WO2020234426A1 - Procédés pour améliorer le rendement en grains de riz - Google Patents

Procédés pour améliorer le rendement en grains de riz Download PDF

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WO2020234426A1
WO2020234426A1 PCT/EP2020/064218 EP2020064218W WO2020234426A1 WO 2020234426 A1 WO2020234426 A1 WO 2020234426A1 EP 2020064218 W EP2020064218 W EP 2020064218W WO 2020234426 A1 WO2020234426 A1 WO 2020234426A1
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plant
nucleic acid
mutation
seq
promoter
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PCT/EP2020/064218
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English (en)
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Xiaorong FAN
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Nanjing Agricultural University
Williams, Andrea
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Priority to CN202080037035.6A priority Critical patent/CN113924367A/zh
Publication of WO2020234426A1 publication Critical patent/WO2020234426A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to methods for increasing rice grain yield and nitrogen use efficiency by altering the splicing of a nitrate transporter gene, as well as genetically altered plants with increased yield and methods of making such plants.
  • Rice is one of the most important staples in the world, being consumed by more than 50% of the worlds people particularly the population of Asia (FAO, 2015). It is also nutritionally one of the most important food crops. However, in the field, rice may be subjected to a number of adverse conditions, which can cause huge losses in yield. One of the major limiting factors that affects yield is low nitrogen availability.
  • N Nitrogen
  • N is fundamental to crop development as it forms the basic component of many organic molecules, nucleic acids and proteins. N nutrition affects all levels of plant function, from metabolism to resource allocation, growth, and development.
  • the most abundant source for N acquisition by plant roots is nitrate (N03-) in natural aerobic soils, due to intensive nitrification of applied organic and fertilizer N.
  • ammonium (NH4+) is the main form of available N in flooded paddy soils due to the anaerobic soil conditions (Sasakawa and Yamamoto, 1978).
  • soil inorganic nitrogen is predominantly available for plants as nitrate in aerobic uplands and well-drained soils and as ammonium in poorly drained soils and flooded anaerobic paddy fields.
  • N soil inorganic nitrogen
  • the nitrate acquired by roots is transported to the shoots before being assimilated.
  • ammonium derived from nitrate reduction or directly from ammonium uptake is preferentially assimilated in the root and then transported in an organic form to the shoot.
  • plant roots have developed at least three nitrate uptake systems, two high-affinity transport systems (HATS) and one low-64 affinity transport system (LATS), responsible for the acquisition of nitrate.
  • HATS high-affinity transport systems
  • LATS low-64 affinity transport system
  • the constitutive HATS (cHATS) and nitrate-inducible HATS (iHATS) operate to take up nitrate at a low nitrate concentration in external medium with saturation in a range of 0.2-0.5 mM.
  • LATS functions in nitrate acquisition at a higher external nitrate concentration.
  • the uptake by LATS and HATS is mediated by nitrate transporters belonging to the families of NRT1 and NRT2, respectively. Uptake by roots is regulated by negative feedback, linking the expression and activity of nitrate uptake to the N status of the plant (Miller et al., 2007).
  • N03 and NH4 Plants vary substantially in their relative adaptations to these two sources of N.
  • NH4 should be the preferred N source, since its metabolism requires less energy than that of N03 , only a few species actually perform well when NH4 is provided as the only N source. Among the latter are boreal conifers, ericaceous species, some vegetable crops, and rice ( ' Oryza sativa L.).
  • Rice differs from other crop plants in that it is capable of growing exclusively on NH4 as the only N source.
  • Rice has been traditionally cultivated under flooded anaerobic soil conditions where ammonium is the main N source.
  • the specialized aerenchyma cells in rice roots can transfer oxygen from the shoots to the roots and release it to the rhizosphere, where bacterial conversion of ammonium to nitrate (nitrification) can take place.
  • Nitrification in the waterlogged paddy rhizosphere can result in a 25-40% of the total crop N being taken up in the form of nitrate, mainly through a high affinity transport system (HATS).
  • HATS high affinity transport system
  • nitrate uptake is mediated by cotransport with protons (H + ) that can be extruded from the cell by plasma membrane H + -ATPases.
  • H + protons
  • the molecular mechanisms of nitrate uptake and translocation in rice are not fully understood. Since the nitrate concentration in the rhizosphere of paddy fields is estimated to be less than 10 mM (Kirk and Kronzucker, 2005), NRT2 family members play a major role in nitrate uptake in rice (Araki and Hasegawa, 2006; Yan et al., 2011).
  • OsNRT2.1 and OsNRT2.2 share an identical coding region sequence with different 5'- and 3'-untranscribed regions (UTRs) and have high similarity to the NRT2 genes of other monocotyledons, while OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 genes.
  • OsNRT2.3 mRNA is actually spliced into two gene products, OsNRT2.3a (AK109776) and OsNRT2.3b (AK072215), with 94.2% similarity in their putative amino acid sequences (Feng et al., 2011 , Yan et al., 2011).
  • OsNRT2.3a is expressed mainly in roots and this pattern is enhanced by nitrate supply, while OsNRT2.3b is expressed weakly in roots and relatively abundantly in shoots with no effect of the N form and concentration on the amount of transcript (Feng et al., 2011 , Feng 2012).
  • the overexpression of OsNRT2.3b has been shown to increase yield, growth and NUE of transgenic plants (Fan et al., 2016).
  • Cis-acting elements on promoters also play an important regulatory role in gene expression and transcriptional translation.
  • Our detailed analyses demonstrate that the TATA-box is the key cis regulation element for OsNRT2.3 to be transcribed into OsNRT2.3a and OsNRT2.3b.
  • OsTBP2.1 a TATA-box binding protein, which binds to a TATA-box motif on the OsNRT2.3 promoter.
  • Our results show that the TATA-box mutant in the 5’UTR of OsNRT2.3b and the binding protein OsTBP2.1 together increase the ratio of OsNRT2.3b to OsNRT2.3a, consequently increasing both yield and NUE.
  • a method for increasing at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content of a plant comprising introducing at least one mutation into a nucleic acid sequence encoding a NRT2.3 promoter.
  • the plant is rice.
  • nucleic acid sequence encoding a NRT2.3 promoter comprises SEQ ID NO: 9 or a functional variant thereof. In a further preferred embodiment, the nucleic acid sequence encoding a NRT2.3 promoter comprises SEQ ID NO: 1 or a functional variant thereof.
  • the mutation is introduced using mutagenesis.
  • the mutation is introduced using TILLING or T-DNA insertion.
  • the mutation is introduced using targeted genome modification, preferably ZFNs, TALENs or CRISPR/Cas9.
  • the mutation is introduced into SEQ ID NO:1 , and preferably into SEQ ID NO: 9. More preferably, the mutation is an insertion, deletion and/or substitution. Even more preferably, the mutation is substitution of at least one nucleotide.
  • substitution is at position 160, position 201 or position 222 of SEQ ID NO: 1.
  • the mutation is a substitution at position 160.
  • the mutation is a T to C substitution.
  • the mutation is the deletion of at least one nucleotide. More preferably, the mutation is the deletion of at least fifty, more preferably sixty 5’ nucleotides of SEQ ID NO: 1. In another embodiment, said mutation is the deletion of at least ninety, more preferably one hundred 5’nucleotides of SEQ ID NO: 1. In a further embodiment, the method further comprises regenerating a plant and screening said plant for an increase in at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content.
  • NUE nitrogen use efficiency
  • a genetically altered plant part thereof of plant cell, wherein said plant comprises at least one mutation in at least one nucleic acid sequence encoding a NRT2.3 promoter.
  • the nucleic acid sequence comprises SEQ ID NO: 9 or a functional variant thereof. In a further embodiment, the nucleic acid sequence comprises SEQ ID NO: 1 or a functional variant thereof.
  • the plant is characterised by an increase in at least one of increase in at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content.
  • NUE nitrogen use efficiency
  • the mutation is introduced into SEQ ID NO: 1 , and preferably into SEQ ID NO: 9.
  • the mutation is an insertion, deletion and/or substitution.
  • the mutation is substitution of at least one nucleotide, and in one example, is a substitution is at position 160, position 201 or position 222 of SEQ ID NO: 1.
  • the mutation is a substitution at position 160.
  • the mutation is a T to C substitution.
  • the mutation is the deletion of at least one nucleotide. In one embodiment, the mutation is the deletion of at least fifty, more preferably sixty 5’ nucleotides of SEQ ID NO: 1. In an alternative embodiment, said mutation is the deletion of at least ninety, more preferably one hundred 5’nucleotides of SEQ ID NO: 1.
  • the genetically altered plant is rice.
  • a method of identifying and/or selecting a plant that has or will have an increased yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content, preferably compared to a control or wild-type plant comprising detecting in the plant or plant germplasm at least one polymorphism in the NRT2.3 promoter gene sequence and selecting said plant or progeny thereof.
  • the NRT2.3 promoter gene sequence comprises SEQ ID NO: 9, and more preferably SEQ ID NO: 1 or a functional variant thereof.
  • the polymorphism is at least one substitution at a least position 160 of SEQ ID NO: 1.
  • the polymorphism is the deletion of at least one 5’ nucleotide of SEQ ID NO: 1 , more preferably the deletion of at least the first sixty 5’ nucleotides of SEQ ID NO: 1.
