CN113924367A

CN113924367A - Method for improving rice grain yield

Info

Publication number: CN113924367A
Application number: CN202080037035.6A
Authority: CN
Inventors: 范晓荣
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-05-23
Filing date: 2020-05-21
Publication date: 2022-01-11
Also published as: WO2020234426A1

Abstract

The present invention relates to methods of increasing rice grain yield and nitrogen use efficiency by increasing the expression of nitrate transporter genes, as well as transgenic plants expressing increased gene expression and methods of making such plants.

Description

Method for improving rice grain yield

Technical Field

The present invention relates to methods of increasing rice grain yield and nitrogen use efficiency by altering splicing of nitrate transporter genes, as well as genetically altered plants having increased yield and methods of making such plants.

Background

In the natural environment, the growth and development of plants are adversely affected by biotic and abiotic stresses. Environmental changes are also important factors affecting the normal growth of crops. Thus, plants have developed a number of unique mechanisms to cope with environmental fluctuations.

Rice is one of the most important major food products in the world and is consumed by more than 50% of the world population, especially asia (grain and agriculture organization (FAO), 2015). It is also one of the most nutritionally important food crops. However, in the field, rice may be affected by a number of adverse conditions, which can result in a significant loss of yield. One of the major limiting factors affecting yield is low nitrogen availability.

Nitrogen (N) is the basis for crop development because it is an essential component of many organic molecules, nucleic acids and proteins. Nitrogen nutrition affects all aspects of plant function, from metabolism to resource allocation, growth and development. The most abundant source of N available to plant roots is nitrate in naturally aerobic soils (NO3-), due to the strong nitrification of the applied organic and fertilizer N. In contrast, ammonium (NH4+) is the predominant form of N available in flooded rice fields due to anaerobic soil conditions (Sasakawa and Yamamoto, 1978).

Thus, the soil inorganic nitrogen (N) is mainly available to plants in the form of nitrate in aerobic high lands and well-drained soils, and in the form of ammonium in poorly-drained soil and flooded anaerobic rice fields. In many plants, nitrate obtained from roots is transported to the stem before being assimilated. In contrast, ammonium from nitrate reduction or directly from ammonium uptake is preferentially assimilated in the roots and then transported to the ground shoots in organic form. To cope with different concentrations of nitrate in the soil, 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 nitrate uptake. Constitutive hats (chats) and nitrate-inducible hats (ihats) take up nitrate in the external medium at low nitrate concentrations, with saturation in the range of 0.2-0.5 mM. In contrast, LATS plays a role in nitrate capture at higher external nitrate concentrations. The uptake of LATS and HATS is mediated by nitrate transporters belonging to the NRT1 and NRT2 families, respectively. Uptake in roots is regulated by negative feedback, linking the expression and activity of nitrate uptake to the N state of the plant (Miller et al, 2007).

Although higher plants have the ability to utilize organic nitrogen, the main source of nitrogen available to the root system is believed to be NO3^-And NH 4. The relative adaptability of plants to these two N sources varies widely. Although NH4 should be the preferred N source because of the energy ratio required for its metabolism to NO3^-Few, but only a few species actually perform well when NH4 is provided as the sole N source. The latter are northern conifers, ericaceae, some vegetable crops, and rice (Oryza sativa L.). In contrast to these species, most agricultural species sometimes have severe toxicity symptoms on NH4, and thus these species are seen in NO3^-The growth rate is faster. However, when both N sources are provided, this is not the case with NH4 or NO3 alone^-Growth and yield are often significantly improved compared to overgrowth (Kronzucker et al, 1999).

Rice differs from other crops in that it can grow entirely on NH4 as the sole nitrogen source. Rice is traditionally grown under flooded anaerobic soil conditions, where ammonium is the primary nitrogen source. However, the specialized aerated tissue cells of rice roots can transfer oxygen from the stem to the roots and release it to the rhizosphere where bacteria convert ammonium salts to nitrates (nitrification) can occur. Nitrification at the waterlogged rice rhizosphere can result in 25-40% of the total crop N being absorbed as nitrate, primarily through the High Affinity Transport System (HATS). The nitrate is absorbed by the proton (H)⁺) Mediated by co-transport of protons (H)⁺) Can pass through the plasma membrane H⁺The ATPase is excreted from the cells. The molecular mechanisms of nitrate uptake and transport in rice are not fully understood. Since nitrate concentrations in the rhizosphere of rice fields are estimated to be less than 10 μ M (Kirk and Kronzucker, 2005), NRT2 family members play a major role in the uptake of nitrate by rice (Araki and Hasegawa, 2006; Yan et al, 2011).Thus, up to 40% of the total N absorbed by the roots of rice grown under wetland conditions is likely to be present as nitrate and has an uptake rate comparable to that of ammonium (Kronzucker et al, 2000; Kirk and Kronzucker, 2005).

In the rice genome, five NRT2 genes have been identified (Araki and Hasegawa, 2006; Feng et al, 2011). Osnrt2.1 and osnrt2.2 have the same coding region sequence, but have different 5 '-and 3' -non-transcribed regions (UTRs), with high similarity to the NRT2 gene of other monocotyledons, whereas osnrt2.3 and osnrt2.4 are more closely related to the arabidopsis thaliana NRT2 gene. OsNRT2.3mRNA is actually spliced into two gene products, OsNRT2.3a (AK109776) and OsNRT2.3b (AK072215), whose putative amino acid sequences are 94.2% similar (Feng et al, 2011; Yan et al, 2011). Osnrt2.3a is expressed mainly in roots and this pattern is enhanced by nitrate supply, whereas osnrt2.3b is less expressed in roots and relatively abundant in shoots, with no effect on transcript numbers in N form and concentration (Feng et al, 2011, Feng 2012). Interestingly, overexpression of OsNRT2.3b has been shown to improve yield, growth and NUE in transgenic plants (Fan et al, 2016).

In summary, there remains a need to provide crop plants with more nutritionally efficient genotypes to ensure sustainable crop production for global food safety and to reduce the cost of mineral fertilizer input and negative environmental impacts, such as loss of air and water quality and biodiversity. The present invention addresses this need.

Disclosure of Invention

We describe the generation of rice osnrt2.3 mutant lines using target-induced local damage to the genome (TILLING). We identified that the resulting mutant lines shared a single mutation at position-83 upstream of the translation start codon of the osnrt2.3 gene. Interestingly, mutation of the NRT2.3 promoter at this position increased the relative expression of NRT2.3b and NRT2.3a, and in addition, significantly increased the growth, yield and Nitrogen Use Efficiency (NUE) of the mutant lines.

Cis-acting elements on promoters also play important regulatory roles in gene expression and transcriptional translation. Our detailed analysis shows that the TATA-box is a key cis-regulatory element for the transcription of OsNRT2.3 to OsNRT2.3a and OsNRT2.3b. In this study, we identified a TATA-box binding protein, OsTBP2.1, which binds to the TATA-box motif on the OsNRT2.3 promoter. The 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, thereby increasing both yield and NUE.

Therefore, the results revealed that the-83 bp region upstream of the translation initiation codon of the OsNRT2.3 gene is important for differential transcription of OsNRT2.3a and OsNRT2.3b and ultimately crop growth.

In one aspect of the present invention, there is provided a method for increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content in a plant, comprising introducing at least one mutation into a nucleic acid sequence encoding an NRT2.3 promoter. In a preferred embodiment, the plant is rice.

In a preferred embodiment, the nucleic acid sequence encoding the NRT2.3 promoter comprises SEQ ID NO 9 or a functional variant thereof. In a further preferred embodiment, the nucleic acid sequence encoding the NRT2.3 promoter comprises SEQ ID NO 1 or a functional variant thereof.

In one embodiment, mutagenesis is used to introduce mutations. In a preferred embodiment, the mutations are introduced using TILLING or T-DNA insertion. In an alternative embodiment, mutations are introduced using targeted genome modifications, preferably ZFNs, TALENs or CRISPR/Cas 9.

Preferably, 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 a substitution of at least one nucleotide.

The method of claim 10, wherein the substitution is at position 160, position 201, or position 222 of SEQ ID NO 1.

In one embodiment, the mutation is a substitution at position 160. Preferably, the mutation is a T to C substitution.

In another embodiment, the mutation is a deletion of at least one nucleotide. More preferably, the mutation is a deletion of at least 50, more preferably 60, nucleotides 5' of SEQ ID NO. 1. In another embodiment, the mutation is a deletion of at least 90, more preferably 100, nucleotides 5' of SEQ ID NO. 1.

