcgcttggtttcttcattagcagcttctta gtatcggttatcaagaaggtgaccggaggcaatggtactgatgaaggctggcttgcagacaacataaacaatgga aggctagacttgttctactggcttcttgctgtgttgggagttataaactttgtgatttacctaatttgcgcaact tggtacaagccaaggaaggctaagtcagcaatacagatggagaatccgcacactaaaaatgcagctgaagagaag tgctaggatttatgctgttcaagttgcccttttcttaattttattagccaatatacagtgaaaaagctgttgttt cagtttagtttctgttctctatatatcgtgtttggatcagtaaattggttttcggttttgcactatcctaataat cttatttctatcttgataaaatgtctgtactccactggctttatcataacctttgtaatgcttcgatgatgggaa atgctcactcactaaattca
SEQ ID NO: 2 - NtNtp2-S transcript sequence (start and stop codons are shown in bold) aagagtgtgactacatacaagagaaagatcagtttcaaaaacattatagtcttttcttttcaatggggaaagaaa tggttagtagaattaatgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaa actggtggttgggtacctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagca gtaaatctggtgacgtacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttc atgggcacatcattcctcctctgcctcctcggcggatttcttgctgactctttccttggcagatacaaaaccatt gtaatctttgcccttatacagacactgggaactgggatgttaacactggccgttagtctaccgcagctgcggcca cctctttgccatcgtcatgatgtcaattgccaaccagcaaaaggttctcaaatgggaatcctatacatggccttg tacctcatagcattaggtactggtggcttaaaatcaagtgtctcaggatttggaacagaccagtttgatgagaaa gacgacaaggaaaaggcgcaaatggcgtatttcttcaacagattctttttcttcatcagcatagggactttgaca gcagttacagtgcttgtttacatccaagatgaagtcggacggagctgggcttacggtgtttgctctgtatccatg ctcattgccatcctaatcttccttctaggtactagaaggtaccgatacaagaaaagctccggaagtcctattgta cacatttttcaagtcatttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctat gagacaactaattcagaggctacaagaatccaacacacacaagaattcagagtactggacaaggctgctatagta gctgagggagattttgaggaccaatatggatcatgctctgctccaaatccatggaagcttagcacagtgacaaga gttgaggaagtgaaaatgatggcaaggctgctaccaatttgggccaccaccataattttctggaccacctacgca caaatgattactttttctgttgaacaagcttcaaccatggaaagatcaattggcaatttccaaattccagcaggg tctctcactgtcttctttgttgcggccatcttgattaccttggctgtttatgaccgaattattatgccactttgg aagaaatggaagggaaaaccaggttttagcagcctacaaaaaatatccattggccttgtactttccataatagga atggcagcagctgccttagcggagaagaaaagattgacagttgcaaaatccgtggggcgccacagctcttcaact aatttgccaataagtgtattcttcttgatcccacaattcttcttggtaggagcaggggaggccttcatatacact ggccaactcgatttcttcataacacaatcgccgaaagggatgaagactatgagcactggccttttcttgacaacc ctttcgcttggtttcttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgat gaaggctggcttgcagacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagtt ataaactttgtgatttacctaatttgcgcaacttggtacaagccaaggaaggctaagtcagcaatacagatggag aatccgcacactaaaaatgcagctgaagagaagtgctaggatttatgctgttcaagttgcccttttcttaatttt attagccaatatacagtgaaaaagctgttgtttcagtttagtttctgttctctatatatcgtgtttggatcagta aattggttttcggttttgcactatcctaataatcttatttctatcttgataaaatgtctgtactccactggcttt atcataacctttgtaatgcttcgatgatgggaaatgctcactcactaaattca
SEQ ID NO: 3 - NtNtp2-S gene sequence atgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaaactggtggttgggta cctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagcagtaaatctggtgacg tacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttcatgggcacatcattc ctcctctgcctcctcggcggatttcttgctgactctttccttggcagatacaaaaccattgtaatctttgccctt atacagacactgggaactgggatgttaacactggccgttagtctaccgcagctgcggccacctctttgccatcgt catgatgtcaattgccaaccagcaaaaggttctcaaatgggaatcctatacatggccttgtacctcatagcatta ggtactggtggcttaaaatcaagtgtctcaggatttggaacagaccagtttgatgagaaagacgacaaggaaaag gcgcaaatggcgtatttcttcaacagattctttttcttcatcagcatagggactttgacagcagttacagtgctt gtttacatccaagatgaagtcggacggagctgggcttacggtgtttgctctgtatccatgctcattgccatccta atcttccttctaggtactagaaggtaccgatacaagaaaagctccggaagtcctattgtacacatttttcaagtc atttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctatgagacaactaattca gaggctacaagaatccaacacacacaagaattcagagtactggacaaggctgctatagtagctgagggagatttt gaggaccaatatggatcatgctctgctccaaatccatggaagcttagcacagtgacaagagttgaggaagtgaaa atgatggcaaggctgctaccaatttgggccaccaccataattttctggaccacctacgcacaaatgattactttt tctgttgaacaagcttcaaccatggaaagatcaattggcaatttccaaattccagcagggtctctcactgtcttc tttgttgcggccatcttgattaccttggctgtttatgaccgaattattatgccactttggaagaaatggaaggga aaaccaggttttagcagcctacaaaaaatatccattggccttgtactttccataataggaatggcagcagctgcc ttagcggagaagaaaagattgacagttgcaaaatccgtggggcgccacagctcttcaactaatttgccaataagt gtattcttcttgatcccacaattcttcttggtaggagcaggggaggccttcatatacactggccaactcgatttc ttcataacacaatcgccgaaagggatgaagactatgagcactggccttttcttgacaaccctttcgcttggtttc ttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgatgaaggctggcttgca gacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagttataaactttgtgatt tacctaatttgcgcaacttggtacaagccaaggaaggctaagtcagcaatacagatggagaatccgcacactaaa aatgcagctgaagagaagtgctag
SEQ ID NO: 4 - NtNtp2-S gene sequence of SEQ ID NO: 3 with g to a mutation shown in bold (g to a mutation at nucleotide 632) atgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaaactggtggttgggta cctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagcagtaaatctggtgacg tacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttcatgggcacatcattc ctcctctgcctcctcggcggatttcttgctgactctttccttggcagatacaaaaccattgtaatctttgccctt atacagacactgggaactgggatgttaacactggccgttagtctaccgcagctgcggccacctctttgccatcgt catgatgtcaattgccaaccagcaaaaggttctcaaatgggaatcctatacatggccttgtacctcatagcatta ggtactggtggcttaaaatcaagtgtctcaggatttggaacagaccagtttgatgagaaagacgacaaggaaaag gcgcaaatggcgtatttcttcaacagattctttttcttcatcagcatagggactttgacagcagttacagtgctt gtttacatccaagatgaagtcggacggagctaggcttacggtgtttgctctgtatccatgctcattgccatccta atcttccttctaggtactagaaggtaccgatacaagaaaagctccggaagtcctattgtacacatttttcaagtc atttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctatgagacaactaattca gaggctacaagaatccaacacacacaagaattcagagtactggacaaggctgctatagtagctgagggagatttt gaggaccaatatggatcatgctctgctccaaatccatggaagcttagcacagtgacaagagttgaggaagtgaaa atgatggcaaggctgctaccaatttgggccaccaccataattttctggaccacctacgcacaaatgattactttt tctgttgaacaagcttcaaccatggaaagatcaattggcaatttccaaattccagcagggtctctcactgtcttc tttgttgcggccatcttgattaccttggctgtttatgaccgaattattatgccactttggaagaaatggaaggga aaaccaggttttagcagcctacaaaaaatatccattggccttgtactttccataataggaatggcagcagctgcc ttagcggagaagaaaagattgacagttgcaaaatccgtggggcgccacagctcttcaactaatttgccaataagt gtattcttcttgatcccacaattcttcttggtaggagcaggggaggccttcatatacactggccaactcgatttc ttcataacacaatcgccgaaagggatgaagactatgagcactggccttttcttgacaaccctttcgcttggtttc ttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgatgaaggctggcttgca gacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagttataaactttgtgatt tacctaatttgcgcaacttggtacaagccaaggaaggctaagtcagcaatacagatggagaatccgcacactaaa aatgcagctgaagagaagtgctag