  • the method further comprises introgressing the chromosomal region comprising at least one polymorphism in the NRT2.3 promoter into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.
  • a method of increasing at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content of a plant comprising introducing and expressing in said plant a nucleic acid construct comprising a NRT2.3 promoter sequence operably linked to a NRT2.3 gene sequence, wherein the NRT2.3 promoter sequence is selected from the group comprising SEQ ID NO: 2, 3, 4 or 5 or a functional variant thereof.
  • a method of making a plant having an increased yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content comprising introducing an expressing in a plant or plant cell a nucleic acid construct comprising a NRT2.3 promoter sequence operably linked to a NRT2.3 gene sequence, wherein the NRT2.3 promoter sequence is selected from the group comprising SEQ ID NO: 2, 3, 4 or 5 or a functional variant thereof.
  • the NRT2.3 gene sequence comprises SEQ ID NO: 8 or a functional variant thereof.
  • the plant is rice.
  • the plant is rice.
  • nucleic acid construct comprising a NRT2.3 promoter sequence operably linked to a NRT2.3 gene sequence, wherein the NRT2.3 promoter sequence is selected from the group comprising SEQ ID NO: 2, 3, 4 or 5 or a functional variant thereof.
  • the NRT2.3 gene sequence comprises SEQ ID NO: 8 or a functional variant thereof.
  • a vector comprising the nucleic acid construct described above.
  • a host cell comprising the vector or the nucleic acid construct.
  • a transgenic plant expressing the vector or the nucleic acid construct.
  • the plant is rice.
  • NUE nitrogen use efficiency
  • a method of altering the splicing of the NRT2.3 gene comprising introducing at least one mutation into a nucleic acid sequence encoding a NRT2.3 promoter.
  • nucleic acid construct comprising a nucleic acid sequence encoding at least one DNA-binding domain that can bind to at least one NRT2.3 promoter.
  • the nucleic acid sequence encodes at least one protospacer element, and wherein the sequence of the protospacer element is selected from SEQ ID Nos 16 to 23 or a sequence that is at least 90% identical to SEQ ID Nos 16 to 23.
  • the construct further comprises a nucleic acid sequence encoding a CRISPR RNA (crRNA) sequence, wherein said crRNA sequence comprises the protospacer element sequence and additional nucleotides.
  • the construct further comprises a nucleic acid sequence encoding a transactivating RNA (tracrRNA), wherein preferably the tracrRNA is defined in SEQ ID NO. 24 or a functional variant thereof. More preferably, said construct encodes at least one single-guide RNA (sgRNA), wherein said sgRNA comprises the tracrRNA sequence and the crRNA sequence.
  • sgRNA single-guide RNA
  • the construct is operably linked to a promoter. More preferably, the promoter is a constitutive promoter.
  • the nucleic acid construct further comprises a nucleic acid sequence encoding a CRISPR enzyme.
  • the CRISPR enzyme is a Cas protein or Cpf1 protein.
  • the Cas protein is Cas9 or a functional variant thereof.
  • the nucleic acid construct encodes a TAL effector.
  • the nucleic acid construct further comprises a sequence encoding an endonuclease or DNA-cleavage domain thereof.
  • the endonuclease is Fokl.
  • an isolated plant cell transfected with at least one nucleic acid construct as described above in another aspect of the invention there is provided an isolated plant cell transfected with at least one nucleic acid construct as described above.
  • the second nucleic acid construct is transfected before, after or concurrently with the first nucleic acid construct.
  • a genetically modified plant wherein said plant comprises the transfected cell described above.
  • the nucleic acid encoding the sgRNA and/or the nucleic acid encoding a Cas protein is integrated in a stable form.
  • a method of increasing at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content of a plant comprising introducing and expressing in a plant the nucleic acid construct as described above, wherein preferably said increase is relative to a control or wild-type plant.
  • NUE nitrogen use efficiency
  • nucleic acid construct as described above to increase at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content in a plant.
  • a method for obtaining the genetically modified plant as defined above comprising: a. selecting a part of the plant;
  • said increase is relative to a control or wild-type plant.
  • Figure 1 shows the biomass, NUE and nitrogen content in the -83 bp mutation lines at maturity stage in the field
  • (a) Characterization of the -83 bp mutation lines of OsNRT2.3 mutation in a field experiment. Wild type, Zhonghua 11 (WT), mutation lines of T8, T11 , T12 and T20. (Bar 30 cm);
  • Figure 2 shows the characterization and identification of the -83 bp mutation lines in hydroponics (a) phenotypes of WT and -83bp mutation lines.
  • Figure 3 shows the effect of mutation of the OsNRT2.3 promoter on expression of OsNRT2.3a/b; (a,c) Schematic representation of the OsNRT2.3 promoter and the different mutation sites that alter expression of OsNRT2.3a and OsNRT2.3b.
  • P is the WT promoter of OsNRT2.3
  • P1 is the -665bp mutation in the OsNRT2.3 promoter
  • P2 is the -44bp mutation in the OsNRT2.3 promoter
  • mP is the -83bp mutation in the OsNRT2.3 promoter
  • Figure 4 shows that the -83bp mutation changes the translation pattern of OsNRT2.3 in the backcross lines;
  • Bar 30 cm)
  • AA is the control, - 83 bp no mutation; aa is the homozygous -83 bp mutation; Aa is the heterozygous -83 bp mutation;
  • B1 F3 T11 and B1 F3 T12 are the B1 F3 generation of OsNRT2.3 mutation backcross lines.
  • WT and the mutation backcross homozygous lines of T11 and T12 were grown in 1.25 mM NH4N03 for 3 weeks and nitrogen starved for 1 week.
  • 15N influx rate was then measured at 2.5 mM 15N03-, 1.25 mM NH415N03, and 1.25 mM 15NH4N03 over a 5 minute period; (d) The shoot 15N influx rate; (e) The root 15N influx rate; (f) the 15N ratio of shoot to root.
  • Error bars: SE(n 5) The different letters indicate a significant difference between the transgenic line and the WT.
  • Figure 5 shows that the -83bp mutation at different length promoters influence the transcription of OsNRT2.3 in rice;
  • 141 bp and 697 bp are the original OsNRT2.3 promoter;
  • 141 M and 697M carry the -83 bp mutation;
  • FIG. 6 shows that OsTBP2.1 binds to the OsNRT2.3 promoter fragments and activates OsNRT2.3 expression
  • Yeast cells were co-transformed with pTATA-box::AbAi and OsTBP2/2.1/2.2::pGADT7. Cells were grown on media to select for interaction (SD,- Ura, -Leu) and (800 nM).
  • AbA was used to suppress background growth
  • Figure 7 shows the expression of OsTBP2.1 and OsNRT2.3a/b in the OsTBP2.1 overexpression and T-DNA mutant lines
  • (Bar 20 cm)
  • Figure 8 shows a schematic diagram of a model of transcription of OsNRT2.3 when the TATA-box is mutated.
  • the data shows that when mutated, OsTBP2.1 enhances the expression of OsNRT2.3b, consequently altering the ratio of OsNRT2.3b to OsNRT2.3a and resulting in higher levels of OsNRT2.3b translation.
  • Figure 9 shows characterization of the TILLING lines in the field.
  • Figure 10 shows the expression of OsNRT2.3a and OsNRT2.3b in the -83 bp mutation lines.
  • Figure 11 shows the identification of the -83 bp mutation backcross lines.
  • Figure 12 shows the yield, dry weight and N-use efficiency of -83 bp mutation backcross lines at the mature stage.
  • Figure 13 shows the effect of different OsNRT2.3 promoter lengths on OsNRT2.3a/b expression in rice.
  • Figure 14 shows the expression of OsNRT2.3a and OsNRT2.3b in the OEOsTBP2.1 and ostbp2.1 lines.
  • Figure 15 shows the influx of 15 NO 3 - and 15 NH4 + in the OsNRT2.3 mutation lines over a 5 minute period.
  • WT and the OsNRT2.3 mutation seedlings were grown in 1.25 mM NH4NO3 for 3 weeks and nitrogen starved for 1 week.
  • 15 N influx rate was then measured at 2.5 mM 15 NO 3 - , 1.25 mM NH4 15 NO 3 , and 1.25 mM 15 NH 4 NO 3 over a 5 minute period
  • b The shoot 15 N influx rate.
  • Figure 16 shows the influx of 15 NO 3 - and 1 5 NH4 + in the OsNRT2.3 mutation backcross lines over a 5 minute period.
  • WT and the mutation backcross homozygosis lines of T11 and T12 were grown in 1.25 mM NH4NO3 for 3 weeks and nitrogen starved for 1 week.
  • 15 N influx rate was then measured at 2.5 mM 15 NO 3 - , 1.25 mM NH4 1 5 NO 3 , and 1.25 mM 1 5 NH 4 NO 3 over a 5 minute period
  • Figure 17 shows the N content in backcross lines of -83 bp mutation at the mature stage.
  • the rice transporter OsNRT2.3 has two spliced forms - OsNRT2.3a and OsNRT2.3b. Some nitrate transporters require two genes for function; the second much smaller component (OsNAR21) is required for the correct targeting of the transporter protein to the plasma membrane. One of the two spliced forms, OsNRT2.3a, requires this second component for function, while the other form, OsNRT2.3b, does not. We have previously demonstrated that overexpression of OsNRT2.3b in rice improves growth and NUE.