In further embodiments, the method further comprises regenerating the plant and screening the plant for an increase in at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content.

In another aspect of the present invention, there is provided a genetically altered plant, a part of a plant cell thereof, wherein said plant comprises at least one mutation in at least one nucleic acid sequence encoding an NRT2.3 promoter.

In a preferred embodiment, the nucleic acid sequence comprises SEQ ID NO 9 or a functional variant thereof. In another embodiment, the nucleic acid sequence comprises SEQ ID NO 1 or a functional variant thereof.

In one embodiment, the plant is characterized by an increase in at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content.

In one embodiment, the mutation is introduced into SEQ ID NO.1, preferably into SEQ ID NO. 9. Preferably, the mutation is an insertion, deletion and/or substitution. In one embodiment, the mutation is a substitution of at least one nucleotide, and in one example, a substitution at position 160, position 201, or position 222 of SEQ ID No. 1. In another example, the mutation is a substitution at position 160. In one embodiment, the mutation is a T to C substitution.

In an alternative embodiment, the mutation is a deletion of at least one nucleotide. In one embodiment, the mutation is a deletion of at least 50, more preferably 60, nucleotides 5' of SEQ ID NO. 1.

Preferably, the genetically altered plant is rice.

In another aspect of the present invention, there is provided a method of identifying and/or selecting for plants having or to have increased yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content, preferably as compared to a control or wild type plant, comprising detecting at least one polymorphism in the NRT2.3 promoter gene sequence in a plant or plant germplasm and selecting for said plant or progeny thereof.

In one embodiment, the NRT2.3 promoter gene sequence comprises SEQ ID NO 9, and more preferably SEQ ID NO 1 or a functional variant thereof.

In one embodiment, the polymorphism is at least one substitution at least position 160 of SEQ ID NO. 1. In an alternative embodiment, the polymorphism is a deletion of at least one 5 'nucleotide of SEQ ID NO.1, more preferably of at least the first 60' nucleotide of SEQ ID NO. 1.

In another embodiment, the method further comprises introgressing the chromosomal region comprising the at least one polymorphism in the NRT2.3 promoter into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.

In another aspect of the present invention, there is provided a method of increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content in a plant, the method comprising introducing and expressing in said plant a nucleic acid construct comprising an NRT2.3 promoter sequence operably linked to an 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.

In another aspect, there is provided a method for producing a plant with increased yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content, the method comprising introducing and expressing in a plant or plant cell a nucleic acid construct comprising an NRT2.3 promoter sequence operably linked to an 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.

Preferably, the NRT2.3 gene sequence comprises SEQ ID NO 8 or a functional variant thereof.

In a preferred embodiment, the plant is rice.

In another aspect of the invention, there is provided a plant obtained or obtainable by any of the methods described above. In a preferred embodiment, the plant is rice.

In another aspect of the invention, a nucleic acid construct is provided comprising an NRT2.3 promoter sequence operably linked to an 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 functional variants thereof. In one embodiment, the NRT2.3 gene sequence comprises SEQ ID NO 8 or a functional variant thereof.

In another aspect, there is provided a vector comprising the above-described nucleic acid construct. Also provided are host cells comprising the vectors or nucleic acid constructs. Finally, transgenic plants expressing the vector or nucleic acid construct are also provided. Preferably, the plant is rice.

In another aspect of the present invention, there is provided the use of a vector or nucleic acid construct for increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content in a plant.

In yet another aspect of the invention, there is provided a method of altering splicing of an NRT2.3 gene, the method comprising introducing at least one mutation into a nucleic acid sequence encoding an NRT2.3 promoter.

In another aspect of the invention, there is provided a 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.

In one embodiment, the nucleic acid sequence encodes at least one protospacer element, wherein the sequence of the protospacer element is selected from SEQ ID NOs 16 to 23 or sequences at least 90% identical to SEQ ID NOs 16 to 23.

In a further embodiment, the construct further comprises a nucleic acid sequence encoding CRISPR RNA (crRNA) sequence, wherein the crRNA sequence comprises the pro-spacer element sequence and additional nucleotides.

Preferably, 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, the construct encodes at least one single guide rna (sgRNA), wherein the sgRNA comprises a tracrRNA sequence and a crRNA sequence.

In a preferred embodiment, the construct is operably linked to a promoter. More preferably, the promoter is a constitutive promoter.

In one embodiment, the nucleic acid construct further comprises a nucleic acid sequence encoding a CRISPR enzyme. Preferably, the CRISPR enzyme is a Cas protein or a Cpf1 protein. In one embodiment, the Cas protein is Cas9 or a functional variant thereof.

In alternative embodiments, the nucleic acid construct encodes a TAL effector. In this embodiment, the nucleic acid construct further comprises a sequence encoding an endonuclease or a DNA cleavage domain thereof. In one embodiment, the endonuclease is Fokl.

In another aspect of the invention, there is provided an isolated plant cell transfected with at least one nucleic acid construct as described above. In an alternative aspect, an isolated plant cell is provided, which cell is transfected with a first nucleic acid construct comprising at least one sgRNA as described above and a second nucleic acid construct, wherein the second nucleic acid construct comprises a nucleic acid sequence encoding a Cas protein, preferably a Cas9 protein or a functional variant thereof. Preferably, the second nucleic acid construct is transfected before, after or simultaneously with the first nucleic acid construct.

In another aspect of the invention, a genetically modified plant is provided, wherein the plant comprises a transfected cell as described above. In one embodiment, the nucleic acid encoding the sgRNA and/or the nucleic acid encoding the Cas protein are integrated in a stable form.

In another aspect of the present invention, there is provided a method of increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content in a plant, the method comprising introducing and expressing in the plant a nucleic acid construct as described above, wherein preferably the increase is relative to a control or wild type plant. Also provided are plants obtained or obtainable by the above methods.

In another aspect, there is provided a use of a nucleic acid construct as described above for increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content in a plant.

In a final aspect of the invention, there is provided a method of obtaining a genetically modified plant as defined above, the method comprising:

a. selecting a part of a plant;

b. transfecting at least one cell of the plant part of paragraph (a) with the nucleic acid construct;

c. regenerating at least one plant derived from the transfected cell or cells;

selecting one or more plants obtained according to paragraph (c) that show increased expression of nrt 2.3b.

In any of the aspects of the invention described above, the increase is relative to a control or wild type plant.

Drawings

The invention is further described in the following non-limiting figures:

FIG. 1 shows biomass, NUE and nitrogen content of field maturity-83 bp mutant lines. (a) Characterization of OsNRT2.3 mutant-83 bp mutant line in field experiments. Wild type, chinese 11(WT), mutant lines of T8, T11, T12 and T20. (bar 30 cm); (b) -nucleotide sequence of an 83bp mutant line; (c) grain yield and dry weight per plant of field grown mutant lines and WT plants. The average dry weight represents aboveground biomass, excluding grain yield; (d) NUE of the mutant line; (e) the total N concentration of shoots and roots in the OsNRT2.3TILLING line and (f) the total N content of shoots and roots in the OsNRT2.3TILLING line. Error line: SE (n ═ 5). Significant differences between mutant and WT are indicated by different letters. (P <0.05, student's t-test).

FIG. 2 shows the characterization and identification of the-83 bp mutant line in hydroponics. (a) Phenotype of WT and-83 bp mutant lines. (10 cm in length); (b) -dry weight of 83bp mutant line; (c) total N concentration of stems and roots; (d) total nitrogen content of stems and roots. Error line: SE (n ═ 6). Seedlings of wild type china 11(WT) and mutant lines were grown for 2 weeks in a solution of IRRI containing 1.25mm NH4NO 3. Then extracting RNA for analyzing OsNRT2.3a/b expression; (e) qRT-PCR analysis of OsNRT2.3b to OsNRT2.3a expression ratio. Error line: SE (n ═ 3). Different letters indicate significant differences between transgenic lines and WT (P <0.05, student t-test).

FIG. 3 shows the effect of OsNRT2.3 promoter mutations on OsNRT2.3a/b expression; (a, c) representation of the OsNRT2.3 promoter and different mutation sites altering expression of OsNRT2.3a and OsNRT2.3b. P is a WT promoter of OsNRT2.3, P1 is a-665 bp mutation in the OsNRT2.3 promoter, P2 is a-44 bp mutation in the OsNRT2.3 promoter, and mP is a-83 bp mutation in the OsNRT2.3 promoter; (b) the expression of OsNRT2.3b in different promoter systems promotes OsNRT2.3b; (d) expression of OsNRT2.3b and OsNRT2.3a in the mp: OsNRT2.3 line; (e) mp: the ratio of OsNRT2.3b to OsNRT2.3a in OsNRT2.3a.