SEQ ID NO: 5 - NtNtp2-S gene sequence of SEQ ID NO: 3 with g to a mutation shown in bold (g to a mutation at nucleotide 633) atgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaaactggtggttgggta cctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagcagtaaatctggtgacg tacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttcatgggcacatcattc ctcctctgcctcctcggcggatttcttgctgactctttccttggcagatacaaaaccattgtaatctttgccctt atacagacactgggaactgggatgttaacactggccgttagtctaccgcagctgcggccacctctttgccatcgt catgatgtcaattgccaaccagcaaaaggttctcaaatgggaatcctatacatggccttgtacctcatagcatta ggtactggtggcttaaaatcaagtgtctcaggatttggaacagaccagtttgatgagaaagacgacaaggaaaag gcgcaaatggcgtatttcttcaacagattctttttcttcatcagcatagggactttgacagcagttacagtgctt gtttacatccaagatgaagtcggacggagctgagcttacggtgtttgctctgtatccatgctcattgccatccta atcttccttctaggtactagaaggtaccgatacaagaaaagctccggaagtcctattgtacacatttttcaagtc atttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctatgagacaactaattca gaggctacaagaatccaacacacacaagaattcagagtactggacaaggctgctatagtagctgagggagatttt gaggaccaatatggatcatgctctgctccaaatccatggaagcttagcacagtgacaagagttgaggaagtgaaa atgatggcaaggctgctaccaatttgggccaccaccataattttctggaccacctacgcacaaatgattactttt tctgttgaacaagcttcaaccatggaaagatcaattggcaatttccaaattccagcagggtctctcactgtcttc tttgttgcggccatcttgattaccttggctgtttatgaccgaattattatgccactttggaagaaatggaaggga aaaccaggttttagcagcctacaaaaaatatccattggccttgtactttccataataggaatggcagcagctgcc ttagcggagaagaaaagattgacagttgcaaaatccgtggggcgccacagctcttcaactaatttgccaataagt gtattcttcttgatcccacaattcttcttggtaggagcaggggaggccttcatatacactggccaactcgatttc ttcataacacaatcgccgaaagggatgaagactatgagcactggccttttcttgacaaccctttcgcttggtttc ttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgatgaaggctggcttgca gacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagttataaactttgtgatt tacctaatttgcgcaacttggtacaagccaaggaaggctaagtcagcaatacagatggagaatccgcacactaaa aatgcagctgaagagaagtgctag
SEQ ID NO: 6 - NtNtp2-S gene sequence of SEQ ID NO: 3 with g to a mutation shown in bold (g to a mutation at nucleotides 632 and 633) atgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaaactggtggttgggta cctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagcagtaaatctggtgacg tacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttcatgggcacatcattc ctcctctgcctcctcggcggatttcttgctgactctttccttggcagatacaaaaccattgtaatctttgccctt atacagacactgggaactgggatgttaacactggccgttagtctaccgcagctgcggccacctctttgccatcgt catgatgtcaattgccaaccagcaaaaggttctcaaatgggaatcctatacatggccttgtacctcatagcatta ggtactggtggcttaaaatcaagtgtctcaggatttggaacagaccagtttgatgagaaagacgacaaggaaaag gcgcaaatggcgtatttcttcaacagattctttttcttcatcagcatagggactttgacagcagttacagtgctt gtttacatccaagatgaagtcggacggagctaagcttacggtgtttgctctgtatccatgctcattgccatccta atcttccttctaggtactagaaggtaccgatacaagaaaagctccggaagtcctattgtacacatttttcaagtc atttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctatgagacaactaattca gaggctacaagaatccaacacacacaagaattcagagtactggacaaggctgctatagtagctgagggagatttt gaggaccaatatggatcatgctctgctccaaatccatggaagcttagcacagtgacaagagttgaggaagtgaaa atgatggcaaggctgctaccaatttgggccaccaccataattttctggaccacctacgcacaaatgattactttt tctgttgaacaagcttcaaccatggaaagatcaattggcaatttccaaattccagcagggtctctcactgtcttc tttgttgcggccatcttgattaccttggctgtttatgaccgaattattatgccactttggaagaaatggaaggga aaaccaggttttagcagcctacaaaaaatatccattggccttgtactttccataataggaatggcagcagctgcc ttagcggagaagaaaagattgacagttgcaaaatccgtggggcgccacagctcttcaactaatttgccaataagt gtattcttcttgatcccacaattcttcttggtaggagcaggggaggccttcatatacactggccaactcgatttc ttcataacacaatcgccgaaagggatgaagactatgagcactggccttttcttgacaaccctttcgcttggtttc ttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgatgaaggctggcttgca gacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagttataaactttgtgatt tacctaatttgcgcaacttggtacaagccaaggaaggctaagtcagcaatacagatggagaatccgcacactaaa aatgcagctgaagagaagtgctag
SEQ ID NO: 7 - NtNtp2-S polypeptide sequence encoded by SEQ ID NO: 3
MSWTVSDAVDYKGSPADKSKTGGWVPAALILGIEIVERLSTMGIAVNLVTYLGGTMHLPSSTSANIVTDFMGTSF LLCLLGGFLADSFLGRYKTIVIFALIQTLGTGMLTLAVSLPQLRPPLCHRHDVNCQPAKGSQMGILYMALYLIAL GTGGLKSSVSGFGTDQFDEKDDKEKAQMAYFFNRFFFFIS IGTLTAVTVLVYIQDEVGRSWAYGVCSVSMLIAIL IFLLGTRRYRYKKSSGSP IVHIFQVIFAAIRKRKMDLPYDVGMLYETTNSEATRIQHTQEFRVLDKAAIVAEGDF EDQYGSCSAPNPWKLSTVTRVEEVKMMARLLP IWATTI IFWTTYAQMITFSVEQASTMERS IGNFQIPAGSLTVF FVAAILITLAVYDRI IMPLWKKWKGKPGFSSLQKIS IGLVLS I IGMAAAALAEKKRLTVAKSVGRHSSSTNLP IS VFFLIPQFFLVGAGEAFIYTGQLDFFITQSPKGMKTMSTGLFLTTLSLGFFISSFLVSVIKKVTGGNGTDEGWLA DNINNGRLDLFYWLLAVLGVINFVIYLICATWYKPRKAKSAIQMENPHTKNAAEEKC*
SEQ ID NO: 8 - NtNtp2-S truncated polypeptide encoded by SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6; stop codon at position W211 of SEQ ID NO: 7
MSWTVSDAVDYKGSPADKSKTGGWVPAALILGIEIVERLSTMGIAVNLVTYLGGTMHLPSSTSANIVTDFMGTSF LLCLLGGFLADSFLGRYKTIVIFALIQTLGTGMLTLAVSLPQLRPPLCHRHDVNCQPAKGSQMGILYMALYLIAL GTGGLKSSVSGFGTDQFDEKDDKEKAQMAYFFNRFFFFIS IGTLTAVTVLVYIQDEVGRS-