  • the backcross mutation lines increased total biomass by approximately 28% and NUE by approximately 75% compared with control plants in the field.
  • the weight per panicle of the backcross mutation lines also increased about 60% in the field.
  • the backcross mutation lines further improved nitrogen uptake from the field compared with the control.
  • nucleic acid As used herein, the words “nucleic acid”, “nucleic acid sequence”, “nucleotide”, “nucleic acid molecule” or “promoter sequence” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene.
  • genes or“gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function.
  • genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.
  • the aspects of the invention involve recombination DNA technology and exclude embodiments that are solely based on generating plants by traditional breeding methods and plants obtained by traditional breeding methods.
  • a method of increasing yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content of a plant comprising introducing at least one mutation into a nucleic acid sequence upstream of the NRT2.3 gene.
  • the method comprises introducing a mutation in at least one nucleotide in the 224 nucleotides upstream (e.g. in the 5’ direction) of the ATG start codon of the NRT2.3 gene.
  • the NRT2.3 gene is defined in SEQ ID NO: 8 or a variant thereof.
  • yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content may be increased by at least 5%-50% or more compared to a control plant, for example by up to or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.
  • yield in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight. The actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square metres.
  • the term“increased yield” as defined herein can be taken to comprise any or at least one of the following and can be measured by assessing one or more of (a) increased biomass (weight) of one or more parts of a plant, aboveground (harvestable parts), or increased root biomass, increased root volume, increased root length, increased root diameter or increased root length or increased biomass of any other harvestable part.
  • Increased biomass may be expressed as g/plant or kg/hectare (b) increased seed yield per plant, which may comprise one or more of an increase in seed biomass (weight) per plant or an individual basis, (c) increased seed filling rate, (d) increased number of filled seeds, (e) increased harvest index, which may be expressed as a ratio of the yield of harvestable parts such as seeds over the total biomass, (f) increased viability/germination efficiency, (g) increased number or size or weight of seeds or pods or beans or grain (h) increased seed volume (which may be a result of a change in the composition (i.e.
  • lipid also referred to herein as oil
  • protein and carbohydrate total content and composition
  • i increased (individual or average) seed area
  • j increased (individual or average) seed length
  • k increased (individual or average) seed perimeter
  • I increased growth or increased branching, for example inflorescences with more branches
  • n increased fresh weight or grain fill
  • o increased ear weight
  • TKW thousand kernel weight
  • All parameters are relative to a wild-type or control plant.
  • an increase in yield comprises an increase in weight, preferably dry weight (g/plant) of the plant.
  • an increase in yield comprises an increase in weight per panicle.
  • the increase in weight per panicle is at least or up to 40%, more preferably 50% and even more preferably 60% compares to a control plant.
  • the yield is increased by at least or up to 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to a control or wild-type plant.
  • Yield may alternatively be increased by between 20-70%, more preferably between 25 and 75% compared to a control plant.
  • NUE nitrogen use efficiency
  • yield of crop e.g. yield of grain
  • NUE can be defined as agricultural NUE that means grain yeild/ N .
  • the overall N use efficiency of plants comprises both uptake and utilization efficiencies and can be calculated as UpE.
  • NUE is increased by 5%-80% or more compared to a control plant, for example by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% compared to a control plant.
  • NUE is increased by at least 60%, more preferably 70% and even more preferably 75% compared to a control or wild-type plant.
  • nitrogen transport encompasses nitrogen acquisition or nitrogen influx or uptake.
  • nitrogen uptake may refer to the uptake of ammonium, nitrate and/or ammonium nitrate.
  • nitrogen influx is increased in the shoots and/or roots of the plant. Such increase is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70% or more compared to a control or wild-type plant.
  • N content is increased in the shoot and/or root of a plant.
  • N content is increased in at least one of the leaf, sheath, culm and panicle.
  • total N content in the plant is not increased.
  • At least one mutation is meant that where the NRT2.3 promoter gene is present as more than one copy or homoeologue (with the same or slightly different sequence) there is at least one mutation in at least one gene. Preferably all genes are mutated.
  • the NRT2.3a gene comprises a 43bp 5’UTR (Untranslated Region), while the NRT2.3b gene comprises a 223bp 5’UTR.
  • the method comprises introducing at least one mutation into the, preferably endogenous, NRT2.3 promoter.
  • A“promoter” as used herein encompasses the nucleic acid sequence 1.5kbp upstream of the ATG (the initiation or start codon).
  • the term“promoter” includes the 5’UTR (untranslated region) of the NRT2.3a and NRT2.3b gene.
  • the mutation is in NRT2.3a promoter.
  • the mutation is in the 5’ UTR of the NRT2.3b gene.
  • the NRT2.3 promoter comprises a TATA box. More preferably the NRT2.3 promoter comprises SEQ ID NO: 1 or a functional variant thereof. In another embodiment, the NRT2.3 promoter comprises SEQ ID NO: 9 or a functional variant thereof.
  • the mutation affects splicing of the NRT2.3 gene, and in particular, the mutation increases the relative expression of NRT2.3b to NRT2.3a. Said increase in relative expression is at least 2-fold, more preferably at least 4-fold, more preferably at least 6-fold, and even more preferably at least 8-fold higher compared to a wild-type plant. In an alternative embodiment, the mutation increases the expression of NRT2.3b at least 5-fold, more preferably 6-fold and even more preferably 7-fold compared to the level of expression in a wild-type plant. In a further preferred embodiment, the mutation is in SEQ ID NO: 1 or a variant thereof.
  • the expression of NRT2.3b is increased in the shoots and/or the expression of NRT2.3a is decreased in the roots of the plant.
  • the mutation is in the TATA box, and more preferably in SEQ ID NO:9 or a functional variant thereof.
  • an‘endogenous’ nucleic acid may refer to the native or natural sequence in the plant genome.
  • the sequence of the NRT2.3 promoter comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 1 , which is the 5’UTR of the NRT2.3b gene or a functional variant thereof.
  • the term“functional variant of a nucleic acid sequence” as used herein with reference to any of SEQ ID NOs: 1 to 8 refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant sequence. In the context of SEQ ID Nos 1 to 4, this may mean that the sequence is able to initiate or otherwise result in transcription of the NRT2.3 gene.
  • a functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example in non- conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active.
  • Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
  • a“variant” or a“functional variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,
  • nucleic acid sequences are said to be “identical” if the sequence of nucleotides in the two sequences is the same when aligned for maximum correspondence as described below.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.
  • a variant as used herein can comprise a nucleic acid sequence encoding a NRT2.3 promoter as defined herein that is capable of hybridising under stringent conditions as defined herein to a nucleic acid sequence as defined in SEQ ID NO: 1.
  • stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing).
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30oC for short probes (e.g., 10 to 50 nucleotides) and at least about 60oC for long probes (e.g., greater than 50 nucleotides).
  • Duration of hybridization is generally less than about 24 hours, usually about 4 to 12.
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • the mutation that is introduced into the NRT2.3 promoter thereof may be selected from the following mutation types:
  • substitution mutation resulting in the substitution of at least one nucleotide for at least one different nucleotide.
  • the mutation is a deletion of at least one nucleotide in the NRT2.3 promoter, wherein preferably the NRT2.3 promoter comprises or consists of SEQ ID NO: 1 or a functional variant thereof.
  • the mutation is the deletion of at least one nucleotide from the 5’ end of SEQ ID NO: 1. More preferably, the mutation is the deletion of at least the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 nucleotides from the 5’ end of SEQ ID NO: 1.
  • the mutation is the deletion of at least the first 50, more preferably the first 60, and even more preferably, the first 62 nucleotides from the 5’ end of SEQ ID NO: 1.
  • the mutation is the deletion of the first 90, more preferably the first 100 and even more preferably the first 101 nucleotides from the 5’ end of SEQ ID NO:1. In an alternative embodiment, the mutation is the deletion of either SEQ I D NO: 6 or 7 from SEQ I D NO: 1.
  • the mutation is the substitution of at least one nucleotide at position 160 of SEQ ID NO: 1 (this may also be referenced herein as being a mutation at position -83 with respect to the ATG start codon of the NRT2.3 gene), position 201 of SEQ ID NO: 1 (again this may be referred to herein as position -42) and/or position 222 of SEQ ID NO: 1 (again, this may be referred to herein as position -21).
  • OSE2ROOTNODULE At position -83 (with respect to the ATG start codon), there are two putative motifs named OSE2ROOTNODULE(-82bp to -86bp) (SEQ ID NO:10) and ASF 1 MOTI FCAMV (-76bp to -83bp) (SEQ ID NO: 11).
  • OSE2ROOTNODULE motif may control the tillering process and that the ASF1MOTIFCAMV motif is a suppressor binding motif. It may be that the loss of gene suppressor binding function of this motif in the mutant lines results in the change in expression ratio seen in these lines.
  • Position 201 (of SEQ ID NO: 1): A to C;
  • the mutation is a T to C substitution at position 160 of SEQ ID NO: 1.
  • the at least one mutation is a substitution, deletion and/or insertion of at least one nucleotide in the TATA box of the NRT2.3 promoter.