FIG. 4 shows that the-83 bp mutation alters the translational pattern of OsNRT2.3 in the backcross line; (a, B) characterization of the OsNRT2.3 mutant B1F2 line in field trials. B1F2 of two independent lines T11 and T12 demonstrated the-83 bp mutant hyper-phenotype. (bar 30cm) AA as control, -83bp no mutation; aa is homozygous-83 bp mutation; aa is heterozygous-83 bp mutation; (c) western blot analysis of osnrt2.3b and HSP expression in leaves and roots. B1F 3T 11 and B1F 3T 12 are B1F3 generations of the osnrt2.3 mutant backcross line. Mutant backcross clones of WT and T11 and T12 were grown in 1.25mM NH4NO3 for 3 weeks and nitrogen starved for 1 week. Then the 15N influx rates of 2.5mM 15NO 3-, 1.25mM NH415NO3, and 1.25mM 15NH4NO3 were measured over 5 minutes; (d) stem 15N influx rate; (e) root 15N inflow rate; (f) 15N ratio of stem to root. Error line: SE (n ═ 5). Different letters indicate significant differences between transgenic lines and WT. (P <0.05, student's t-test).

FIG. 5 shows the effect of-83 bp mutations of promoters of different lengths on OsNRT2.3 transcription in rice; (a) schematic representation of OsNRT2.3 promoter fragments and-83 bp mutant fragments of different lengths, which drive the expression of the 437bp ORF and ZIIIB reporter genes of OsNRT2.3. 141bp and 697bp are the original OsNRT2.3 promoter; 141M and 697M carry a-83 bp mutation; (b-d) transgenic rice seedlings of the control line (NO mutation) and the mutant line were grown for 2 weeks in a solution containing 1.25mm NH4NO3 IRRI. RNA was then extracted to analyze OsNRT2.3a/b expression. (b) Shows the expression of OsNRT2.3a in transgenic lines; (c) shows the expression of OsNRT2.3b in transgenic lines; (d) expression ratios of OsNRT2.3b/OsNRT2.3a in transgenic lines are shown. Error line: SE (n ═ 3). Different letters indicate significant differences between transgenic lines and WT. (P <0.05, student's t-test).

Fig. 6 shows that ostnp2.1 binds to the osnrt2.3 promoter fragment and activates osnrt2.3 expression. (a) OsTBP2.1 binds to the TATA-box of OsNRT2.3b 5' UTR. Yeast cells were co-transformed with pTATA-cassette: AbAi and OsTBP2/2.1/2.2: pGADT 7. Cells were grown on medium to screen for interactions (SD, -Ura, -Leu) and (800 nM). AbA was used to inhibit background growth. (b) Constructs for transient assays of rice protoplasts. The OsNRT2.3 promoter or the-83 bp mutant promoter is used to drive the expression of the reporter. pNRT2.3: Luc, and pmNRT2.3: Luc. (c) OsTBP2.1 activates OsNRT2.3 promoter. Expression of OsTBP2.1 is driven by the Ubi promoter. The reporter and effector were co-transformed into rice protoplasts. (d) A stable rice genetic vector constructs the framework. Reporter proteins eGFP and mCherry are constructed in one vector, and 141bp and 697bp promoters promote the expression of eGFP and mCherry. (e) The level of eGFP. (f) Level of mCherry. Error line: SE (n ═ 3). (P <0.05, student's t-test).

FIG. 7 shows the expression of OsTBP2.1 and OsNRT2.3a/b in OsTBP2.1 overexpression and T-DNA mutant lines. (a) Characterization of OsTBP2.1 overexpression in field trials. Wild type, Wuyunjing27 (WT-W27). (b) Characterization of the OsTBP2.1T-DNA mutant line in field experiments. Wild type, Huang (WT-HY). (bar 20 cm). (c) Expression of OsTBP2.1 in OEOsTBP2.1 and OsTBP2.1 lines. (d) OsTBP2.1 overexpression and the ratio of OsNRT2.3b to OsNRT2.3a in T-DNA mutation lines.

FIG. 8 shows a schematic representation of the OsNRT2.3 transcription model when TATA-box is mutated. The data show that, when mutated, ostbp2.1 enhances the expression of osnrt2.3b, thereby altering the ratio of osnrt2.3b to osnrt2.3a and resulting in higher levels of osnrt2.3b translation.

Fig. 9 shows characteristics of the field TILLING lines.

FIG. 10 shows the expression of OsNRT2.3a and OsNRT2.3b in a-83 bp mutant line.

FIG. 11 shows the identification of a-83 bp mutant backcross line.

FIG. 12 shows yield, dry weight and nitrogen use efficiency of the-83 bp mutant backcross line at the maturation stage.

FIG. 13 shows the effect of different OsNRT2.3 promoter lengths on OsNRT2.3a/b expression in rice.

FIG. 14 shows the expression of OsNRT2.3a and OsNRT2.3b in OEOsTBP2.1 and OsTBP2.1 lines.

FIG. 15 shows¹⁵NO3^-And¹⁵NH4⁺the osnrt2.3 mutant line was flowed in within 5 minutes. WT and OsNRT2.3 mutant seedlings were grown at 1.25mM NH₄NO₃Medium growth for 3 weeks and nitrogen starvation for 1 week. Then 2.5mM was measured in 5 minutes¹⁵NO3^–、1.25mM NH₄ ¹⁵NO₃And 1.25mM NH₄ ¹⁵NO₃Is/are as follows¹⁵And (N) inflow rate. (a) Root of a tree¹⁵And (N) inflow rate. (b) Stem¹⁵And (N) inflow rate. Error line: SE (n ═ 5). Different letters indicate significant differences (P) between transgenic lines and WT<0.05, one-way analysis of variance).

FIG. 16 shows¹⁵NO3^-And¹⁵NH4⁺osnrt2.3 mutant backcross lines were flowed in 5 min. Mutant backcross homozygous lines for WT and T11 and T12 at 1.25mM NH₄NO₃Medium growth for 3 weeks and nitrogen starvation for 1 week. Then 2.5mM was measured in 5 minutes¹⁵NO3^–、1.25mM NH₄ ¹⁵NO₃And 1.25mM NH₄ ¹⁵NO₃Is/are as follows¹⁵And (N) inflow rate. (a) Root of a tree¹⁵And (N) inflow rate. (b) Stem¹⁵And (N) inflow rate. Error line: SE (n ═ 5). Different letters indicate significant differences between transgenic lines and WT (P)<0.05, one-way analysis of variance).

FIG. 17 shows nitrogen content in mature-83 bp mutant backcross lines. Nitrogen content of mutant backcross lines and control leaves, leaf sheaths, stems and ears.

Detailed Description

The rice transport protein OsNRT2.3 has two splicing forms, OsNRT2.3a and OsNRT2.3b. Some nitrate transporters require two genes to function; the second smaller module (OsNAR21) is required for proper targeting of the transporter to the plasma membrane. One of the two forms of stitching osnrt2.3a requires the second component to function, while the other form of osnrt2.3b does not. We have previously demonstrated that overexpression of osnrt2.3b in rice can improve growth and NUE.

We have now surprisingly shown that a mutation in the nucleic acid sequence 5' upstream of the NRT2.3 gene, i.e. upstream of the ATG start codon of the NRT2.3 gene, affects the splicing of the gene, resulting in an increased expression ratio of osnrt2.3b to osnrt 2.3a. Furthermore, by targeting the local lesions induced in the genome, we obtained a number of lines carrying mutations at the-83 bp position of the osnrt2.3 gene (relative to the ATG start codon of the NRT2.3 gene). Compared with wild type, the mutation of the sequence at the upstream of the position changes the transcription of OsNRT2.3 gene and increases the ratio of OsNRT2.3b to OsNRT2.3a. In addition, the backcross mutant increased total biomass by about 28% and NUE by about 75% compared to field control plants. The weight per ear was also increased by about 60% in the field backcrossed mutant lines. At the same time, the backcross mutant line further improved nitrogen uptake in the field compared to the control.

Therefore, we concluded that a mutation at-83 in the sequence upstream of the osnrt2.3 gene is critical for controlling splicing of the osnrt2.3 gene, resulting in increased relative expression of osnrt2.3b, with positive effects on rice growth, yield and NUE.

The invention will now be further described. In the following paragraphs, the different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are within the skill of the art. These techniques are explained fully in the literature.