SEQ ID NO: 9 - NtNtp2-T genomic sequence aagagtgtgaaaataaagagtttcaataactttatatctctggggtcttttcttttcgactgttgacactaatta gtcgggaaagaaatggttagtagaattagttaatactattattacattatctacgtgtttcctgtcttaatattc tcttcatctcaccaattactttgtttcagatgagttggactgtctcagatgctgtggactacaaggggtccccag ctgataagtctaaaactggtggttgggtacctgctgctcttatcttaggtttggactgtattattattttatatt ttgaaaccatttggctttaataagttggtatagcgatacaacttggaagtcatataaaagtgtttactctttaag tttttaagattttaaaagtctttatttatttttttaaatttcgtgccgagtcaattttattggagtttttaactc gatctcagtgtttcctcgtgctttataagatagtgattgagaaacttagcatgcattttttgaattcattttcat atggttatgcttattatggtaagtaatggaaattaagagttaagttgcttctgtaactaaacattaaaggcaatg acaagtgatgttaataattaaagatatactattaactaatgaaaaagtgattgatcttcgttggtaaatgaaatt ttgcagggattgaaattgttgaaaggttgtcgacaatgggaatagcagtaaatctggtgacatacttaggtggaa ctatgcatttgcctagttcaacttcagccaatattgtaacagatttcatgggcacatcattcctcctctgcctcc tcggcggctttcttgctgactctttccttggcagatacaaaaccattgcaatctttgctcttatacagacactgg taattaaactaataatcatgaggccgtcaatattaataatgttttcaactttatattaataaaaacataaaattc tactcagggaactgggatgttaacactggccgttagcctaccgcagctgcggccacctccttgccatcgtcatga tgtcagttgtcaaccagcaaaaggttctcaaatgggaatcctatacatggccttgtatctcatagcattaggtac tggtggcttaaaatcaagtgtctcgggatttggaacagaccagttcgatgagaaagacgacaaggaaaaggcgca aatggcgtatttcttcaacagattcttcttcttcatcagcatagggactttgacagcagtaaccgtgcttgttta catccaagatgaagtcggacggagctgggcttatggtgtttgctctgtatccatgctcattgccatcctaatctt ccttctaggtactaaaaagtaccgatacaagaaaagctcgggaagtcctatagtacacatttttcaagtcatttt tgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctatgagactactaattcagaggc tacaagaatccaacacacacaagaatttaggttagttttctttttatatcaaaattatgttcggctaaacttact cgaagatactcttaacatggtgtgatattgtctgctttgggccaaattcgtacggtttttcccaaaaggtctcac accattatgagaaccatattatcttctcagctactaatatgggactacgttcacacacctaacaaattcaaatat tggactctttaactaacgatgtttactaatttggttgtagagtactggacaaggctgcaattgtagctgagggag attttgaggaccaatatggatcatgctctgatccaaatccatggaagcttagcacagtgacaagagttgaggaag tgaaaatgatggcaaggctgctcccaatttgggccaccactataattttctggaccacctacgcacaaatgatta cattttctgttgaacaagcttccaccatggaaagatcaattggcaatttccaaattccagcagggtctctcactg tcttctttgttgcggccatcttgatcaccttggctgtttatgaccgaattattatgccactttggaagaaatgga agggaaaaccaggtataacttttatgccttttaaatattttgaattgttaagtattgtaactaatgatgtacttt ccaaatatgaattttatttcgcaaaatttaaagattctatgtttgaattcgcgtcaaaattaagaagtttgactc tcaaaatccgaactgcatcacataaattcgggcagagggagggagtatatctgagtggagaattaagctgaaggt gcttcatcaaccctaaacatgcatggttaattttgcaggttttagcagcctacaaagaatttccattgggcttgt attttccataataggaatggcagcagctgccttagcggagaagaaaagattgacagttgcaaaatccatggggcg ccacaactcttcaactaatttgccaattagtgtattcttcttgatcccacaattcttcttggtaggagcagggga agccttcatatacactggccaactcgatttcttcataacacaatcgcccaaagggatgaagactatgagcactgg ccttttcttgacaaccctttcgcttggtttcttcattagcagcttcttagtatcggttatcaagaaggtgaccgg aggcaatggtactgatgaaggctggcttgcagacaacataaacaatggaaggctagacttgttctactggcttct tgctgtgttgggagttataaacattgtgatttacctaatttgcgcaacatggtacaagccaaggaagcctaagtc agecat acagatggagaatccgcacactaaaaatgcagctgaagagaagt get aggatt tat get gttcaagttg cccttttcttaattttattagccaatatacagtgaaagagctgttgtttcagtttagtttctgttctctatatat cgtgtttggatcagtaaattggttttcggttttgcagtatcctaataatcttatttctatcttgataaaatgtct atccactggctttatcatgacctttgtaatgcttcgatgggaaatgtttactcactaaattcat
SEQ ID NO: 10 - NtNtp2-T transcript sequence (start and stop codons are shown in bold) aagagtgtgaaaataaagagtttcaataactttatatctctggggtcttttcttttcgactgttgacactaatta gtcgggaaagaaatgatgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaa actggtggttgggtacctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagca gtaaatctggtgacatacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttc atgggcacatcattcctcctctgcctcctcggcggctttcttgctgactctttccttggcagatacaaaaccatt gcaatctttgctcttatacagacactgggaactgggatgttaacactggccgttagcctaccgcagctgcggcca cctccttgccatcgtcatgatgtcagttgtcaaccagcaaaaggttctcaaatgggaatcctatacatggccttg tatctcatagcattaggtactggtggcttaaaatcaagtgtctcgggatttggaacagaccagttcgatgagaaa gacgacaaggaaaaggcgcaaatggcgtatttcttcaacagattcttcttcttcatcagcatagggactttgaca gcagtaaccgtgcttgtttacatccaagatgaagtcggacggagctgGgcttatggtgtttgctctgtatccatg ctcattgccatcctaatcttccttctaggtactaaaaagtaccgatacaagaaaagctcgggaagtcctatagta cacatttttcaagtcatttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctat gagactactaattcagaggctacaagaatccaacacacacaagaatttagagtactggacaaggctgcaattgta gctgagggagattttgaggaccaatatggatcatgctctgatccaaatccatggaagcttagcacagtgacaaga gttgaggaagtgaaaatgatggcaaggctgctcccaatttgggccaccactataattttctggaccacctacgca caaatgattacattttctgttgaacaagcttccaccatggaaagatcaattggcaatttccaaattccagcaggg tctctcactgtcttctttgttgcggccatcttgatcaccttggctgtttatgaccgaattattatgccactttgg aagaaatggaagggaaaaccaggttttagcagcctacaaagaatttccattgggcttgtattttccataatagga atggcagcagctgccttagcggagaagaaaagattgacagttgcaaaatccatggggcgccacaactcttcaact aatttgccaattagtgtattcttcttgatcccacaattcttcttggtaggagcaggggaagccttcatatacact ggccaactcgatttcttcataacacaatcgcccaaagggatgaagactatgagcactggccttttcttgacaacc ctttcgcttggtttcttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgat gaaggctggcttgcagacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagtt at aaacattgt gat tt acct aatttgcgcaacatggtacaagccaaggaagcctaagt cagecat acagatggag aatccgcacactaaaaatgcagctgaagagaagtgctaggatttatgctgttcaagttgcccttttcttaatttt attagccaatatacagtgaaagagctgttgtttcagtttagtttctgttctctatatatcgtgtttggatcagta aattggttttcggttttgcagtatcctaataatcttatttctatcttgataaaatgtctatccactggctttatc atgacctttgtaatgcttcgatgggaaatgtttactcactaaattcat