  • the TATA box is defined in SEQ ID NO: 9 or a variant thereof. Accordingly, in one embodiment the mutation affects the binding of transcription factors or histones to the NRT2.3 promoter and therefore affects transcription of the NRT2.3 gene. In a preferred embodiment, the mutation alters the binding ability of TATA-box binding factors such as TBP2.1 , which also affects the expression of the NRT2.3 gene.
  • the mutation is a substitution, deletion and/or deletion of at least one nucleotide in the OSE2ROOTNODULE motif and/or the ASF1MOTIFCAMV motif.
  • the mutation results in a loss or a partial loss of function of one or both motifs.
  • the OSE2ROOTNODULE motif may be involved in the control of tillering.
  • the ASF1 MOTIFCAMV motif is a suppressor binding motif.
  • the mutation is in the ASF1MOTIFCAMV motif and prevents or reduces the ability of its suppressor to bind. More preferably, the OSE2ROOTNODULE motif is defined in SEQ ID NO: 10 or a variant thereof, and the ASF1MOTIFCAMV motif is defined in SEQ ID NO: 11 or a variant thereof. A variant is defined herein.
  • the mutation is a substitution of at least one nucleotide in the TATA box. Even more preferably, the mutation is a substitution at position 12 of SEQ ID NO: 9. In one embodiment, the substitution is a T to C substitution. Other major changes such as deletions that remove functional regions of the promoter or enhancers are also included as these can affect splicing of the NRT2 gene. For example, a mutation may result in the deletion of the TATA box. In other words, the deletion of SEQ ID NO: 9.
  • the mutation is introduced using mutagenesis or targeted genome editing. That is, in one embodiment, the invention relates to a method and plant that has been generated by genetic engineering methods as described above, and does not encompass naturally occurring varieties.
  • Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events.
  • DSBs DNA double-strand breaks
  • HR homologous recombination
  • customisable DNA binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcription factors, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats).
  • ZF and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate nuclease and DNA-binding domains, ZF and TALE proteins consist of individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of Fokl to direct nucleolytic activity toward specific genomic loci.
  • TAL effectors Upon delivery into host cells via the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in host gene promoters and activate transcription. Their targeting specificity is determined by a central domain of tandem, 33-35 amino acid repeats. This is followed by a single truncated repeat of 20 amino acids. The majority of naturally occurring TAL effectors examined have between 12 and 27 full repeats. These repeats only differ from each other by two adjacent amino acids, their repeat- variable di-residue (RVD).
  • RVD repeat- variable di-residue
  • the RVD that determines which single nucleotide the TAL effector will recognize one RVD corresponds to one nucleotide, with the four most common RVDs each preferentially associating with one of the four bases.
  • Naturally occurring recognition sites are uniformly preceded by a T that is required for TAL effector activity.
  • TAL effectors can be fused to the catalytic domain of the Fokl nuclease to create a TAL effector nuclease (TALEN) which makes targeted DNA double-strand breaks (DSBs) in vivo for genome editing.
  • TALEN TAL effector nuclease
  • the Golden Gate method uses Type IIS restriction endonucleases, which cleave outside their recognition sites to create unique 4 bp overhangs. Cloning is expedited by digesting and ligating in the same reaction mixture because correct assembly eliminates the enzyme recognition site. Assembly of a custom TALEN or TAL effector construct and involves two steps: (i) assembly of repeat modules into intermediary arrays of 1-10 repeats and (ii) joining of the intermediary arrays into a backbone to make the final construct. Accordingly, using techniques known in the art it is possible to design a TAL effector that targets a NRT2.3 promoter sequence as described herein.
  • CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids.
  • CRISPR loci in microbial hosts contain a combination of CRISPR- associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA).
  • Cas CRISPR-associated genes
  • sgRNA CRISPR-mediated nucleic acid cleavage
  • I- III Three types (I- III) of CRISPR systems have been identified across a wide range of bacterial hosts.
  • each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers).
  • the non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer).
  • the Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus.
  • tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences.
  • the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition.
  • Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.
  • CRISPR-Cas9 compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene.
  • the intervening section can be deleted or inverted.
  • Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
  • the Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases.
  • the HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA.
  • sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms.
  • DSBs site-specific double strand breaks
  • codon optimized versions of Cas9 which is originally from the bacterium Streptococcus pyogenes, have been used.
  • the single guide RNA is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease.
  • sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA.
  • the sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities.
  • the canonical length of the guide sequence is 20 bp.
  • sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3. Accordingly, using techniques known in the art it is possible to design sgRNA molecules that targets a NRT2.3 promoter sequence as described herein.
  • Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.
  • mutagenesis methods can be used to introduce at least one mutation into the NRT2.3 promoter sequence. These methods include both physical and chemical mutagenesis. A skilled person will know further approaches can be used to generate such mutants, and methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein.
  • insertional mutagenesis is used, for example using T-DNA mutagenesis (which inserts pieces of the T-DNA from the Agrobacterium tumefaciens T-Plasmid into DNA causing either loss of gene function or gain of gene function mutations), site-directed nucleases (SDNs) or transposons as a mutagen. Insertional mutagenesis is an alternative means of disrupting gene function and is based on the insertion of foreign DNA into the gene of interest (see Krysan et al, The Plant Cell, Vol. 11 , 2283-2290, December 1999). Accordingly, in one embodiment, T-DNA is used as an insertional mutagen to disrupt the activity of the NRT2.3 promoter.
  • T-DNA may be inserted into SEQ ID NO: 1 or a functional variant thereof.
  • T-DNA not only disrupts the expression of the gene into which it is inserted, but also acts as a marker for subsequent identification of the mutation. Since the sequence of the inserted element is known, the gene in which the insertion has occurred can be recovered, using various cloning or PCR-based strategies. The insertion of a piece of T- DNA in the order of 5 to 25 kb in length generally produces a disruption of gene function. If a large enough population of T-DNA transformed lines is generated, there are reasonably good chances of finding a transgenic plant carrying a T-DNA insert within any gene of interest.
  • Transformation of spores with T-DNA is achieved by an Agrobacterium-medIiated method which involves exposing plant cells and tissues to a suspension of Agrobacterium cells.
  • Agrobacterium-medIiated method which involves exposing plant cells and tissues to a suspension of Agrobacterium cells.
  • plant transformation by Agrobacterium results in the integration into the nuclear genome of a sequence called T-DNA, which is carried on a bacterial plasmid.
  • T-DNA transformation leads to stable single insertions.
  • Further mutant analysis of the resultant transformed lines is straightforward and each individual insertion line can be rapidly characterized by direct sequencing and analysis of DNA flanking the insertion.
  • Gene expression of NRT2.3b or the relative expression of NRT2.3a to NRT2.3b in the mutant is compared to expression in a wild type plant. A phenotypic analysis is also carried out.
  • mutagenesis is physical mutagenesis, such as application of ultraviolet radiation, X-rays, gamma rays, fast or thermal neutrons or protons.
  • the targeted population can then be screened to identify a mutant with a mutation in the NRT 2.3 promoter, and preferably a mutation in SEQ ID NO: 1 or a variant thereof.
  • the method comprises mutagenizing a plant population with a mutagen.
  • the mutagen may be a fast neutron irradiation or a chemical mutagen, for example selected from the following non-limiting list: ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N- nitrosurea (ENU), triethylmelamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N'-nitro- Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimethyl- benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoronate, ethylene oxide,
  • the method used to create and analyse mutations is targeting induced local lesions in genomes (TILLING), reviewed in Henikoff et al, 2004.
  • TILLING induced local lesions in genomes
  • seeds are mutagenised with a chemical mutagen, for example EMS.
  • the resulting M1 plants are self-fertilised and the M2 generation of individuals is used to prepare DNA samples for mutational screening.
  • DNA samples are pooled and arrayed on microtiter plates and subjected to gene specific PCR.
  • the PCR amplification products may be screened for mutations in the NRT2.3 promoter using any method that identifies heteroduplexes between wild type and mutant genes.
  • dHPLC denaturing high pressure liquid chromatography
  • DCE constant denaturant capillary electrophoresis
  • TGCE temperature gradient capillary electrophoresis
  • the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences.
  • Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program.
  • Any primer specific to the NRT2.3 promoter sequence may be utilized to amplify the NRT2.3 promoter sequence within the pooled DNA sample.
  • the primer is designed to amplify the regions of the NRT2.3 promoter where useful mutations are most likely to arise, specifically in the areas of the NRT2.3 promoter that are highly conserved and/or confer activity as explained elsewhere.
  • the PCR primer may be labelled using any conventional labelling method.
  • the method used to create and analyse mutations is EcoTILLING.
  • EcoTILLING is molecular technique that is similar to TILLING, except that its objective is to uncover natural variation in a given population as opposed to induced mutations. The first publication of the EcoTILLING method was described in Comai et al.2004.
  • Rapid high-throughput screening procedures allow the analysis of amplification products for identifying a mutation in the NRT2.3 promoter, and in particular in SEQ ID NO: 1 as compared to a corresponding non-mutagenised wild type plant. Once a mutation is identified, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with a mutation in the NRT2.3 promoter as described herein.