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), naturally occurring, mutant, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It may be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, antisense sequences, and non-coding regulatory sequences that do not encode mRNA or protein products. These terms also encompass genes. The term "gene" or "gene sequence" is used broadly to refer to a DNA nucleic acid that is associated with a biological function. Thus, a gene may include introns and exons as in genomic sequence, or may comprise only cDNA coding sequences, and/or may include cDNA in combination with regulatory sequences.

Aspects of the present invention relate to recombinant DNA technology and exclude embodiments based solely on the generation of plants by traditional breeding methods and the obtainment of plants by traditional breeding methods.

Methods for increasing yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content

In a first aspect of the present invention, there is provided a method for increasing yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content in a plant, the method comprising introducing at least one mutation into the nucleic acid sequence upstream of the NRT2.3 gene.

In one embodiment, the method comprises introducing a mutation in at least one nucleotide of 224 nucleotides upstream (e.g., in the 5' direction) of the ATG initiation codon of the NRT2.3 gene. Preferably, the NRT2.3 gene is defined in SEQ ID NO 8 or a variant thereof.

As used herein, the terms "increase", "improve" or "enhance" as used in accordance with various aspects of the present invention are interchangeable. In one embodiment, yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content may be increased by at least 5% -50% or more, e.g., by up to or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% as compared to control plants.

The term "yield" generally refers to a measurable product of economic value, typically associated with a particular crop, area, and period of time. Individual plant parts directly affect yield according to their number, size and/or weight. Actual yield is the yield per square meter for the crop and year, which is determined by dividing the total yield (including harvest and evaluation yields) by the square meter of planting.

The term "increased yield" as defined herein may be considered to include any one or at least one of the following, and may be measured by assessing one or more of the following: (a) increased biomass (weight) of one or more parts of the 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 include one or more of increased seed biomass (weight) per plant or individual basis, (c) increased seed filling, (d) increased number of filled seeds, (e) increased harvest index, which may be expressed as a ratio of yield of harvestable parts (e.g. seeds) to total biomass, (f) increased viability/germination efficiency, (g) increased number or size or weight of seeds or pods or legumes or grains, (h) increased seed volume (which may be the result of changes in composition (i.e. lipid (also referred to herein as oil)), total protein and carbohydrate content and composition), (i) increased (individual or average) seed area, (j) increased (individual or average) seed length, (k) increased (individual or average) seed circumference, (l) increased growth or increased branching, e.g. inflorescences with more branches, (m) increased fresh weight or grain filling, (n) increased ear weight, (o) increased Thousand Kernel Weight (TKW), which may be derived from the number of filled seeds and their total weight, and which may be the result of an increase in seed size and/or seed weight, (p) decreased number of sterile tillers per plant and (q) stronger or stronger stalks or stems. All parameters are relative to wild type or control plants.

In one embodiment, the increase in yield comprises an increase in plant weight, preferably dry weight (g/plant). In another alternative or additional embodiment, the increase in yield comprises an increase in weight per ear. In one embodiment, the weight per ear is increased by at least or up to 40%, more preferably 50%, even more preferably 60% compared to control plants.

In one example, 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% as compared to a control or wild type plant. Alternatively, yield may be increased by 20-70%, more preferably 25-75% compared to control plants.

The term "nitrogen utilization efficiency" or NUE can be defined as crop yield (e.g., grain yield). Alternatively, NUE can be defined as agricultural NUE, meaning grain yield/N. The overall N utilization efficiency of the plant includes absorption and utilization efficiency and can be calculated as UpE. In one embodiment, the NUE is increased by 5% -80% or more as compared to a control plant, e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% as compared to a control plant. In preferred embodiments, the NUE is increased by at least 60%, more preferably 70%, even more preferably 75% as compared to a control or wild type plant.

As used herein, the term "nitrogen transport" encompasses nitrogen acquisition or nitrogen influx or absorption. In one embodiment, nitrogen absorption may refer to absorption of ammonium, nitrate, and/or ammonium nitrate. In one embodiment, nitrogen influx in the stem and/or root of the plant is increased. Such an increase is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70% compared to a control or wild type plant.

In one embodiment, the nitrogen content in the stem and/or root of the plant is increased. In further embodiments, the nitrogen content in at least one of the leaf, leaf sheath, stem and ear is increased. In a further embodiment, the total nitrogen content in the plant is not increased.

By "at least one mutation" is meant that when the NRT2.3 promoter gene is present in more than one copy or homologue (having the same or slightly different sequence), there is at least one mutation in at least one gene. Preferably, all genes are mutated.

As shown in FIG. 5a, the NRT2.3a gene contains a 43bp 5'UTR (untranslated region), while the NRT2.3b gene contains a 223bp 5' UTR.

In one embodiment, the method comprises introducing at least one mutation into the preferably endogenous NRT2.3 promoter. As used herein, "promoter" encompasses a nucleic acid sequence (start or initiation codon) 1.5kbp upstream of the ATG. As used herein, the term "promoter" includes the 5' UTR (untranslated region) of the nrt2.3a and nrt2.3b genes. In one embodiment, the mutation is in the nrt2.3a promoter. In an alternative embodiment, the mutation is located in the 5' UTR of the nrt2.3b gene.

In another embodiment, 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.

In a further preferred embodiment, the mutation affects splicing of the NRT2.3 gene, in particular the mutation increases the relative expression of NRT2.3b and NRT 2.3a. The increase in relative expression is at least 2-fold, more preferably at least 4-fold, more preferably at least 6-fold, even more preferably at least 8-fold compared to wild type plants. In an alternative embodiment, the mutation increases expression of nrt2.3b by at least 5-fold, more preferably 6-fold, even more preferably 7-fold, compared to the expression level in wild type plants. In a further preferred embodiment, the mutation is in SEQ ID NO 1 or a variant thereof. In one embodiment, expression of nrt2.3b in the shoot of the plant is increased and/or expression of nrt2.3a in the root of the plant is decreased.

In a further embodiment, the mutation is in the TATA-box, more preferably in SEQ ID NO 9 or a functional variant thereof.

In the above embodiments, "endogenous" nucleic acid may refer to a native or native sequence in the genome of a plant. In one embodiment, 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 one of SEQ ID NOs 1 to 8 refers to a variant gene sequence or a portion of the gene sequence that retains the biological function of the entire non-variant sequence. In the context of SEQ ID No.1 to 4, this may mean that the sequence is capable of initiating or otherwise causing transcription of the NRT2.3 gene.

Functional variants (or "variants" — such terms are used interchangeably) also include variants of the target gene that have sequence changes that do not affect function, for example in non-conserved residues. Also included are variants that are substantially identical compared to the wild-type sequences set forth herein, i.e., have only some sequence variation, e.g., in non-conserved residues, and are biologically active. Each of the modifications proposed is within the routine skill in the art, as is the determination of retention of biological activity of the encoded product.

As used in any aspect of the invention described herein, a "variant" or "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%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% >, and a non-variant nucleic acid or amino acid sequence, 95%, 96%, 97%, 98% or at least 99% overall sequence identity.

Two nucleic acid sequences are said to be "identical" if the nucleotide sequences in the two sequences are identical at the maximum correspondence alignment described below. The term "identical" or "percent identity" in the context of two or more nucleic acids refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When sequences differ in conservative substitutions, the percentage of sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. For sequence comparison, typically one sequence acts as a reference sequence, which is compared to the test sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Non-limiting examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.

In a further embodiment, a variant as used herein may comprise a nucleic acid sequence encoding an NRT2.3 promoter as defined herein, which is capable of hybridising to a nucleic acid sequence as defined by SEQ ID No.1 under stringent conditions as defined herein.

"stringent conditions" or "stringent hybridization conditions" refer to conditions under which a probe hybridizes to its target sequence to a detectably greater degree (e.g., at least 2-fold over background) than to other sequences. Stringent conditions depend on the sequence and may be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified that are 100% complementary to the probe (homologous probing).

Alternatively, stringency conditions can be adjusted to allow for some mismatches in the sequence, so that a lower degree of similarity is detected (heterologous probing). Typically, stringent conditions are those at a pH of 7.0 to 8.3 with a salt concentration of less than about 1.5M Na ion, typically about 0.01 to 1.0M Na ion concentration (or other salt) and a temperature of at least about 30 ℃ for short probes (e.g., 10 to 50 nucleotides) and at least about 60 ℃ for long probes (e.g., greater than 50 nucleotides). The duration of hybridization is generally less than about 24 hours, usually about 4 to 12 hours. Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide.