SEQ ID NO: 11 - NtNtp2-T gene sequence atgatgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaaactggtggttgg gtacctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagcagtaaatctggtg acatacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttcatgggcacatca ttcctcctctgcctcctcggcggctttcttgctgactctttccttggcagatacaaaaccattgcaatctttgct cttatacagacactgggaactgggatgttaacactggccgttagcctaccgcagctgcggccacctccttgccat cgtcatgatgtcagttgtcaaccagcaaaaggttctcaaatgggaatcctatacatggccttgtatctcatagca ttaggtactggtggcttaaaatcaagtgtctcgggatttggaacagaccagttcgatgagaaagacgacaaggaa aaggcgcaaatggcgtatttcttcaacagattcttcttcttcatcagcatagggactttgacagcagtaaccgtg cttgtttacatccaagatgaagtcggacggagctgggcttatggtgtttgctctgtatccatgctcattgccatc ctaatcttccttctaggtactaaaaagtaccgatacaagaaaagctcgggaagtcctatagtacacatttttcaa gtcatttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctatgagactactaat tcagaggctacaagaatccaacacacacaagaatttagagtactggacaaggctgcaattgtagctgagggagat tttgaggaccaatatggatcatgctctgatccaaatccatggaagcttagcacagtgacaagagttgaggaagtg aaaatgatggcaaggctgctcccaatttgggccaccactataattttctggaccacctacgcacaaatgattaca ttttctgttgaacaagcttccaccatggaaagatcaattggcaatttccaaattccagcagggtctctcactgtc ttctttgttgcggccatcttgatcaccttggctgtttatgaccgaattattatgccactttggaagaaatggaag ggaaaaccaggttttagcagcctacaaagaatttccattgggcttgtattttccataataggaatggcagcagct gccttagcggagaagaaaagattgacagttgcaaaatccatggggcgccacaactcttcaactaatttgccaatt agtgtattcttcttgatcccacaattcttcttggtaggagcaggggaagccttcatatacactggccaactcgat ttcttcataacacaatcgcccaaagggatgaagactatgagcactggccttttcttgacaaccctttcgcttggt ttcttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgatgaaggctggctt gcagacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagttataaacattgtg at tt acct aatttgcgcaacatggtacaagccaaggaagcctaagt cagecat acagatggagaatccgcacact aaaaatgeagetgaagagaagtgetag
SEQ ID NO: 12 - NtNtp2-T polypeptide sequence encoded by SEQ ID NO: 11
MMSWTVSDAVDYKGSPADKSKTGGWVPAALILGIEIVERLSTMGIAVNLVTYLGGTMHLPSSTSANIVTDFMGTS FLLCLLGGFLADSFLGRYKTIAIFALIQTLGTGMLTLAVSLPQLRPPPCHRHDVSCQPAKGSQMGILYMALYLIA LGTGGLKSSVSGFGTDQFDEKDDKEKAQMAYFFNRFFFFIS IGTLTAVTVLVYIQDEVGRSWAYGVCSVSMLIAI LIFLLGTKKYRYKKSSGSP IVHIFQVIFAAIRKRKMDLPYDVGMLYETTNSEATRIQHTQEFRVLDKAAIVAEGD FEDQYGSCSDPNPWKLSTVTRVEEVKMMARLLP IWATTI IFWTTYAQMITFSVEQASTMERS IGNFQIPAGSLTV FFVAAILITLAVYDRI IMPLWKKWKGKPGFSSLQRIS IGLVFS I IGMAAAALAEKKRLTVAKSMGRHNSSTNLP I SVFFLIPQFFLVGAGEAFIYTGQLDFFITQSPKGMKTMSTGLFLTTLSLGFFISSFLVSVIKKVTGGNGTDEGWL ADNINNGRLDLFYWLLAVLGVINIVIYLICATWYKPRKPKSAIQMENPHTKNAAEEKC*
SEQ ID NO: 13 - NtNtp2-T gene sequence of SEQ ID NO: 11 with g to a mutation shown in bold (g to a mutation at nucleotide 636 resulting in a tga stop codon) atgatgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaaactggtggttgg gtacctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagcagtaaatctggtg acatacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttcatgggcacatca ttcctcctctgcctcctcggcggctttcttgctgactctttccttggcagatacaaaaccattgcaatctttgct cttatacagacactgggaactgggatgttaacactggccgttagcctaccgcagctgcggccacctccttgccat cgtcatgatgtcagttgtcaaccagcaaaaggttctcaaatgggaatcctatacatggccttgtatctcatagca ttaggtactggtggcttaaaatcaagtgtctcgggatttggaacagaccagttcgatgagaaagacgacaaggaa aaggcgcaaatggcgtatttcttcaacagattcttcttcttcatcagcatagggactttgacagcagtaaccgtg cttgtttacatccaagatgaagtcggacggagctgagcttatggtgtttgctctgtatccatgctcattgccatc ctaatcttccttctaggtactaaaaagtaccgatacaagaaaagctcgggaagtcctatagtacacatttttcaa gtcatttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctatgagactactaat tcagaggctacaagaatccaacacacacaagaatttagagtactggacaaggctgcaattgtagctgagggagat tttgaggaccaatatggatcatgctctgatccaaatccatggaagcttagcacagtgacaagagttgaggaagtg aaaatgatggcaaggctgctcccaatttgggccaccactataattttctggaccacctacgcacaaatgattaca ttttctgttgaacaagcttccaccatggaaagatcaattggcaatttccaaattccagcagggtctctcactgtc ttctttgttgcggccatcttgatcaccttggctgtttatgaccgaattattatgccactttggaagaaatggaag ggaaaaccaggttttagcagcctacaaagaatttccattgggcttgtattttccataataggaatggcagcagct gccttagcggagaagaaaagattgacagttgcaaaatccatggggcgccacaactcttcaactaatttgccaatt agtgtattcttcttgatcccacaattcttcttggtaggagcaggggaagccttcatatacactggccaactcgat ttcttcataacacaatcgcccaaagggatgaagactatgagcactggccttttcttgacaaccctttcgcttggt ttcttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgatgaaggctggctt gcagacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagttataaacattgtg at tt acct aatttgcgcaacatggtacaagccaaggaagcctaagt cagecat acagatggagaatccgcacact aaaaatgeagetgaagagaagtgetag
SEQ ID NO: 14 - NtNtp2-T gene sequence of SEQ ID NO: 11 with g to a mutation shown in bold (g to a mutation at nucleotide 635 resulting in a tag stop codon) atgatgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaaactggtggttgg gtacctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagcagtaaatctggtg acatacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttcatgggcacatca ttcctcctctgcctcctcggcggctttcttgctgactctttccttggcagatacaaaaccattgcaatctttgct cttatacagacactgggaactgggatgttaacactggccgttagcctaccgcagctgcggccacctccttgccat cgtcatgatgtcagttgtcaaccagcaaaaggttctcaaatgggaatcctatacatggccttgtatctcatagca ttaggtactggtggcttaaaatcaagtgtctcgggatttggaacagaccagttcgatgagaaagacgacaaggaa aaggcgcaaatggcgtatttcttcaacagattcttcttcttcatcagcatagggactttgacagcagtaaccgtg cttgtttacatccaagatgaagtcggacggagctaggcttatggtgtttgctctgtatccatgctcattgccatc ctaatcttccttctaggtactaaaaagtaccgatacaagaaaagctcgggaagtcctatagtacacatttttcaa gtcatttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctatgagactactaat tcagaggctacaagaatccaacacacacaagaatttagagtactggacaaggctgcaattgtagctgagggagat tttgaggaccaatatggatcatgctctgatccaaatccatggaagcttagcacagtgacaagagttgaggaagtg aaaatgatggcaaggctgctcccaatttgggccaccactataattttctggaccacctacgcacaaatgattaca ttttctgttgaacaagcttccaccatggaaagatcaattggcaatttccaaattccagcagggtctctcactgtc ttctttgttgcggccatcttgatcaccttggctgtttatgaccgaattattatgccactttggaagaaatggaag ggaaaaccaggttttagcagcctacaaagaatttccattgggcttgtattttccataataggaatggcagcagct gccttagcggagaagaaaagattgacagttgcaaaatccatggggcgccacaactcttcaactaatttgccaatt agtgtattcttcttgatcccacaattcttcttggtaggagcaggggaagccttcatatacactggccaactcgat ttcttcataacacaatcgcccaaagggatgaagactatgagcactggccttttcttgacaaccctttcgcttggt ttcttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgatgaaggctggctt gcagacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagttataaacattgtg at tt acct aatttgcgcaacatggtacaagccaaggaagcctaagt cagecat acagatggagaatccgcacact aaaaatgeagetgaagagaagtgetag