  • Plants obtained or obtainable by such method which carry a mutation in the endogenous NRT2.3 promoter, and in particular in SEQ ID NO: 1 or 9 or a variant thereof are also within the scope of the invention
  • aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and in a preferred embodiment exclude embodiments that are solely based on generating plants by traditional breeding methods.
  • a method of altering splicing of the NRT2.3 gene and/or increasing the relative expression of NRT2.3b to NRT2.3a and/or increasing the expression of NRT2.3b and/or decreasing the expression of NRT2.3a comprising introducing at least one mutation into a nucleic acid sequence encoding a NRT2.3 promoter, as described herein.
  • a method of increasing yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content of a plant comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a TATA-binding protein (TBP), preferably TBP2.1.
  • TBP TATA-binding protein
  • the nucleic acid sequence encodes a TBP2.1 protein as defined in SEQ ID NO: 32 or a functional variant thereof.
  • the TBP2.1 nucleic acid comprises or consists of SEQ ID NO: 33 or a functional variant thereof.
  • the nucleic acid sequence comprises a regulatory sequence operably linked to the nucleic acid sequence encoding a TBP.
  • transgenic plant characterised by an increase in NRT2.3b expression levels compared to a wild-type plant, where the plant expresses the above described nucleic acid construct.
  • a genetically altered plant, part thereof or plant cell characterised in that the plant has an increased relative expression of NRT2.3b to NRT2.3a compared to a wild-type or control plant.
  • said plant is characterised by an increase in NRT2.3b expression compared to a wild-type or control plant.
  • the plant is characterised by a decrease in NRT2.3a expression compared to a wild-type or control plant.
  • said increase or decrease may be at least 1-fold, 2-fold, 3-fold, -fold, 5-fold, 6-fold, 7- fold, 8-fold or 9-fold higher than the level of expression in a wild-type or control plant.
  • said increase or decrease may be by up to or more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a wild-type or control plant.
  • a genetically altered plant, part thereof or plant cell wherein said plant comprises at least one mutation, as described above, in the nucleic acid sequence upstream of the NRT2.3 gene.
  • the mutation is in the 5’UTR of the NRT2.3b gene.
  • the mutation is in the NRT2.3a promoter.
  • the genetically altered plant, part thereof or plant cell plant is characterised by an increase in at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content, as described above.
  • NUE nitrogen use efficiency
  • the plant may be produced by introducing a mutation, preferably a deletion, insertion and/or substitution into the NRT2.3b 5’UTR or NRT2.3a promoter sequence by any of the above described methods.
  • a mutation preferably a deletion, insertion and/or substitution into the NRT2.3b 5’UTR or NRT2.3a promoter sequence by any of the above described methods.
  • said mutation is introduced into a least one plant cell and a plant regenerated from the at least one mutated plant cell.
  • the method comprises introducing at least one mutation into the NRT2.3b 5’UTR or NRT2.3a promoter of preferably at least one plant cell using any mutagenesis technique described herein. Preferably said method further comprising regenerating a plant from the mutated plant cell.
  • the method may further comprise selecting one or more mutated plants, preferably for further propagation.
  • said selected plants comprise at least one mutation in the NRT2.3b 5’UTR or NRT2.3a promoter.
  • said plants are characterised by an increase in the relative expression of NRT2.3b to NRT2.3a, an increase in the level of NRT2.3b and/or a decrease in the expression of NRT2.3a, as described herein. Expression levels of the NRT2.3b 5’UTR or NRT2.3a promoter can be measured by any standard technique known to the skilled person.
  • the selected plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
  • a“genetically altered plant” or“mutant plant” is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant.
  • a mutant plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant using a mutagenesis method, such as any of the mutagenesis methods described herein.
  • the mutagenesis method is targeted genome modification or genome editing.
  • the plant genome has been altered compared to wild type sequences using a mutagenesis method.
  • Such plants have an altered phenotype as described herein, such as an increased yield, biomass, NUE, nitrogen transport and/or N content.
  • increased yield, biomass, NUE, nitrogen transport and/or N content is conferred by the presence of an altered plant genome, for example, a mutated NRT2.3b 5’UTR or NRT2.3a promoter sequence.
  • the endogenous promoter sequence is specifically targeted using targeted genome modification and the presence of a mutated gene or promoter sequence is not conferred by the presence of transgenes expressed in the plant.
  • the genetically altered plant can be described as transgene-free.
  • mutating the NRT2.3 promoter and in particular mutating the 5’UTR of the NRT2.3b gene (defined in SEQ ID NO: 1) alters splicing of the NRT2.3 gene, resulting in an increased expression or relative expression of NRT2.3b.
  • a mutated promoter operably linked to the NRT2.3 gene in a wild-type or control plant will also increase yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content.
  • NUE nitrogen use efficiency
  • a nucleic acid construct comprising a NRT2.3 promoter sequence operably linked to a NRT2.3 gene sequence, wherein the NRT2.3 promoter sequence is selected from the group comprising SEQ ID NO: 2, 3, 4 or 5 or a functional variant thereof.
  • a functional variant is defined above.
  • the NRT2.3 gene sequence comprises SEQ ID NO: 8 or a functional variant thereof.
  • operably linked refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
  • a host cell comprising the nucleic acid construct.
  • the host cell may be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell.
  • the invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described below.
  • transgenic plant expressing the nucleic acid construct as described above.
  • said nucleic acid construct is stably incorporated into the plant genome.
  • the nucleic acid sequence is introduced into said plant through a process called transformation.
  • Transformation of plants is now a routine technique in many species.
  • any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like.
  • Transgenic plants, including transgenic crop plants are preferably produced via Agrobacterium tumefaciens mediated transformation.
  • the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
  • a further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
  • the transformed plants are screened for the presence of a selectable marker such as the ones described above.
  • putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation.
  • expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
  • the various aspects of the invention described herein clearly extend to any plant cell or any plant produced, obtained or obtainable by any of the methods described herein, and to all plant parts and propagules thereof unless otherwise specified.
  • the present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
  • the invention relates to the use of a nucleic acid construct as described herein to increase at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content.
  • NUE nitrogen use efficiency
  • a method of increasing at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content comprising introducing and expressing in said plant the nucleic acid construct described herein.
  • a method of producing a plant with an increase in at least one of yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content comprising introducing and expressing in said plant the nucleic acid construct described herein.
  • Said increase is relative to a control or wild-type plant.
  • a plant according to the various aspects of the invention is preferably rice.
  • plant as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), tissues and organs, wherein each of the aforementioned comprise at least one mutation in a NRT2.3 promoter as described herein.
  • plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises least one mutation in a NRT2.3 promoter as described herein.
  • the invention also extends to harvestable parts of a plant of the invention as described herein, such as the grain.
  • the aspects of the invention also extend to products derived, preferably directly derived, from a harvestable part of such a plant, namely the grain, such as, but not limited to, rice bran, rice bran oil, rice flour, rice hulls, rice starch, ash from hulls, broken rice and brewers rice.
  • a product derived from a plant as described herein or from a part thereof is provided.
  • a control plant as used herein according to all of the aspects of the invention is a plant which has not been modified according to the methods of the invention. Accordingly, in one embodiment, the control plant does not have at least one mutation in a NRT2.3 promoter. In an alternative embodiment, the plant has not been genetically modified, as described above. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.
  • crRNA or CRISPR RNA is meant the sequence of RNA that contains the protospacer element and additional nucleotides that are complementary to the tracrRNA.
  • tracrRNA transactivating RNA
  • RNA transactivating RNA
  • protospacer element is meant the portion of crRNA (or sgRNA) that is complementary to the genomic DNA target sequence, usually around 20 nucleotides in length. This may also be known as a spacer or targeting sequence.
  • sgRNA single-guide RNA
  • sgRNA single-guide RNA
  • linker loop that links the tracrRNA and crRNA into a single molecule.
  • sgRNA may also be referred to as“gRNA” and in the present context, the terms are interchangeable.
  • the sgRNA or gRNA provide both targeting specificity and scaffolding/binding ability for a Cas nuclease.
  • a gRNA may refer to a dual RNA molecule comprising a crRNA molecule and a tracrRNA molecule.
  • TAL effector transcription activator-like (TAL) effector
  • TALE transcription activator-like (TAL) effector
  • genomic DNA target sequence a sequence within the NRT2.3 PROMOTER gene or promoter sequence
  • a TALE protein is composed of a central domain that is responsible for DNA binding, a nuclear-localisation signal and a domain that activates target gene transcription.
  • the DNA-binding domain consists of monomers and each monomer can bind one nucleotide in the target nucleotide sequence.
  • Monomers are tandem repeats of 33-35 amino acids, of which the two amino acids located at positions 12 and 13 are highly variable (repeat variable diresidue, RVD). It is the RVDs that are responsible for the recognition of a single specific nucleotide.
  • HD targets cytosine; Nl targets adenine, NG targets thymine and NN targets guanine (although NN can also bind to adenine with lower specificity).
  • nucleic acid construct wherein the nucleic acid construct encodes at least one DNA-binding domain, wherein the DNA- binding domain can bind to a sequence in the NRT2.3 promoter gene, wherein said sequence is selected from SEQ ID Nos 12 to 15 or a variant thereof, as defined herein.
  • said construct further comprises a nucleic acid encoding a (SSN) sequence-specific nuclease, such as Fokl or a Cas protein.