In one embodiment, the mutation introduced into its NRT2.3 promoter may be selected from the following types of mutations:

1. "insertion mutations" of one or more nucleotides;

2. "deletion mutations" of one or more nucleotides;

a "substitution mutation" resulting in the substitution of at least one nucleotide by at least one different nucleotide.

An "inverted" mutation, i.e., one hundred and eighty degrees of rotation of a nucleic acid sequence.

In one embodiment, 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. In a preferred embodiment, the mutation is the deletion of at least one nucleotide from the 5' end of SEQ ID NO. 1. More preferably, the mutation is a deletion of at least the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 nucleotides of the 5' end of SEQ ID NO. 1. In a more preferred embodiment, the mutation is a deletion of at least the first 50, more preferably the first 60, and even more preferably the first 62 nucleotides of the 5' end of SEQ ID NO. 1. In an alternative embodiment, the mutation is a deletion of the first 90, more preferably the first 100, and even more preferably the first 101 nucleotides of the 5' end of SEQ ID NO. 1. In an alternative embodiment, the mutation is a deletion of

SEQ ID NO

6 or 7 in SEQ ID NO 1.

In a further embodiment, the mutation is a substitution of at least one nucleotide at position 160 of SEQ ID NO:1 (which may also be referred to herein as a mutation at position-83 relative to the ATG start codon of the NRT2.3 gene), position 201 of SEQ ID NO:1 (which may also be referred to herein as position-42), and/or position 222 of SEQ ID NO:1 (which may also be referred to herein as position-21). At position-83 (relative to the ATG start codon), there are two putative motifs, named OSE2ROOTNODULE (-82bp to-86 bp) (SEQ ID NO:10) and ASF1MOTIFCAMV (-76bp to-83 bp) (SEQ ID NO: 11). We believe that the OSE2 rootonduplex motif may control the tillering process, and the ASF1MOTIFCAMV motif is an inhibitor binding motif. It is likely that loss of the gene repressor binding function of this motif in the mutant lines results in a change in the expression ratio observed in these lines.

In a further preferred embodiment, the substitutions may be as follows:

position 160 (of SEQ ID NO: 1): t to C;

position 201: A to C (of SEQ ID NO: 1);

position 222 (of SEQ ID NO: 1): t to C.

In a most preferred embodiment, the mutation is a T to C substitution at position 160 of SEQ ID NO. 1.

In a preferred embodiment, 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. In one embodiment, the TATA-box is defined in SEQ ID NO 9 or a variant thereof. Thus, in one embodiment, the mutation affects the binding of a transcription factor or histone to the NRT2.3 promoter and thus affects the transcription of the NRT2.3 gene. In a preferred embodiment, the mutation alters the binding capacity of a TATA-box binding factor, such as TBP2.1, which also affects the expression of the NRT2.3 gene.

In another embodiment, the mutation is a substitution, deletion and/or deletion of at least one nucleotide in the OSE2 rootonduplex motif and/or the ASF1MOTIFCAMV motif. Preferably, the mutation results in the loss of function or partial loss of one or both motifs. As described above, we believe that the OSE2 rootodule motif may be involved in the control of tillering. We also believe that the ASF1MOTIFCAMV motif is a repressor binding motif. In one embodiment, the mutation is in the ASF1MOTIFCAMV motif and prevents or reduces its ability to inhibit binding. 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. Variants are defined herein.

In a preferred embodiment, 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 are also included, such as deletion of promoter or enhancer functional regions, as these affect splicing of the NRT2 gene. For example, mutations may result in the deletion of the TATA-box. In other words, the deletion of SEQ ID NO 9.

In one embodiment, mutations are introduced using mutagenesis or targeted genome editing. That is, in one embodiment, the present invention relates to methods and plants produced by the above-described genetic engineering methods, 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. To achieve efficient genome editing by introducing site-specific DNA DSBs, four broad classes of customizable DNA binding proteins can be used: meganucleases derived from microbial mobile genetic elements, eukaryotic transcription factor-based ZF nucleases, transcription activator-like effectors (TALEs) from xanthomonas bacteria, and RNA-guided DNA endonucleases Cas9 from type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats). Meganucleases, ZF and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate a nuclease and a DNA binding domain, ZF and TALE proteins consist of separate modules that target 3 or 1 nucleotide (nt) of DNA, respectively. ZFs and TALEs can be assembled in the desired combination and ligated to the nuclease domain of fokl to direct the nucleolytic activity to a specific genomic site.

After delivery to the host cell by the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in the host gene promoter and activate transcription. Their targeting specificity is determined by the central domain of 33-35 amino acid repeats in tandem. Followed by a single truncated repeat of 20 amino acids. Most of the naturally occurring TAL effectors examined had 12 to 27 complete repeats.

These repeats are distinguished only by two adjacent amino acids, their Repeat Variable Diresidue (RVD). RVD determines which mononucleotide the TAL effector will recognize: one RVD corresponds to one nucleotide and each of the four most common RVDs preferentially binds to one of the four bases. The naturally occurring recognition site is preceded by a T required for TAL effector activity. TAL effectors can be fused to the catalytic domain of FokI nucleases to create TAL effector nucleases (TALENs) to generate targeted DNA Double Strand Breaks (DSBs) in vivo for genome editing. The use of this technique in genome editing is well described in the art, for example in US8,440,431, US8,440,432 and US8,450,471. Cerak T et al describe a set of custom plasmids that can be used with the Golden Gate cloning method to assemble multiple DNA fragments. As described therein, the Golden Gate method uses a type IIS restriction endonuclease that cleaves outside of its recognition site to create a unique 4bp overhang. Cloning can be accelerated by digestion and ligation in the same reaction mixture, since the enzyme recognition sites are eliminated by correct assembly. The assembly of a custom TALEN or TAL effector construct comprises two steps: (i) assembling the repeating modules into an intermediate array of 1-10 repeats and (ii) ligating the intermediate array into a scaffold to make a final construct. Thus, TAL effectors targeting NRT2.3 promoter sequences as described herein can be designed using techniques known in the art.

Another genome editing method that can be used according to various aspects of the invention is CRISPR. The use of this technique in genome editing is also well described in the art, for example in US8,697,359 and the references cited therein. Briefly, CRISPR is a microbial nuclease system, a bacteriophage and plasmid involved in defense against invasion. A CRISPR locus in a microbial host comprises a combination of a CRISPR-associated (Cas) gene and a non-coding RNA element capable of programming CRISPR-mediated nucleic acid cleavage (sgRNA) specificity. Three types (I-III) of CRISPR systems have been identified in a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of a series of repeated sequences (direct repeats) separated by a short non-repeated sequence (spacer). The non-coding CRISPR array is transcribed and cleaved in direct repeats into short crrnas comprising a single spacer sequence that directs the Cas nuclease to a target site (protospacer). Type II CRISPR is one of the most well characterized systems that target DNA double strand breaks in four sequential steps. First, two non-coding RNAs, pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, the tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates the processing of the pre-crRNA into mature crRNA comprising a single spacer sequence. Third, the mature crRNA tracrRNA complex directs Cas9 to the target DNA through watson-crick base pairing between a spacer on the crRNA and a protospacer on the target DNA next to a Protospacer Adjacent Motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of the target DNA, creating a double-stranded break in the protospacer.

Compared with traditional gene targeting and other programmable endonucleases, one main advantage of the CRISPR-Cas9 system is that it is easy to multiplex, and multiple genes can be mutated simultaneously by using only multiple sgrnas, each targeting a different gene. Furthermore, if two sgrnas are used flanking the genomic region, the middle part may be deleted or inverted.

Thus, Cas9 is a marker protein for type II CRISPR-Cas system and is a large monomeric DNA nuclease, guided by a complex of two non-coding RNAs to a DNA target sequence adjacent to a PAM (protospacer adjacent motif) sequence motif: CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves complementary DNA strands, while the RuvC-like domain cleaves non-complementary strands, thus introducing blunt cleavage in the target DNA. Heterologous expression of Cas9 with sgrnas can introduce site-specific Double Strand Breaks (DSBs) into genomic DNA of living cells from various organisms. For use in eukaryotes, a codon optimized version of Cas9, originally from the bacterium streptococcus pyogenes, has been used.

Single guide rna (sgrna) is the second component of the CRISPR/Cas system, which forms a complex with Cas9 nuclease. sgRNA is a synthetic RNA chimera produced by fusing crRNA to tracrRNA. The sgRNA guide sequence at its 5' end confers DNA target specificity. Thus, by modifying the guide sequence, sgrnas with different target specificities can be created. The guide sequence has a standard length of 20 bp. In plants, sgrnas have been expressed using plant RNA polymerase III promoters, e.g., U6 and U3. Accordingly, sgRNA molecules targeting NRT2.3 promoter sequences as described herein can be designed using techniques known in the art.