SEQ ID NO: 15 - NtNtp2-T gene sequence of SEQ ID NO: 11 with gg to aa mutation shown in bold (g to a mutation at nucleotide 635 and g to a mutation at nucleotide 636 resulting in a taa stop codon) atgatgagttggactgtctcagatgctgtggactacaaggggtccccagctgataagtctaaaactggtggttgg gtacctgctgctcttatcttagggattgaaattgttgaaaggttgtcgacaatgggaatagcagtaaatctggtg acatacttaggtggaactatgcatttgcctagttcaacttcagccaatattgtaacagatttcatgggcacatca ttcctcctctgcctcctcggcggctttcttgctgactctttccttggcagatacaaaaccattgcaatctttgct cttatacagacactgggaactgggatgttaacactggccgttagcctaccgcagctgcggccacctccttgccat cgtcatgatgtcagttgtcaaccagcaaaaggttctcaaatgggaatcctatacatggccttgtatctcatagca ttaggtactggtggcttaaaatcaagtgtctcgggatttggaacagaccagttcgatgagaaagacgacaaggaa aaggcgcaaatggcgtatttcttcaacagattcttcttcttcatcagcatagggactttgacagcagtaaccgtg cttgtttacatccaagatgaagtcggacggagctaagcttatggtgtttgctctgtatccatgctcattgccatc ctaatcttccttctaggtactaaaaagtaccgatacaagaaaagctcgggaagtcctatagtacacatttttcaa gtcatttttgctgctataaggaaaaggaaaatggatcttccatatgatgttggcatgctctatgagactactaat tcagaggctacaagaatccaacacacacaagaatttagagtactggacaaggctgcaattgtagctgagggagat tttgaggaccaatatggatcatgctctgatccaaatccatggaagcttagcacagtgacaagagttgaggaagtg aaaatgatggcaaggctgctcccaatttgggccaccactataattttctggaccacctacgcacaaatgattaca ttttctgttgaacaagcttccaccatggaaagatcaattggcaatttccaaattccagcagggtctctcactgtc ttctttgttgcggccatcttgatcaccttggctgtttatgaccgaattattatgccactttggaagaaatggaag ggaaaaccaggttttagcagcctacaaagaatttccattgggcttgtattttccataataggaatggcagcagct gccttagcggagaagaaaagattgacagttgcaaaatccatggggcgccacaactcttcaactaatttgccaatt agtgtattcttcttgatcccacaattcttcttggtaggagcaggggaagccttcatatacactggccaactcgat ttcttcataacacaatcgcccaaagggatgaagactatgagcactggccttttcttgacaaccctttcgcttggt ttcttcattagcagcttcttagtatcggttatcaagaaggtgaccggaggcaatggtactgatgaaggctggctt gcagacaacataaacaatggaaggctagacttgttctactggcttcttgctgtgttgggagttataaacattgtg at tt acct aatttgcgcaacatggtacaagccaaggaagcctaagt cagecat acagatggagaatccgcacact aaaaatgeagetgaagagaagtgetag
SEQ ID NO: 16 - NtNtp2-T truncated polypeptide sequence encoded by SEQ ID NO: 13 or
SEQ ID NO: 14 or SEQ ID NO: 15; stop codon at position W212 of SEQ ID NO: 12
MMSWTVSDAVDYKGSPADKSKTGGWVPAALILGIEIVERLSTMGIAVNLVTYLGGTMHLPSSTSANIVTDFMGTS
FLLCLLGGFLADSFLGRYKTIAIFALIQTLGTGMLTLAVSLPQLRPPPCHRHDVSCQPAKGSQMGILYMALYLIA
LGTGGLKSSVSGFGTDQFDEKDDKEKAQMAYFFNRFFFFIS IGTLTAVTVLVYIQDEVGRS- SEQ ID NO: 17 - NtNtp2-S forward primer gaatagcagtaaatctggtgacg
SEQ ID NO: 18 - NtNtp2-S reverse primer cacaccatgttaagagtattttaag
SEQ ID NO: 19 - NtNtp2-T forward primer tgaggccgtcaatattaataatgt
SEQ ID NO: 20 - NtNtp2-T reverse primer acaccatgttaagagtatcttcga
SEQ ID NO: 21 - sequence in 5’ position of ntp2-T W212stop SNP mutation acggagctg
SEQ ID NO: 22 - sequence in 3’ position of ntp2-T W212stop SNP mutation gcttatggtg
SEQ ID NO: 23 - sequence in 5’ position of ntp2-T W212stop SNP mutation acggagct
SEQ ID NO: 24 - sequence in 3’ position of ntp2-T W212stop SNP mutation ggcttatggtg
SEQ ID NO: 26 NtNTP2-S wt reverse primer cggagcttttcttgtatcggtacc
SEQ ID NO: 27 - NtNTP2-S wt forward primer agcatagggactttgacagcagtt
SEQ ID NO: 28 NtNTP2-S wt probe agctgggcttacgg
SEQ ID NO: 29 NtNTP2-S mut tAg forward primer agcatagggactttgacagcagtt
SEQ ID NO: 30 NtNTP2-S mut tAg reverse primer cggagcttttcttgtatcggtacc
SEQ ID NO: 31 NtNTP2-S mut tAg probe agctaggcttacggtgt
SEQ ID NO: 32 NtNTP2-S mut tgA forward primer agcatagggactttgacagcagtt
SEQ ID NO: 33 NtNTP2-S mut tgA reverse primer cggagcttttcttgtatcggtacc
SEQ ID NO: 34 NtNTP2-S mut tga probe agctgagcttacggtgt
SEQ ID NO: 35 NtNTP2-T wt forward primer catagggactttgacagcagtaacc
SEQ ID NO: 36 NtNTP2-T wt reverse primer ccgagcttttcttgtatcggtact
SEQ ID NO: 37 NtNTP2-T wt probe agctgggcttatgg
SEQ ID NO: 38 NtNTP2-T mut forward primer catagggactttgacagcagtaacc
SEQ ID NO: 39 NtNTP2-T mut reverse primer ccgagcttttcttgtatcggtact
SEQ ID NO: 40 NtNTP2-T mut probe agctgagcttatggtg
SEQ ID NO: 41 sequence in 5’ position of ntp2-S W211stop SNP mutation acggagctg
SEQ ID NO: 42 - sequence in 3’ position of ntp2-S W211stop SNP mutation gcttacggtg
SEQ ID NO: 43 - sequence in 5’ position to ntp2-S W211 stop SNP mutation acggagct
SEQ ID NO: 44 - sequence in 3’ position of ntp2-S W211 stop SNP mutation ggcttacggtg SEQ ID NO: 45 DNA sequence of synthetized RNAi loop with a 35S CaMV terminator ggatccaagctttggaagatccattttccttttccttatagcagcaaaaatgacttgaaaaatgtgtactatagg acttcccgagcttttcttgtatcggtactttttagtacctagaaggaagattaggatggcaatgagcatggatac agagcaaacaccataagcccagctccgtccgact teat ct tggatgtaaacaagcacggt tact get gtcaaagt ccctatggtaacctttaatgtttaaccgttcacatttctaatatttacttatttgtaacatgtcgtcacgtgtta gtttcattctttttatgaaccaaacatgcatgcaaagatatttttagatatttggacggcgagtgagatttgaaa ctaggaccgtttgcctgatacaatattaaaatatgtaaccattttatgtacaagtttaaactgttgatagtagca tattttttacttttatttaagtatactatattccaacaggtaagttaaccatagggactttgacagcagtaaccg tgcttgtttacatccaagatgaagtcggacggagctgggcttatggtgtttgctctgtatccatgctcattgcca tcctaatcttccttctaggtactaaaaagtaccgatacaagaaaagctcgggaagtcctatagtacacatttttc aagtcatttttgctgctataaggaaaaggaaaatggatcttccatacgtacctgaaatcaccagtctctctctac aaatctatctctctctattttctccataaataatgtgtgagtagtttcccgataagggaaattagggttcttata gggtttcgctcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataa aatttctaattcctaaaaccaaaatccagtactaaaatccagatctcctaaagtccctatagatctttgtcgtga atataaaccagacacgagacgactaaacctggagcccagacgccgttcgaagctagaagtaccgcttaggcagga ggccgttagggaaaagatgctaaggcagggttggttacgttgactcccccgtaggtttggtttaaatatgatgaa gtggacggaaggaaggaggaagacaaggaaggataaggttgcaggccctgtgcaaggtaagaagatggaaatttg atagaggtacgctactatacttatactatacgctaagggaatgcttgtatttataccctataccccctaataacc ccttatcaatttaagaaataatccgcataagcccccgcttaaaaattggtatcagagccatgaataggtctatga ccaaaactcaagaggataaaacctcaccaaaatacgaaagagttcttaactctaaagataaaagatcctaggcaa tteeg