  • SSN sequence-specific nuclease
  • the nucleic acid construct encodes at least one protospacer element wherein the sequence of the protospacer element is selected from SEQ ID No 16 to 23 or a variant thereof.
  • the nucleic acid construct comprises a crRNA-encoding sequence.
  • a crRNA sequence may comprise the protospacer elements as defined above and preferably additional nucleotides that are complementary to the tracrRNA.
  • An appropriate sequence for the additional nucleotides will be known to the skilled person as these are defined by the choice of Cas protein.
  • the nucleic acid construct further comprises a tracrRNA sequence. Again, an appropriate tracrRNA sequence would be known to the skilled person as this sequence is defined by the choice of Cas protein. Nonetheless, in one embodiment said sequence comprises or consists of a sequence as defined in SEQ ID NO: 24 or a variant thereof.
  • the nucleic acid construct comprises at least one nucleic acid sequence that encodes a sgRNA (or gRNA).
  • sgRNA typically comprises a crRNA sequence, a tracrRNA sequence and preferably a sequence for a linker loop.
  • the nucleic acid construct comprises at least one nucleic acid sequence that encodes a sgRNA sequence as defined herein.
  • the nucleic acid construct may further comprise at least one nucleic acid sequence encoding an endoribonuclease cleavage site.
  • the endoribonuclease is Csy4 (also known as Cas6f).
  • the nucleic acid construct comprises multiple sgRNA nucleic acid sequences the construct may comprise the same number of endoribonuclease cleavage sites.
  • the cleavage site is 5’ of the sgRNA nucleic acid sequence. Accordingly, each sgRNA nucleic acid sequence is flanked by a endoribonuclease cleavage site.
  • variant refers to a nucleotide sequence where the nucleotides are substantially identical to one of the above sequences.
  • the variant may be achieved by modifications such as insertion, substitution or deletion of one or more nucleotides.
  • the variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of the above described sequences.
  • sequence identity is at least 90%.
  • sequence identity is 100%. Sequence identity can be determined by any one known sequence alignment program in the art.
  • the invention also relates to a nucleic acid construct comprising a nucleic acid sequence operably linked to a suitable plant promoter.
  • a suitable plant promoter may be a constitutive or strong promoter or may be a tissues-specific promoter.
  • suitable plant promoters are selected from, but not limited to, cestrum yellow leaf curling virus (CmYLCV) promoter or switchgrass ubiquitin 1 promoter (PvUbil) U6 RNA polymerase III (TaU6) CaMV35S, U6 or maize ubiquitin (e.g. Ubi1) promoters.
  • CmYLCV cestrum yellow leaf curling virus
  • PvUbil switchgrass ubiquitin 1 promoter
  • TaU6 switchgrass ubiquitin 1 promoter
  • CaMV35S RNA polymerase III
  • U6 or maize ubiquitin e.g. Ubi1 promoters.
  • expression can be specifically directed to particular tissues of rice seeds through gene expression-regulating sequences.
  • the promoter is selected from the U3 promoter (SEQ ID NO: 25), the U6a promoter (SEQ ID NO: 26), the U6b promoter (SEQ ID NO: 27), the U3b promoter in dicot plants (SEQ ID NO: 28) and the U6-1 promoter in dicot plants (SEQ ID NO: 29).
  • the nucleic acid construct of the present invention may also further comprise a nucleic acid sequence that encodes a CRISPR enzyme.
  • CRISPR enzyme is meant an RNA-guided DNA endonuclease that can associate with the CRISPR system. Specifically, such an enzyme binds to the tracrRNA sequence.
  • the CRIPSR enzyme is a Cas protein (“CRISPR associated protein), preferably Cas 9 or Cpf1 , more preferably Cas9.
  • Cas9 is codon-optimised Cas9, and more preferably, has the sequence described in SEQ ID NO: 30 or a functional variant or homolog thereof.
  • the CRISPR enzyme is a protein from the family of Class 2 candidate x proteins, such as C2c1 , C2C2 and/or C2c3.
  • the Cas protein is from Streptococcus pyogenes.
  • the Cas protein may be from any one of Staphylococcus aureus, Neisseria meningitides, Streptococcus thermophiies or Treponema denticola.
  • the term“functional variant” as used herein with reference to Cas9 refers to a variant Cas9 gene sequence or part of the gene sequence which retains the biological function of the full non-variant sequence, for example, acts as a DNA endonuclease, or recognition and/or binding to DNA.
  • a functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example non-conserved residues.
  • Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active.
  • a functional variant of SEQ ID No.30 has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the amino acid represented by SEQ ID NO: 30.
  • the Cas9 protein has been modified to improve activity. Suitable homologs or orthologs can be identified by sequence comparisons and identifications of conserved domains. The function of the homolog or ortholog can be identified as described herein and a skilled person would thus be able to confirm the function when expressed in a plant.
  • the Cas9 protein has been modified to improve activity.
  • the Cas9 protein may comprise the D10A amino acid substitution, this nickase cleaves only the DNA strand that is complementary to and recognized by the gRNA.
  • the Cas9 protein may alternatively or additionally comprise the H840A amino acid substitution, this nickase cleaves only the DNA strand that does not interact with the sRNA.
  • Cas9 may be used with a pair (i.e. two) sgRNA molecules (or a construct expressing such a pair) and as a result can cleave the target region on the opposite DNA strand, with the possibility of improving specificity by 100-1500 fold.
  • the cas9 protein may comprise a D1135E substitution.
  • the Cas 9 protein may also be the VQR variant.
  • the Cas protein may comprise a mutation in both nuclease domains, HNH and RuvC-like and therefore is catalytically inactive. Rather than cleaving the target strand, this catalytically inactive Cas protein can be used to prevent the transcription elongation process, leading to a loss of function of incompletely translated proteins when co-expressed with a sgRNA molecule.
  • An example of a catalytically inactive protein is dead Cas9 (dCas9) caused by a point mutation in RuvC and/or the HNH nuclease domains (Komor et al. , 2016 and Nishida et al., 2016).
  • a Cas protein such as Cas9 may be further fused with a repression effector, such as a histone-modifying/DNA methylation enzyme or a Cytidine deaminase ( Komor et al.2016) to effect site-directed mutagenesis.
  • a repression effector such as a histone-modifying/DNA methylation enzyme or a Cytidine deaminase ( Komor et al.2016) to effect site-directed mutagenesis.
  • the cytidine deaminase enzyme does not induce dsDNA breaks, but mediates the conversion of cytidine to uridine, thereby effecting a C to T (or G to A) substitution.
  • the nucleic acid construct comprises an endoribonuclease.
  • the endoribonuclease is Csy4 (also known as Cas6f) and more preferably a rice codon optimised csy4.
  • the nucleic acid construct may comprise sequences for the expression of an endoribonuclease, such as Csy4 expressed as a 5’ terminal P2A fusion (used as a self-cleaving peptide) to a cas protein, such as Cas9.
  • the cas protein, the endoribonuclease and/or the endoribonuclease-cas fusion sequence may be operably linked to a suitable plant promoter.
  • suitable plant promoters are already described above, but in one embodiment, may be the Zea Mays Ubiquitin 1 promoter.
  • Suitable methods for producing the CRISPR nucleic acids and vectors system are known, and for example are published in Molecular Plant (Ma et al., 2015, Molecular Plant, D0l:10.1016/j.molp.2015.04.007), which is incorporated herein by reference.
  • the nucleic acid construct comprises at least one nucleic acid sequence that encodes a TAL effector, wherein said effector targets a NRT2.3 promoter sequence selected from SEQ ID NO 12 to 15.
  • said nucleic acid construct comprises two nucleic acid sequences encoding a TAL effector, to produce a TALEN pair.
  • the nucleic acid construct further comprises a sequence-specific nuclease (SSN).
  • SSN sequence-specific nuclease
  • the TALENs are assembled by the Golden Gate cloning method in a single plasmid or nucleic acid construct.
  • a sgRNA molecule comprising a crRNA sequence and a tracrRNA sequence and wherein the crRNA sequence can bind to at least one sequence selected from SEQ ID Nos 12 to 15 or a variant thereof.
  • A“variant” is as defined herein.
  • the sgRNA molecule may comprise at least one chemical modification, for example that enhances its stability and/or binding affinity to the target sequence or the crRNA sequence to the tracrRNA sequence. Such modifications would be well known to the skilled person, and include for example, but not limited to, the modifications described in Rahdar et al., 2015, incorporated herein by reference.
  • the crRNA may comprise a phosphorothioate backbone modification, such as 2’-fluoro (2’-F), 2’-0- methyl (2’-0-Me) and S-constrained ethyl (cET) substitutions.
  • a phosphorothioate backbone modification such as 2’-fluoro (2’-F), 2’-0- methyl (2’-0-Me) and S-constrained ethyl (cET) substitutions.
  • Cas9 and sgRNA may be combined or in separate expression vectors (or nucleic acid constructs, such terms are used interchangeably).
  • an isolated plant cell is transfected with a single nucleic acid construct comprising both sgRNA and Cas9 as described in detail above.
  • an isolated plant cell is transfected with two nucleic acid constructs, a first nucleic acid construct comprising at least one sgRNA as defined above and a second nucleic acid construct comprising Cas9 or a functional variant or homolog thereof.