The Cas9 expression plasmid used in the methods of the invention can be constructed as described in the art.

Alternatively, at least one mutation may be introduced into the NRT2.3 promoter sequence using more conventional mutagenesis methods. These methods include physical mutagenesis and chemical mutagenesis. The skilled person will know that other methods may be used to generate such mutants, and methods for mutagenesis and polynucleotide alteration are well known in the art. See, e.g., Kunkel (1985) Proc. Natl. Acad. Sci. USA82: 488-Asan 492; kunkel et al, (1987) Methods in enzymol.154: 367-; U.S. Pat. nos. 4,873,192; walker and Gaastra, eds (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein.

In one embodiment, insertional mutagenesis, for example using T-DNA mutagenesis (which inserts T-DNA fragments from the agrobacterium tumefaciens T-plasmid into DNA resulting in loss of gene function or gain of gene function mutation), site-directed nucleases (SDNs) or transposons as mutagens. Insertional mutagenesis is another method of disrupting gene function, and is based on The insertion of foreign DNA into a gene of interest (see Krysan et al, The Plant Cell, Vol. 11, 2283-2290, 12 months 1999). Thus, in one embodiment, T-DNA is used as an insertional mutagen to disrupt the activity of the NRT2.3 promoter. In one example, the 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 serves as a marker for the subsequent identification of mutations. Since the sequence of the inserted element is known, various cloning or PCR-based strategies can be used to recover the inserted gene. Insertion of a T-DNA of the order of 5 to 25kb in length usually disrupts gene function. If enough T-DNA transformants are generated, it is likely that transgenic plants carrying the T-DNA insertion will be found in any gene of interest. Transformation of spores with T-DNA is accomplished by Agrobacterium-mediated methods, which involve exposing plant cells and tissues to a suspension of Agrobacterium cells.

The details of this method are well known to the skilled person. Briefly, transformation of plants by Agrobacterium results in the integration of a sequence called T-DNA, carried by a bacterial plasmid, into the nuclear genome. The use of T-DNA transformation results in a stable single insertion. Further mutation analysis of the resulting transformed lines is simple and each individual insertion line can be rapidly characterized by direct sequencing and analysis of the DNA flanking the insertion. The gene expression of nrt2.3b or the relative expression of nrt2.3a and nrt2.3b in the mutant was compared with that in wild type plants. Phenotypic analysis was also performed.

In another embodiment, the mutagenesis is physical mutagenesis, for example, application of ultraviolet radiation, X-rays, gamma rays, fast or thermal neutrons or protons. The target population can then be screened to identify mutants having a mutation in the NRT2.3 promoter, preferably a mutation in SEQ ID No.1 or variants thereof.

In another embodiment of the various aspects of the invention, the method comprises mutagenizing a population of plants with a mutagen. The mutagen may be a fast neutron radiation or a chemical mutagen, for example selected from the following non-limiting list: ethyl Methanesulfonate (EMS), Methyl Methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), triethylmelamine (1' EM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, mechlorethamine, vincristine, dimethylnitrosamine, N-methyl-N' -nitro-nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7, 12-dimethylbenzene (a) anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, disulfane, diepoxyalkane (diepoxyoctane (DEO), diepoxybutane (BEB), etc.), 2-methoxy-6-chloro-9 [3- (ethyl-2-chloroethyl) aminopropanamino ] acridine dihydrochloride (ICR-170), or formaldehyde. Again, the target population may then be screened to identify NRT2.3 promoter mutants.

In another embodiment, the method used to generate and analyze mutations is to target induced local lesions in the genome (TILLING), reviewed in Henikoff et al, 2004. In this method, seeds are mutagenized with a chemical mutagen, such as EMS. The resulting M1 plants were self-fertilized, and M2 generation individuals were used to prepare DNA samples for mutation screening. DNA samples were pooled and arrayed on microtiter plates and subjected to gene-specific PCR. Any method of identifying heteroduplexes between wild-type and mutant genes can be used to screen PCR amplification products for NRT2.3 promoter mutations. Such as, but not limited to, denaturing high pressure liquid chromatography (dHPLC), Constant Denaturing Capillary Electrophoresis (CDCE), Temperature Gradient Capillary Electrophoresis (TGCE), or fragmentation by using chemical cleavage. Preferably, the PCR amplification product is incubated with an endonuclease that preferentially cleaves mismatches in the heteroduplex between wild type and mutant sequences. The cleavage products were electrophoresed using an automated sequencing gel apparatus and the gel images were analyzed with the aid of standard commercial image processing procedures. Any primer specific for the NRT2.3 promoter sequence can be used to amplify the NRT2.3 promoter sequence in the pooled DNA samples. Preferably, the primers are designed to amplify the NRT2.3 promoter region where useful mutations are most likely to occur, particularly in the highly conserved and/or activity-conferring NRT2.3 promoter region, as described elsewhere. To facilitate detection of the PCR product on the gel, the PCR primers can be labeled using any conventional labeling method. In an alternative embodiment, the method used to generate and analyze the mutations is EcoTILLING. EcoTILLING is a molecular technique similar to TILLING, except that it aims to reveal natural variations of a given population, rather than to induce mutations. The first disclosure of EcoTILLING method is described in Comai et al 2004.

The rapid high-throughput screening procedure allows analysis of the amplification products to identify mutations in the NRT2.3 promoter, in particular in SEQ ID No.1, compared to corresponding non-mutagenized wild-type plants. Once a mutation is identified, seeds of M2 plants carrying the mutation are grown into adult M3 plants and screened for phenotypic characteristics associated with the mutation in the NRT2.3 promoter described herein.

Plants which carry mutations in the endogenous NRT2.3 promoter, in particular in SEQ ID NO 1 or 9 or variants thereof, or which can be obtained by such methods are also within the scope of the present invention.

Thus, aspects of the invention relate to targeted mutagenesis methods, particularly genome editing, and in preferred embodiments exclude embodiments based solely on the generation of plants by traditional breeding methods.

In another aspect of the invention, there is provided 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, the method comprising introducing at least one mutation into a nucleic acid sequence encoding the NRT2.3 promoter, as described herein.

In another aspect of the present invention, there is provided a method for increasing yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content in a plant, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a TATA Binding Protein (TBP), preferably TBP 2.1. In one embodiment, the nucleic acid sequence encodes the TBP2.1 protein as defined in SEQ ID NO:32 or a functional variant thereof. In a further embodiment, the TBP2.1 nucleic acid comprises or consists of SEQ ID NO 33 or a functional variant thereof. In another embodiment, the nucleic acid sequence comprises a regulatory sequence operably linked to a nucleic acid sequence encoding a TBP. The invention also includes a transgenic plant characterized by an increased expression level of nrt2.3b as compared to a wild type plant, wherein the plant expresses the above nucleic acid construct.

Genetically altered or modified plants and methods for producing such plants

In another aspect of the present invention, there is provided a genetically altered plant, part thereof or plant cell, characterized in that the plant has increased relative expression of nrt2.3b to nrt2.3a as compared to a wild type or control plant. In an alternative embodiment, the plant is characterized by increased expression of nrt2.3b as compared to a wild type or control plant. Alternatively, the plant is characterized by reduced nrt2.3a expression as compared to a wild type or control plant. Preferably, the increase or decrease may be 1-fold, 2-fold, 3-fold, 5-fold, 6-fold, 7-fold, 8-fold or 9-fold greater than the expression level in a wild type or control plant. Alternatively, the increase or decrease may be up to or greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% as compared to the level of wild type or control plant.

In another aspect of the present invention, there is provided a genetically altered plant, part thereof or plant cell, wherein said plant comprises at least one mutation as described above in a nucleic acid sequence upstream of the NRT2.3 gene. Preferably, the mutation is in the 5' UTR of the nrt2.3b gene. Alternatively, the mutation is in the nrt2.3a promoter.

In one embodiment, the genetically altered plant, part thereof, or plant cell plant is characterized by an increase in at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content, as described above.

Plants may be produced by introducing a mutation, preferably a deletion, insertion and/or substitution, in the nrt2.3b 5' UTR or nrt2.3a promoter sequence by any of the methods described above. Preferably, the mutation is introduced into at least one plant cell and a plant regenerated from the at least one mutated plant cell.