Claims

1. A mutant, non-naturally occurring or transgenic plant or part of the plant having reduced or inhibited expression or activity of NtNTP2-T, or reduced or inhibited expression or activity of NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of:
(i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or
(ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11; or
(iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or
(iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or
(v) a NtNTP2-T polypeptide having at least 77 % sequence identity to SEQ ID NO:12; wherein the expression or activity of the NtNTP2-T or the expression or activity of the NtNTP2-T and the NtNTP2-S is reduced or inhibited as compared to a control plant.
2. The mutant, non-naturally occurring or transgenic plant or part of the plant according to claim 1, wherein the plant or the part of the plant:
(i) does not have decreased nitrate levels as compared to the control plant grown in the same fertilization conditions; and
(ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and
(iii) has increased NUE response, indicated as biomass per unit of nitrogen applied, as compared to the control plant grown in the same fertilization conditions.
3. The mutant, non-naturally occurring or transgenic plant or part of the plant according to claim 1 or claim 2, wherein the mutant, non-naturally occurring or transgenic plant or part of the plant in which the expression or activity of NtNTP2-T or the expression or activity of NtNTP2-T and NtNTP2-S is reduced or inhibited comprises:
(i) one or more sequence-specific polynucleotides that can interfere with the transcription of NtNTP2-T or NtNTP2-T and NtNTP2-S; and/or
(ii) one or more sequence-specific polypeptides that can interfere with the stability of NtNTP2-T or NtNTP2-T and NtNTP2-S; and/or
(iii) one or more sequence-specific polynucleotides that can interfere with the enzymatic activity of NtNTP2-T or NtNTP2-T and NtNTP2-S or the binding activity of NtNTP2-T or NtNTP2-T and NtNTP2-S with respect to substrates or regulatory proteins; and/or
(iv) gene edited NtNTP2-T or NtNTP2-T and NtNTP2-S; and/or
(v) at least one genetic alteration in the NtNTP2-T polynucleotide sequence or at least one genetic alteration in the NtNTP2-T polynucleotide and the NtNTP2-S polynucleotide sequence or at least one genetic alteration in the NtNTP2-T polypeptide sequence or at least one genetic alteration in the NtNTP2-T polypeptide sequence and the NtNTP2-S polypeptide sequence, suitably at least one genetic alteration that causes the encoded polypeptide(s) to terminate or end translation earlier than in the control plant.
4. The mutant, non-naturally occurring or transgenic plant or part of the plant according to claim 3 (v), wherein the at least one genetic alteration is at least one mutation, suitably, wherein the at least one genetic alteration comprises at least one nonsense mutation in the NtNTP2-T polynucleotide or the NtNTP2-T polypeptide or at least one nonsense mutation in the NtNTP2-T polynucleotide or NtNTP2-T polypeptide and at least one nonsense mutation in the NtNTP2-S polynucleotide or the NtNTP2-S polypeptide.
5. The mutant, non-naturally occurring or transgenic plant or part of the plant according to claim 4, comprising a single nucleotide polymorphism in NtNTP2-S at nucleotide position 632 or 633 or 632 and 633 of SEQ ID NO: 3, suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 632 or 633 of SEQ ID NO: 3 or a ‘g’ to ‘a’ mutation at nucleotide positions 632 and 633 of SEQ ID NO: 3.
6. The mutant, non-naturally occurring or transgenic plant or part of the plant according to claim 5, wherein the mutated NtNTP2-S polynucleotide sequence comprises, consists or consists essentially of SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6.
7. The mutant, non-naturally occurring or transgenic plant or part of the plant according to claim 4, comprising a single nucleotide polymorphism in NtNTP2-T at nucleotide position 636 of SEQ ID NO: 11, suitably, wherein the single nucleotide polymorphism is a ‘g’ to ‘a’ mutation at nucleotide position 635 or 636 of SEQ ID NO: 11 or a ‘g’ to ‘a’ mutation at nucleotide positions 635 and 636 of SEQ ID NO: 11.
8. The mutant, non-naturally occurring or transgenic plant or part of the plant according to claim 7, wherein the mutated NtNTP2-T polynucleotide sequence comprises, consists or consists essentially of SEQ I D NO: 13 or SEQ ID NO: 14 or SEQ I D NO: 15.
9. The mutant, non-naturally occurring or transgenic plant or part of the plant according to claim 4, wherein the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide each have at least one nonsense mutation at position W212 or position W212 and W211, respectively.
10. The mutant, non-naturally occurring or transgenic plant or part of the plant according to claim 9, wherein the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and mutated NtNTP2-S polypeptide comprise(s), consist(s) or consist(s) essentially of either SEQ ID NO: 16 or SEQ ID NO: 8 and SEQ ID NO: 16, respectively; optionally, wherein the mutated NtNTP2-T polypeptide or the mutated NtNTP2-T polypeptide and the mutated NtNTP2-S polypeptide are truncated.
11. The mutant, non-naturally occurring or transgenic plant or part of the plant according to any of the preceding claims, wherein the plant part is selected from: (i) green leaf or part thereof; or (ii) dried leaf or part thereof, suitably, wherein the dried leaf or part thereof is air dried, suitably, sun dried or fire dried; or (iii) cured leaf or part thereof, suitably, wherein the cured leaf is air cured, more suitably, sun cured or fire cured, flue cured.