  • the second nucleic acid construct may be transfected below, after or concurrently with the first nucleic acid construct.
  • the advantage of a separate, second construct comprising a cas protein is that the nucleic acid construct encoding at least one sgRNA can be paired with any type of cas protein, as described herein, and therefore are not limited to a single cas function (as would be the case when both cas and sgRNA are encoded on the same nucleic acid construct).
  • the nucleic acid construct comprising a cas protein is transfected first and is stably incorporated into the genome, before the second transfection with a nucleic acid construct comprising at least one sgRNA nucleic acid.
  • a plant or part thereof or at least one isolated plant cell is transfected with mRNA encoding a cas protein and co-transfected with at least one nucleic acid construct as defined herein.
  • Cas9 expression vectors for use in the present invention can be constructed as described in the art.
  • the expression vector comprises a nucleic acid sequence as defined in SEQ ID NO: 30 or a functional variant or homolog thereof, wherein said nucleic acid sequence is operably linked to a suitable promoter.
  • suitable promoters include the Actin, CaMV35S, U6, U3 or maize ubiquitin (e.g. Ubi1) promoter.
  • CRISPR constructs nucleic acid constructs described above or the sgRNA molecules in any of the above described methods.
  • a method of modulating NRT2.3 promoter activity and/or NRT2.3 gene splicing and/or increasing expression of NRT2.3b comprising introducing and expressing a CRISPR construct as described above or introducing a sgRNA molecule as also described above into a plant.
  • a method of modulating NRT2.3 promoter activity and/or NRT2.3 gene splicing and/or increasing expression of NRT2.3b as described herein, wherein the method comprises introducing at least one mutation into the endogenous NRT2.3 promoter using CRISPR/Cas9, and specifically, the CRISPR constructs described herein.
  • an isolated plant cell transfected with at least one sgRNA molecule as described herein.
  • a genetically modified or edited plant comprising the transfected cell described herein.
  • the nucleic acid construct or constructs may be integrated in a stable form.
  • the nucleic acid construct or constructs are not integrated (i.e. are transiently expressed).
  • the genetically modified plant is free of any sgRNA and/or Cas protein nucleic acid. In other words, the plant is transgene free.
  • introduction encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
  • Plant tissue capable of subsequent clonal propagation may be transformed with a genetic construct of the present invention and a whole plant regenerated there from.
  • the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
  • Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
  • the resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
  • transformation Transformation of plants is now
  • Any of several transformation methods known to the skilled person may be used to introduce the nucleic acid construct or sgRNA molecule of interest into a suitable ancestor cell.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation.
  • Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant (microinjection), gene guns (or biolistic particle delivery systems (bioloistics)) as described in the examples, lipofection, transformation using viruses or pollen and microprojection.
  • Methods may be selected from the calcium/polyethylene glycol method for protoplasts, ultrasound-mediated gene transfection, optical or laser transfection, transfection using silicon carbide fibers, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like.
  • Transgenic plants can also be produced via Agrobacterium tumefaciens mediated transformation, including but not limited to using the floral dip/ Agrobacterium vacuum infiltration method as described in Clough & Bent (1998) and incorporated herein by reference.
  • At least one nucleic acid construct or sgRNA molecule as described herein can be introduced to at least one plant cell using any of the above described methods.
  • any of the nucleic acid constructs described herein may be first transcribed to form a preassembled Cas9- sgRNA ribonucleoprotein and then delivered to at least one plant cell using any of the above described methods, such as lipofection, electroporation or microinjection.
  • the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
  • the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
  • a further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
  • a suitable marker can be bar-phosphinothricin or PPT.
  • the transformed plants are screened for the presence of a selectable marker, such as, but not limited to, GFP, GUS (b-glucuronidase). Other examples would be readily known to the skilled person.
  • putatively transformed plants may also be evaluated, for instance using PCR to detect the presence of the gene of interest, copy number and/or genomic organisation.
  • integration and expression levels of the newly introduced DNA may be monitored using Southern, Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
  • the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
  • a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
  • the method also comprises the step of screening the genetically modified plant for SSN (preferably CRISPR)-induced mutations in the NRT2.3 promoter sequence.
  • the method comprises obtaining a DNA sample from a transformed plant and carrying out DNA amplification to detect a mutation in at least one NRT2.3 promoter sequence.
  • the methods comprise generating stable T2 plants preferably homozygous for the mutation (that is a mutation in at least one NRT2.3 promoter sequence).
  • Plants that have a mutation in at least one NRT2.3 promoter sequence can also be crossed with another plant also containing at least one mutation in at least one NRT2.3 promoter sequence to obtain plants with additional mutations in the NRT2.3 promoter sequence.
  • This method can be used to generate a T2 plants with mutations on all or an increased number of homoelogs, when compared to the number of homoeolog mutations in a single T 1 plant transformed as described above.
  • a genetically altered plant of the present invention may also be obtained by transference of any of the sequences of the invention by crossing, e.g., using pollen of the genetically altered plant described herein to pollinate a wild-type or control plant, or pollinating the gynoecia of plants described herein with other pollen that does not contain a mutation in at least one of the NRT2.3 promoter sequence.
  • the methods for obtaining the plant of the invention are not exclusively limited to those described in this paragraph; for example, genetic transformation of germ cells could be carried out as mentioned, but without having to regenerate a plant afterward. Method of screening plants for naturally occurring increased grain yield phenotypes
  • a method for screening a population of plants and identifying and/or selecting a plant that has an increased yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content is provided.
  • the method comprises detecting in the plant or plant germplasm at least one polymorphism in the NRT2.3 promoter and preferably at least one mutation in SEQ ID NO: 1 or 9 or a variant thereof.
  • said screening comprises determining the presence of at least one polymorphism, wherein said polymorphism is at least one insertion and/or at least one deletion and/or substitution.
  • the polymorphism is a deletion of at least one nucleotide in the NRT2.3 promoter, wherein preferably the NRT2.3 promoter comprises or consists of SEQ ID NO: 1.
  • the polymorphism is the deletion of at least one nucleotide from the 5’ end of SEQ ID NO: 1. More preferably, the polymorphism is the deletion of at least the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 nucleotides from the 5’ end of SEQ ID NO: 1.
  • the polymorphism is the deletion of at least the first 50, more preferably the first 60, and even more preferably, the first 62 nucleotides from the 5’ end of SEQ ID NO: 1.
  • the polymorphism is the deletion of the first 90, more preferably the first 100 and even more preferably the first 101 nucleotides from the 5’ end of SEQ ID NO:1.
  • the polymorphism is the deletion of either SEQ I D NO: 6 or 7 from SEQ I D NO: 1.
  • the polymorphism is a substitution of at least one nucleotide.
  • the polymorphism is the substitution of at least one nucleotide at position 160 of SEQ ID NO: 1 (this may also be referenced herein as being a polymorphism at position -83 with respect to the ATG start codon of the NRT2.3 gene), position 201 of SEQ ID NO: 1 (again this may be referred to herein as position -42) and position 222 of SEQ ID NO: 1 (again, this may be referred to herein as position -21).
  • the polymorphism is a substitution at position 160 of SEQ ID NO: 1.
  • the substitutions may be as follows:
  • the at least one polymorphism is a substitution, deletion and/or insertion of at least one nucleotide in the TATA box of the NRT2.3 promoter.
  • the TATA box is defined in SEQ ID NO: 9 or a variant thereof.
  • the polymorphism affects the binding of transcription factors or histones to the NRT2.3 promoter and therefore affects transcription of the NRT2.3 gene.
  • the at least one polymorphism affects the binding ability of TATA-binding proteins, such as TBP2.1 (which consequently increases the expression levels of NRT2.3b).
  • the polymorphism is a substitution of at least one nucleotide in the TATA box. Even more preferably, the polymorphism is a substitution at position 12 of SEQ ID NO: 9. In one embodiment, the substitution is a T to C substitution.
  • Suitable tests for assessing the presence of a polymorphism would be well known to the skilled person, and include but are not limited to, Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats (SSRs-which are also referred to as Microsatellites), and Single Nucleotide Polymorphisms (SNPs).
  • RFLPs Restriction Fragment Length Polymorphisms
  • RAPDs Randomly Amplified Polymorphic DNAs
  • AP-PCR Arbitrarily Primed Polymerase Chain Reaction
  • DAF Sequence Characterized Amplified Regions
  • AFLPs Am
  • the method comprises
  • the method may further comprise introgressing the chromosomal region comprising a NRT2.3 promoter sequence polymorphism into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.
  • said second plant will display an increase in yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or N content.
  • the OsNRT2.3 TIILLING lines, backcross lines and the transgenic lines were surface sterilized with 30% (v/v) NaCIO for 30 min and then rinsed thoroughly with water.
  • the seedlings were grown in the artificial climate chamber with a 16 hours light (30 ° C) and 8 hours dark (26 ° C).
  • the plants for field experiments were grown in plots at the Nanjing Agricultural University in Nanjing, Jiangsu.
  • the chemical properties of the soils in the plots is as Chen et al. described. (Chen et al., 2016)
  • the DNA samples were collected and stored at -80oC .
  • the DNA samples were extracted by the CTAB method.
  • the product sequences were determined from the company names Genescript®.