In another aspect of the invention, a method for producing a genetically altered plant as described herein is provided. In one embodiment, the method comprises introducing at least one mutation into the nrt2.3b 5' UTR or nrt2.3a promoter, preferably of at least one plant cell, using any of the mutagenesis techniques described herein. Preferably, the method further comprises regenerating a plant from the mutated plant cell.

The method may further comprise selecting one or more mutant plants, preferably for further propagation. Preferably, said selected plant comprises at least one mutation in the nrt2.3b 5' UTR or nrt2.3a promoter. In one embodiment, the plant is characterized by increased relative expression of nrt2.3b to nrt2.3a, increased levels of nrt2.3b, and/or decreased expression of nrt2.3a, as described herein. The expression level 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 can be propagated in a variety of ways, for example by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants can be selfed and homozygous second generation (or T2) transformants selected, and the T2 plants can then be further propagated by classical breeding techniques. The transformed organisms produced may take a variety of forms. For example, they may be chimeras of transformed and non-transformed cells; cloning transformants (e.g., all cells are transformed to contain the expression cassette); grafts of transformed and untransformed tissue (e.g., in plants, a transformed rootstock is grafted onto an untransformed scion).

In another aspect of the present invention, there is provided a plant obtained or obtainable by the above method.

For the purposes of the present invention, a "genetically altered plant" or "mutant plant" is a plant which has been genetically altered in comparison with naturally occurring wild-type (WT) plants. In one embodiment, a mutant plant is a plant that has been altered compared to a naturally occurring Wild Type (WT) plant using a mutagenesis method, e.g., any of the mutagenesis methods described herein. In one embodiment, the mutagenesis method is targeted genome modification or genome editing. In one embodiment, the plant genome has been altered using mutagenesis methods as compared to the wild type sequence. Such plants have altered phenotypes as described herein, such as increased yield, biomass, NUE, nitrogen transport, and/or nitrogen content. Thus, in this example, increased yield, biomass, NUE, nitrogen transport, and/or nitrogen content is conferred by the presence of an altered plant genome, e.g., a mutated nrt2.3b 5' UTR or nrt2.3a promoter sequence. In one embodiment, the endogenous promoter sequence is specifically targeted using targeted genomic modifications, and the presence of the mutant gene or promoter sequence is not conferred by the presence of a transgene expressed in the plant. In other words, a genetically altered plant can be described as not containing a transgene.

Transgenic plants

As discussed throughout, the present inventors have surprisingly identified that a mutant NRT2.3 promoter, and in particular a mutant NRT2.3b gene (defined in SEQ ID NO: 1) 5' UTR, alters splicing of the NRT2.3 gene, resulting in increased expression or relative expression of NRT2.3 b.

Thus, overexpression of a mutant promoter operably linked to the NRT2.3 gene will also increase yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content in wild type or control plants.

Thus, in a further aspect of the invention there is provided a nucleic acid construct comprising an NRT2.3 promoter sequence operably linked to an 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. Functional variants are defined above.

In one embodiment, the NRT2.3 gene sequence comprises SEQ ID NO 8 or a functional variant thereof.

As used herein, the term "operably linked" refers to a functional linkage between a promoter sequence and a gene of interest such that the promoter sequence is capable of initiating transcription of the gene of interest.

In another aspect of the invention, there is provided a vector comprising the nucleic acid sequence described above.

In another aspect of the invention, a host cell comprising the nucleic acid construct is provided. 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 a kit comprising a culture medium and an isolated host cell, as described below.

In another embodiment, transgenic plants expressing the above nucleic acid constructs are provided. In one embodiment, the nucleic acid construct is stably integrated into the plant genome.

The nucleic acid sequence is introduced into the plant by a process known as transformation.

Plant transformation is now a routine technique for many species. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable progenitor cell. The described methods for transforming and regenerating plants from plant tissues or plant cells can be used for transient or stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, direct injection of DNA into plants, particle gun bombardment, transformation with viruses or pollen, and microprojection. The method may be selected from the group consisting of calcium/polyethylene glycol methods for protoplasts, electroporation of protoplasts, microinjection into plant material, bombardment of DNA or RNA-coated particles, infection with (non-integrating) viruses, and the like. Transgenic plants, including transgenic crop plants, are preferably produced by agrobacterium tumefaciens-mediated transformation.

For the selection of transformed plants, the plant material obtained in the transformation is usually subjected to selective conditions in order to distinguish the transformed plants from the untransformed plants. For example, the seeds obtained in the above-described manner may be planted and suitably selected by spraying after the initial growth period. Another possibility consists in growing the seeds on agar plates, if appropriate after sterilization, using suitable selection agents, so that only the transformed seeds can grow into plants. Alternatively, transformed plants are screened for the presence of a selectable marker, such as one of those described above. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for example using Southern analysis, to determine the presence of the gene of interest, copy number and/or genomic tissue. Alternatively or additionally, expression levels of newly introduced DNA can be monitored using Northern and/or Western analysis, both techniques being well known to those of ordinary skill in the art.

The resulting transformed plants can be propagated in a variety of ways, for example, by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants can be selfed and homozygous second generation (or T2) transformants selected, and the T2 plants can then be further propagated by classical breeding techniques. The transformed organisms produced may take a variety of forms. For example, they may be chimeras of transformed and non-transformed cells; cloning transformants (e.g., all cells are transformed to contain the expression cassette); grafts of transformed and untransformed tissue (e.g., in plants, a transformed rootstock is grafted onto an untransformed scion).

Unless otherwise indicated, the various aspects of the invention described herein clearly extend to any plant cell or any plant produced, obtained or obtainable by any method described herein, as well as to all plant parts and propagules thereof. The invention further extends to progeny comprising a primary transformed or transfected cell, tissue, organ or whole plant produced by any of the above methods, the only requirement being that the progeny exhibit the same genotypic and/or phenotypic characteristics as those produced by the parents in the method according to the invention.

In another aspect, the invention relates to the use of a nucleic acid construct as described herein for increasing at least one of yield, biomass, Nitrogen Utilization Efficiency (NUE), nitrogen transport, and/or nitrogen content.

In another aspect of the present invention, there is provided a method of increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content, comprising introducing and expressing in said plant a nucleic acid construct as described herein.

In another aspect of the present invention, there is provided a method of producing a plant having an increase in at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content, comprising introducing and expressing in said plant a nucleic acid construct as described herein.

The increase is relative to a control or wild type plant.

The plant according to the various aspects of the present invention is preferably rice.

As used herein, the term "plant" encompasses whole plants, ancestors and progeny of plants, and plant parts, including seeds, stems, leaves, roots (including tubers), tissues, and organs, each of which includes a mutation in at least one NRT2.3 promoter as described herein. The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, wherein each of the foregoing also comprises at least one mutation of the NRT2.3 promoter as described herein.

The invention also extends to harvestable parts, such as grain, of a plant of the invention as described herein. Aspects of the invention also extend to products derived, preferably directly derived, from harvestable parts of such plants, i.e. cereals, such as, but not limited to, rice bran, rice oil, rice flour, rice hulls, rice starch, hull ash, broken rice and beer rice. In another aspect of the invention, there is provided a product derived from a plant or part thereof as described herein.

In a most preferred aspect of the invention, there is provided a seed produced by a genetically altered plant as described herein.

According to all aspects of the invention, a control plant as used herein is a plant that has not been modified according to the method of the invention. Thus, in one embodiment, the control plant does not have at least one mutation in the NRT2.3 promoter. In an alternative embodiment, the plant is free of genetic modifications 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 with the same genetic background as the modified plant.

Genome editing constructs for use in methods of targeted genome modification described herein

"crRNA" or CRISPR RNA means an RNA sequence containing a protospacer element and additional nucleotides complementary to a tracrRNA.

By "tracrRNA" (transactivating RNA) is meant an RNA sequence that hybridizes to crRNA and binds a CRISPR enzyme, e.g., Cas9, thereby activating the nuclease complex to introduce a double-strand break at a specific site within the genomic sequence of at least one NRT2.3 promoter nucleic acid or promoter sequence.

By "protospacer element" is meant the portion of crRNA (or sgRNA) that is complementary to a genomic DNA target sequence, typically about 20 nucleotides in length. This may also be referred to as a spacer or targeting sequence.

By "sgRNA" (single guide RNA) is meant a combination of tracrRNA and crRNA in a single RNA molecule, preferably further comprising a linker loop (which links the tracrRNA and crRNA into a single molecule). "sgRNA" may also be referred to as "gRNA" and in this context, these terms are interchangeable. sgrnas or grnas provide targeting specificity and scaffold/binding ability for Cas nucleases. A gRNA may refer to a double RNA molecule comprising a crRNA molecule and a tracrRNA molecule.