12. A method of preparing a plant or a part of the plant comprising: (a) providing a plant comprising:
(i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or
(ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or
(iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or
(iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or
(v) a NtNTP2-T polypeptide having at least 77 % sequence identity to SEQ ID NO:12;
(b) reducing the expression or activity of the NtNTP2-T or the combination of the NtNTP2-T and NtNTP2-S in the plant; and
(c) obtaining a plant or part of the plant which: (i) does not have decreased nitrate levels as compared to a control plant grown in the same fertilization conditions; and (ii) has increased biomass as compared to the control plant grown in the same fertilization conditions; and (iii) has increased NUE response as compared to the control plant grown in the same fertilization conditions.
13. A mutant, non-naturally occurring or transgenic plant or a part thereof obtained or obtainable by the method of claim 12; or a mutant, non-naturally occurring or transgenic plant or a part thereof in which there is no significant difference in nitrate levels as compared to a control plant grown in the same fertilization conditions, wherein biomass yield is higher as compared to the control plant grown in the same fertilization conditions, and wherein the NUE of the plant is higher as compared to the control plant grown in the same fertilization conditions.
14. A tobacco product or a smoking article comprising the mutant, non-naturally occurring or transgenic plant or part of the plant according to any of claims 1 to 11 or comprising the plant or part of the plant according to claim 12.
15. A method of improving an agronomic characteristic of a plant, the method comprising reducing or inhibiting the expression or activity of NtNTP2-T or NtNTP2-T and NtNTP2-S, said NtNTP2-T and NtNTP2-S comprising or consisting of:
(i) a NtNTP2-S polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 3; or
(ii) a NtNTP2-T polynucleotide sequence comprising, consisting or consisting essentially of a sequence having at least 70 % sequence identity to SEQ ID NO: 11 ; or
(iii) a polypeptide encoded by the polynucleotide set forth in (i) or (ii); or
(iv) a NtNTP2-S polypeptide having at least 77 % sequence identity to SEQ ID NO: 7; or
(v) a NtNTP2-T polypeptide having at least 77 % % sequence identity to SEQ ID NO:12; wherein the expression or activity of the NtNTP2-T or the expression or activity of the NtNTP2- T and the NtNTP2-S is reduced or inhibited as compared to a control plant.
PCT/EP2023/078084 2022-10-13 2023-10-10 Increasing leaf biomass and nitrogen use efficiency by regulating ntp2 WO2024079137A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP22201409 2022-10-13
EP22201409.4 2022-10-13

Publications (1)

Publication Number Publication Date
WO2024079137A1 true WO2024079137A1 (en) 2024-04-18

Family

ID=83693019

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/078084 WO2024079137A1 (en) 2022-10-13 2023-10-10 Increasing leaf biomass and nitrogen use efficiency by regulating ntp2

Country Status (1)

Country Link
WO (1) WO2024079137A1 (en)

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
US5879918A (en) 1989-05-12 1999-03-09 Pioneer Hi-Bred International, Inc. Pretreatment of microprojectiles prior to using in a particle gun
US5886244A (en) 1988-06-10 1999-03-23 Pioneer Hi-Bred International, Inc. Stable transformation of plant cells
US5932782A (en) 1990-11-14 1999-08-03 Pioneer Hi-Bred International, Inc. Plant transformation method using agrobacterium species adhered to microprojectiles
US5981840A (en) 1997-01-24 1999-11-09 Pioneer Hi-Bred International, Inc. Methods for agrobacterium-mediated transformation
WO2006091194A1 (en) 2005-02-23 2006-08-31 North Carolina State University Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes
WO2008070274A2 (en) 2006-10-13 2008-06-12 North Carolina State University Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes
WO2009064771A2 (en) 2007-11-12 2009-05-22 North Carolina State University Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes
WO2009074325A1 (en) 2007-12-13 2009-06-18 Philip Morris Products S.A. Transgenic plants modified for reduced cadmium transport, derivative products, and related methods
WO2011088180A1 (en) 2010-01-15 2011-07-21 North Carolina State University Compositions and methods for minimizing nornicotine synthesis in tobacco
WO2011117249A1 (en) 2010-03-22 2011-09-29 Philip Morris Products S.A. Modifying enzyme activity in plants
WO2012028309A2 (en) 2010-09-03 2012-03-08 Philip Morris Products S.A. Heavy metal reduction in planta
WO2012098111A1 (en) 2011-01-17 2012-07-26 Philip Morris Products S.A. Vectors for nucleic acid expression in plants
WO2013029800A1 (en) 2011-09-02 2013-03-07 Philip Morris Products S.A Threonine synthase from nicotiana tabacum and methods and uses thereof
WO2013029799A1 (en) 2011-09-02 2013-03-07 Philip Morris Products S.A Isopropylmalate synthase from nicotiana tabacum and methods and uses thereof
WO2013064499A1 (en) 2011-10-31 2013-05-10 Philip Morris Products S.A. Modulating beta-damascenone in plants
WO2014096283A2 (en) 2012-12-21 2014-06-26 Philip Morris Products S.A Tobacco specific nitrosamine reduction in plants
US20140201863A1 (en) 2009-07-08 2014-07-17 Academia Sinica Method for changing nitrogen utilization efficiency in plants
WO2015169927A1 (en) 2014-05-08 2015-11-12 Philip Morris Products S.A. Reduction of nicotine to nornicotine conversion in plants
WO2015189693A1 (en) 2014-06-12 2015-12-17 King Abdullah University Of Science And Technology Targeted viral-mediated plant genome editing using crispr/cas9
WO2015197727A2 (en) 2014-06-25 2015-12-30 Philip Morris Products S.A Modulation of nitrate content in plants
WO2016046288A1 (en) 2014-09-26 2016-03-31 Philip Morris Products S.A Reducing tobacco specific nitrosamines through alteration of the nitrate assimilation pathway
WO2017042162A1 (en) 2015-09-09 2017-03-16 Philip Morris Products S.A. Plants with reduced asparagine content
WO2017129739A1 (en) 2016-01-29 2017-08-03 Philip Morris Products S.A. Reducing cadmium accumulation in field grown tobacco plants
US20190142058A1 (en) 2016-05-27 2019-05-16 Philip Morris Products S.A. Method for the preparation of a cast sheet of homogenized tobacco material

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US5886244A (en) 1988-06-10 1999-03-23 Pioneer Hi-Bred International, Inc. Stable transformation of plant cells
US5879918A (en) 1989-05-12 1999-03-09 Pioneer Hi-Bred International, Inc. Pretreatment of microprojectiles prior to using in a particle gun
US5932782A (en) 1990-11-14 1999-08-03 Pioneer Hi-Bred International, Inc. Plant transformation method using agrobacterium species adhered to microprojectiles
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
US5981840A (en) 1997-01-24 1999-11-09 Pioneer Hi-Bred International, Inc. Methods for agrobacterium-mediated transformation
WO2006091194A1 (en) 2005-02-23 2006-08-31 North Carolina State University Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes
WO2008070274A2 (en) 2006-10-13 2008-06-12 North Carolina State University Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes
WO2009064771A2 (en) 2007-11-12 2009-05-22 North Carolina State University Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes
WO2009074325A1 (en) 2007-12-13 2009-06-18 Philip Morris Products S.A. Transgenic plants modified for reduced cadmium transport, derivative products, and related methods
US20140201863A1 (en) 2009-07-08 2014-07-17 Academia Sinica Method for changing nitrogen utilization efficiency in plants
WO2011088180A1 (en) 2010-01-15 2011-07-21 North Carolina State University Compositions and methods for minimizing nornicotine synthesis in tobacco
WO2011117249A1 (en) 2010-03-22 2011-09-29 Philip Morris Products S.A. Modifying enzyme activity in plants
WO2012028309A2 (en) 2010-09-03 2012-03-08 Philip Morris Products S.A. Heavy metal reduction in planta
WO2012098111A1 (en) 2011-01-17 2012-07-26 Philip Morris Products S.A. Vectors for nucleic acid expression in plants
WO2013029800A1 (en) 2011-09-02 2013-03-07 Philip Morris Products S.A Threonine synthase from nicotiana tabacum and methods and uses thereof
WO2013029799A1 (en) 2011-09-02 2013-03-07 Philip Morris Products S.A Isopropylmalate synthase from nicotiana tabacum and methods and uses thereof
WO2013064499A1 (en) 2011-10-31 2013-05-10 Philip Morris Products S.A. Modulating beta-damascenone in plants
WO2014096283A2 (en) 2012-12-21 2014-06-26 Philip Morris Products S.A Tobacco specific nitrosamine reduction in plants
WO2015169927A1 (en) 2014-05-08 2015-11-12 Philip Morris Products S.A. Reduction of nicotine to nornicotine conversion in plants
WO2015189693A1 (en) 2014-06-12 2015-12-17 King Abdullah University Of Science And Technology Targeted viral-mediated plant genome editing using crispr/cas9
WO2015197727A2 (en) 2014-06-25 2015-12-30 Philip Morris Products S.A Modulation of nitrate content in plants
WO2016046288A1 (en) 2014-09-26 2016-03-31 Philip Morris Products S.A Reducing tobacco specific nitrosamines through alteration of the nitrate assimilation pathway
WO2017042162A1 (en) 2015-09-09 2017-03-16 Philip Morris Products S.A. Plants with reduced asparagine content
WO2017129739A1 (en) 2016-01-29 2017-08-03 Philip Morris Products S.A. Reducing cadmium accumulation in field grown tobacco plants
US20190142058A1 (en) 2016-05-27 2019-05-16 Philip Morris Products S.A. Method for the preparation of a cast sheet of homogenized tobacco material

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Genbank", Database accession no. L22344
"UniProt: the universal protein knowledgebase", NUCLEIC ACIDS RES., vol. 45, 2017, pages D158 - D169
ANAL. BIOCHEM., vol. 126, 1982, pages 131 - 138
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", vol. 1-3, 1993, JOHN WILEY & SONS, INC.
BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 72, no. 2, 2008, pages 615 - 617
BIOTECHNOLOGY ADVANCES, vol. 33, no. 1, 2015, pages 41 - 52
CARLSON ET AL., CHEM RES TOXICOL., vol. 31, no. 5, 21 May 2018 (2018-05-21), pages 358 - 370
CHIU ET AL., PLANT CELL PHYSIOL., vol. 45, no. 9, 2004, pages 1139 - 1148
CROSSWAY ET AL., BIOTECHNIQUES, vol. 4, 1986, pages 320 - 334
CURR. OP. IN PLANT BIOL., vol. 36, 2017, pages 1 - 8
DATABASE UniProt [online] 10 May 2017 (2017-05-10), "SubName: Full=protein NRT1/ PTR FAMILY 6.2-like {ECO:0000313|RefSeq:XP_009778211.1};", XP002808754, retrieved from EBI accession no. UNIPROT:A0A1U7WLP9 Database accession no. A0A1U7WLP9 *
DATABASE UniProt [online] 3 August 2022 (2022-08-03), "SubName: Full=protein NRT1/ PTR FAMILY 6.2-like {ECO:0000313|RefSeq:XP_016460993.1};", XP002808755, retrieved from EBI accession no. UNIPROT:A0A1S3Z9E1 Database accession no. A0A1S3Z9E1 *
FASEB JOURNAL, vol. 6, 1992, pages 3051 - 3064
HECHT, CHEM RES TOXICOL, vol. 11, no. 6, June 1998 (1998-06-01), pages 559 - 603
KHATODIA ET AL., FRONT PLANT SCI, vol. 7, 2016, pages 506
KOVI ET AL., TOXICOL PATHOL., vol. 46, no. 2, February 2018 (2018-02-01), pages 184 - 192
KRIEGLER: "Gene Transfer and Expression: A Laboratory Manual", 1990, STOCKTON PRESS
LIU ET AL., ACTA PHARMACEUTICA SINICA B, vol. 7, no. 3, 2017, pages 292 - 302
LU ET AL., PLANT BIOTECHNOLOGY JOURNAL, vol. 14, 2016, pages 1500 - 1510
MA ET AL., CARCINOGENESIS, vol. 39, no. 2, 9 February 2018 (2018-02-09), pages 232 - 241
MCCABE ET AL., BIOTECHNOLOGY, vol. 6, 1988, pages 923 - 926
MCCALLUM ET AL., NAT BIOTECHNOL, vol. 18, 2000, pages 455 - 457
MCCORMICK ET AL., PLANT CELL REPORTS, vol. 5, 1986, pages 81 - 84
METHODS, vol. 7, 1995, pages 48 - 54
MORALES DE LOS RÍOS LAURA ET AL: "The Arabidopsis protein NPF6.2/NRT1.4 is a plasma membrane nitrate transporter and a target of protein kinase CIPK23", PLANT PHYSIOLOGY AND BIOCHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 168, 11 October 2021 (2021-10-11), pages 239 - 251, XP086887641, ISSN: 0981-9428, [retrieved on 20211011], DOI: 10.1016/J.PLAPHY.2021.10.016 *
NATURE, vol. 407, 2000, pages 319 - 320
NUCLEIC ACIDS RESEARCH, vol. 22, 1994, pages 4673 - 4680
NUCLEIC ACIDS RESEARCH, vol. 24, 1997, pages 4876 - 4882
NUCLEIC ACIDS RESEARCH, vol. 41, no. D1, January 2013 (2013-01-01), pages D36 - 42
PASZKOWSKI ET AL., EMBO J, vol. 3, 1984, pages 2717 - 2722
PLANT J, vol. 27, 2001, pages 581 - 590
RIGGS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 5602 - 5606
SAMBROOK, J.E. F. FRITSCHT. MANIATIS: "Molecular Cloning and Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHIML ET AL., PLANT METHODS, vol. 12, 2016, pages 8
SRIVASTAVA SUDHAKAR ET AL: "MicroRNAs: Tiny, powerful players of metal stress responses in plants", PLANT PHYSIOLOGY AND BIOCHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 166, 5 July 2021 (2021-07-05), pages 928 - 938, XP086756433, ISSN: 0981-9428, [retrieved on 20210705], DOI: 10.1016/J.PLAPHY.2021.07.004 *
STEMPLE, NAT REV GENET, vol. 5, no. 2, 2004, pages 145 - 50

Similar Documents

Publication Publication Date Title
US10415050B2 (en) Reduction of nicotine to nornicotine conversion in plants
US10563215B2 (en) Tobacco specific nitrosamine reduction in plants
JP6225108B2 (en) Threonine synthase from Nicotiana tabacum and methods and uses thereof
US10883114B2 (en) Plants with reduced asparagine content
US11685929B2 (en) Plants with shortened time to flowering
JP2018186820A (en) Isopropyl malate synthase from Nicotiana tabacum and methods and uses thereof
EP3169149B1 (en) Tobacco protease genes
US20170145431A1 (en) Modulation of nitrate content in plants
EP3480314A1 (en) Regulation of alkaloid content
JP7463284B2 (en) Regulation of amino acid content in plants
US11591609B2 (en) Modulating reducing sugar content in a plant
WO2024079137A1 (en) Increasing leaf biomass and nitrogen use efficiency by regulating ntp2
RU2799785C2 (en) Modulation of the content of amino acids in the plant
WO2023117701A1 (en) Modulation of nicotine production by alteration of nicotinamidase expression or function in plants
WO2023117661A1 (en) Increasing anatabine in tobacco leaf by regulating methyl putrescine oxidase