  • the sequences of the amplified TILLING lines were aligned with the sequence of Zhonghuo11 (WT) using DNAMAN.
  • the 437 bp ORF was linked with the sequence of ZIIIB ( cct gca ggt cgc cac att age aat gcc aca tta gca atg ccg act eta gag gat ccc) (SEQ ID NO: 31).
  • OsNRT2.3a and OsNRT2.3b were combined into one vector.
  • the 141 bp fragment of the OsNRT2.3 promoter was used to drive expression of OsNRT2.3a and eGFP and to drive expression of OsNRT2.3b and the mCherry reporter gene.
  • OsTBP2.1 In order to explore the function of the gene OsTBP2.1 with OsNRT2.3, we built a OsTBP2.1 overexpression vector (pUbi::OsTBP2.1), and obtained T-DNA mutation lines.
  • the yeast one-hybrid assay was performed according to the manufacturer’s protocol of the ‘Matchmaker Gold Yeast One-Hybrid Library Screening System User Manual’ (Clontech). Briefly, the vector of pTATA-box-pAbAi was transferred into the yeast strain and grown on medium lacking uracil (Ura). We used different concentrations of Aureobasidin A (AbA) to test the bait strain on medium lacking Ura. The vectors of pGADT7-TBP2, pGADT7-TBP2.1 and pGADT7-TBP2.2 were then transferred into the strains with pTATA-box-pAbAi. The strains were then grown on medium lacking leucine with 800 ng ml -1 AbA r .
  • the reverse transcription of total RNA to the cDNA was done by using a reverse transcription kit, and the synthesized cDNA was used as templates in the real time PCR reactions.
  • the templates were reacted three times with AceQ qPCR SYBR Green Master Mix kit on an Applied Biosystems (ABI) Plus Real-Time PCR System.
  • the specific OsNRT2.3b antibody was described in Yan et al (2011).
  • the total protein is extracted from -83 bp mutation backcross lines for SDS-PAGE. Then the protein is transferred to a PVDF membrane and incubated with OsHSP (1 :5000) and OsNRT2.3b (1 :2000) overnight at 4oC .
  • the PVDF membrane is then incubated with secondary antibody (1 :20000; Pierce). Bound antibody was detected with chemiluminescence (Yan et al., 2011 ; Tang et al., 2012).
  • the lines of WT, T11/WT(aa) and T12/WT(aa) were grown in IRRI nutrient solution for 3 weeks and then under N starvation for 3 days. Initially the plants were moved to 0.1 mM CaS0 4 for 1 minute, then they were separately moved to nutrient solutions containing 2.5 mM 15 NO 3 - and 1.25 mM NH4I5NO 3 for 5 minutes and then again moved to 0.1 mM CaS0 4 for 1 minute. All plants were placed in 105oC for 30 minutes to inactivate enzymes. The roots and shoots were separated. They were then oven dried in 70 ° C for 7 days and the dry samples grounded. About 1 mg of the powder of each sample was analyzed using the Isotope Ratio Mass Spectrometer system (Thermo Fisher Scientific). Influx of 15nO 3 - was calculated from the 15N concentrations of the roots and shoots.
  • the lines were placed in 105oC for 30 minutes to inactivate the enzymes.
  • the samples were oven dried in 75oC for 3 days and dry weights were recorded as biomass values.
  • the 1.5-kb promoter and -83 bp mutation on 1.5-kb promoter fragments of OsNRT2.3 were amplified from rice and inserted into the luciferase reporter.
  • the plasmids were transferred into rice protoplast together with pUbi::OsTBP2.1 , and harvested at 24-h.
  • the protoplasts were analyzed by using the kit Dual-Luciferase Reporter Assay System (Promega) to calculate the ratio of firefly luciferase (LUC) and Renilla (REN) luciferase.
  • the -83 bp region upstream from the translational start codon OsNRT2.3 is essential for rice development
  • the mutant lines T8, T11 , T12 and T20 were obtained using TILLING (Targeting Induced Local Lesions in Genomes) (Tsai et al., 2011). (Fig. 9, Fig. 1a). We extracted DNA from the lines to identify the place of mutation. The results revealed that T8, T11 and T12 all carry a mutation at -83 bp, upstream from the translational start codon OsNRT2.3. (Fig. 1 b) However the T20 line did not carry a mutation upstream from the translational start codon of gene OsNRT2.3. (Fig.1b). In the field, the grain yield, dry weight and NUE were all increased compared to WT and T20. (Fig. 1c, d).
  • OsNRT2.3a and OsNRT2.3b showed that OsNRT2.3a was downregulated in T8,T11 and T12 lines compared to WT and T20, but OsNRT2.3b was upregulated (Fig. 10a, b). Accordingly the mutant at -83 bp upstream from the ATG of OsNRT2.3 increases the ratio of OsNRT2.3b to OsNRT2.3a (Fig. 2e). Further, as shown in Figure 15, the content of 15 N accumulated in the roots (Fig. 15a) and shoots (Fig. 15b) was significantly greater in the T8, T11 and T12 lines compared to WT for all three types of 15 N sources studied. The T20 line didn’t show any significant difference compared to WT in the roots or shoots (Fig. 15a, b).
  • Figure 17 also shows the N content in backcross lines of -83 bp mutation at the mature stage.
  • the -83bp region upstream from the ATG of OsNRT2.3 can increase the ratio of OsNRT2.3b to OsNRT2.3a
  • the -83 bp mutation increases OsNRT2.3b protein levels and nitrogen transport efficiency
  • OsNRT2.3a is mainly responsible for the long-distance transport of nitrate, from root to shoot, and OsNRT2.3b mainly responsible in the shoot.
  • OsNRT2.3a When knocking out OsNRT2.3a, it will increase the accumulation of nitrate in the root, while overexpression of OsNRT2.3b will increase the absorption and transport of nitrate.
  • Figure 16 shows the influx of 15 NO 3 - and 15 NH 4 + in the OsNRT2.3 mutation backcross lines over a 5 minute period.
  • WT and the mutation backcross homozygous lines of T11 and T12 were grown in 1.25 mM NH 4 NO 3 for 3 weeks and nitrogen starved for 1 week.
  • 15 N influx rate was then measured at 2.5 mM 15 NO 3 - , 1.25 mM NH 4 15 NO 3 , and 1.25 mM 15 NH 4 NO 3 over a 5 minute period
  • b The shoot 15 N influx rate.
  • OsNRT2.3b only the shorter promoters - 141 bp and 180 bp, significantly increased expression of OsNRT2.3b compared with the other lines (Fig. 14c).
  • the proportion of OsNRT2.3b to OsNRT2.3a was also increased in the 141 bp and 180 bp promoters compared to other lines (Fig. 14d).
  • the expression ratio of OsNRT2.3b to OsNRT2.3a is strongly correlated promoter length.
  • OsTBP2.1 bound to cis-element TATA-box of OsNRT2.3
  • the level of luciferase expression was lower in the Pnrt2.3::Luc and pUbi: :TBP2.1 lines compared to the pmNRT2.3::Luc and pUbi: :TBP2.1 lines (Fig. 6c).
  • the lowest levels of luciferase expression was observed in the single transformed lines pNRT2.3::Luc and pmNRT2.3::Luc (Fig. 6c).
  • the protein content of OsNRT2.3a and OsNRT2.3b was increased compared to the lines of no mutant at different promoter lengths (Fig. 6e,f). Taken together these results suggest that the transcription factor, OsTBP2.1 , can increase the transcription of OsNRT2.3 to OsNRT2.3b.
  • OsTBP2.1 increases the ratio of OsNRT2.3b to OsNRT2.3a and affects the growth of rice
  • OsTBP2.1 In the OsTBP2.1 overexpression lines, the ratio of OsNRT2.3b to OsNRT2.3a was increased, but in ostbp2.1 T-DNA lines, the ratio decreased. (Fig. 7d). In summary, OsTBP2.1 can enhance the expression of OsNRT2.3b, and decrease the expression of OsNRT2.3a to affect rice development. When the TATA box is mutated to TACA, the binding ability of OsTBP2.1 was increased, resulting in higher expression levels of OsNRT2.3b.
  • the 5’UTR plays a regulatory role in RNA translation, RNA stability, and in RNA transcription.
  • the number and length of introns at the 5’UTR can influence the expression of genes, (Chung et al., 2006) as well as key cis-acting elements on promoters and on the 5’UTR containing the first intron. (Hernandez-Garcia & Finer, 2014; Gallegos & Rose, 2015).
  • the TATA-box mutant in the 5’UTR of OsNRT2.3b increased the ratio of OsNRT2.3b to OsNRT2.3a, resulting in improved yield and growth.
  • Floral dip a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6): 735-43.
  • Rice OsNAR2.1 interacts with OsNRT2.1 , OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges. Plant Cell and Environment 34:1360-1372.

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Abstract

La présente invention concerne des procédés pour augmenter le rendement en grains de riz et l'efficacité d'utilisation d'azote par augmentation de l'expression d'un gène transporteur de nitrate, ainsi que des plantes transgéniques exprimant une expression accrue du gène et des procédés de production de telles plantes.
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CN116391529B (zh) * 2023-04-24 2024-01-19 中国科学院南京土壤研究所 一种促进水稻根系发育的方法

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