By "TAL effector" (transcription activator-like (TAL) effector) or TALE is meant a cleavage domain that can bind to a genomic DNA target sequence (a sequence within the NRT2.3 promoter gene or promoter sequence) and can be fused to an endonuclease, such as fokl, to produce a TAL effector nuclease or TALENS or meganuclease to produce megaTAL. TALE proteins consist of a central domain responsible for DNA binding, a nuclear localization signal, and a domain that activates transcription of a target gene. The DNA binding domain consists of monomers, each of which can bind to one nucleotide in the target nucleotide sequence. The monomer is a tandem repeat of 33-35 amino acids, where the two amino acids at positions 12 and 13 are highly variable (repeat variable diresidue, RVD). RVDs are responsible for identifying a single specific nucleotide. HD-targeted cytosine; NI targets adenine, NG targets thymine, NN targets guanine (although NN can also bind to adenine, but is less specific).

In another aspect of the invention, there is provided a nucleic acid construct, wherein said nucleic acid construct encodes at least one DNA binding domain, wherein said 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 variants thereof, as defined herein. In one embodiment, the construct further comprises a nucleic acid encoding a (SSN) sequence-specific nuclease, such as FokI or Cas protein.

In one embodiment, the nucleic acid construct encodes at least one protospacer element, wherein the sequence of the protospacer element is selected from SEQ ID NOs 16 to 23 or variants thereof.

In a further embodiment, the nucleic acid construct comprises a crRNA coding sequence. As defined above, the crRNA sequence may comprise a protospacer element as defined above and preferably comprises additional nucleotides complementary to the tracrRNA. The skilled person will know the appropriate sequences of additional nucleotides as these are defined by the choice of Cas protein.

In another embodiment, the nucleic acid construct further comprises a tracrRNA sequence. Again, suitable tracrRNA sequences are known to the skilled person, as the sequence is defined by the selection of the Cas protein. Nevertheless, in one embodiment, the sequence comprises or consists of a sequence as defined in SEQ ID NO.24 or a variant thereof.

In a further embodiment, the nucleic acid construct comprises at least one nucleic acid sequence encoding a sgRNA (or gRNA). Again, as already discussed, the sgRNA typically comprises a crRNA sequence, a tracrRNA sequence and preferably a sequence for a linker loop. In a preferred embodiment, the nucleic acid construct comprises at least one nucleic acid sequence encoding a sgRNA sequence as defined herein.

In further embodiments, the nucleic acid construct may further comprise at least one nucleic acid sequence encoding an endoribonuclease cleavage site. Preferably, the endoribonuclease is Csy4 (also known as Cas6 f). When the nucleic acid construct comprises multiple sgRNA nucleic acid sequences, the construct can comprise the same number of endoribonuclease cleavage sites. In another embodiment, the cleavage site is 5' of the sgRNA nucleic acid sequence. Thus, each sgRNA nucleic acid sequence is flanked by endoribonuclease cleavage sites.

The term "variant" refers to a nucleotide sequence in which the nucleotides are substantially identical to one of the sequences described above. Variants may be achieved by modification, for example, by insertion, substitution or deletion of one or more nucleotides. In preferred embodiments, 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 of the above sequences. In one embodiment, the sequence identity is at least 90%. In another embodiment, the sequence identity is 100%. Sequence identity can be determined by any sequence alignment program known in the art.

The invention also relates to nucleic acid constructs comprising a nucleic acid sequence operably linked to a suitable plant promoter. Suitable plant promoters may be constitutive or strong promoters, or may be tissue-specific promoters. In one embodiment, a suitable plant promoter is selected from, but not limited to, the nocturia yellow leaf curl virus (CmYLCV) promoter or switchgrass ubiquitin 1 promoter (PvUbi1) U6 RNA polymerase III (TaU6) CaMV35S, U6 or maize ubiquitin (e.g., Ubi1) promoter. Alternatively, expression may be directed specifically to a particular tissue of rice seeds by gene expression regulatory sequences. In one embodiment, the promoter is selected from the group consisting of the U3 promoter (SEQ ID NO:25) in dicots, the U6a promoter (SEQ ID NO:26), the U6b promoter (SEQ ID NO:27), the U3b promoter (SEQ ID NO:28), and the U6-1 promoter (SEQ ID NO:29) in dicots.

The nucleic acid construct of the invention may further comprise a nucleic acid sequence encoding a CRISPR enzyme. By "CRISPR enzyme" is meant an RNA-guided DNA endonuclease that can bind to a CRISPR system. In particular, this enzyme binds to the tracrRNA sequence. In one embodiment, the CRIPSR enzyme is a Cas protein ("CRISPR-associated protein"), preferably Cas9 or Cpf1, more preferably Cas 9. In a specific embodiment, Cas9 is a codon optimized Cas9 and, more preferably, has the sequence depicted in SEQ ID No.30 or a functional variant or homolog thereof. In another embodiment, the CRISPR enzyme is a protein from the class 2 candidate x protein family, e.g. C2C1, C2C2 and/or C2C 3. In one embodiment, the Cas protein is from Streptococcus pyogenes (Streptococcus pyogenes). In alternative embodiments, the Cas protein may be from any one of Staphylococcus aureus (Staphylococcus aureus), Neisseria meningitidis (Neisseria meningitidis), Streptococcus thermophilus (Streptococcus thermophiles), or Treponema dentata (Treponema dentata).

The term "functional variant" as used herein with respect to Cas9 refers to a variant Cas9 gene sequence or a portion of a gene sequence that retains the biological function of the intact non-variant sequence, e.g., functions as a DNA endonuclease, or recognizes and/or binds to DNA. Functional variants also include variants of the target gene that have sequence changes that do not affect function, such as non-conserved residues. Also included are variants that are substantially identical compared to the wild-type sequences set forth herein, i.e., have only some sequence variation, e.g., in non-conserved residues, and are biologically active. In one embodiment, the 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. In further embodiments, the Cas9 protein has been modified to increase activity.

Suitable homologues or orthologues may be identified by sequence comparison and identification of conserved domains. The function of a homologue or orthologue may be identified as described herein and the skilled person is thus able to confirm the function when expressed in a plant.

In further embodiments, the Cas9 protein has been modified to increase activity. For example, in one embodiment, the Cas9 protein may include a D10A amino acid substitution that cleaves only the DNA strand that is complementary to and recognized by the gRNA. In alternative embodiments, the Cas9 protein may alternatively or additionally comprise H840A amino acid substitutions that cleave only DNA strands that do not interact with sRNA. In this embodiment, Cas9 can be used with a pair (i.e., two) of sgRNA molecules (or constructs expressing such a pair), and thus can cleave target regions on opposite DNA strands, potentially increasing specificity by 100-fold over 1500-fold. In further embodiments, the cas9 protein may comprise a D1135E substitution. The Cas9 protein may also be a VQR variant. Alternatively, the Cas protein may contain mutations in both the HNH and RuvC-like nuclease domains and thus be catalytically inactive. This catalytically inactive Cas protein can be used to prevent the process of transcriptional extension, rather than cleavage of the target strand, which when co-expressed with the sgRNA molecule, would result in loss of function of the incompletely translated protein. An example of a catalytically inactive protein is killed Cas9(dCas9) caused by point mutations in RuvC and/or HNH nuclease domains (Komor et al, 2016 and Nishida et al, 2016).

In further embodiments, a Cas protein, such as Cas9, may be further fused to inhibitory effectors, such as histone modification/DNA methylase or cytidine deaminase (Komor et al, 2016) to effect site-directed mutagenesis. In the latter, cytidine deaminase does not induce double-stranded DNA breaks, but mediates the conversion of cytidine to uridine, thereby effecting a C to T (or G to a) substitution.

In a further embodiment, the nucleic acid construct comprises an endoribonuclease. Preferably, the endoribonuclease is Csy4 (also known as Cas6f), more preferably rice codon optimized Csy 4. In one embodiment, when the nucleic acid construct comprises a Cas protein, the nucleic acid construct may comprise a sequence for expressing an endoribonuclease, e.g., Csy4, expressed as a fusion (used as a self-cleaving peptide) of the 5' end P2A to a Cas protein, e.g., Cas 9.

In one embodiment, the cas protein, endoribonuclease, and/or endoribonuclease-cas fusion sequence may be operably linked to a suitable plant promoter. Suitable plant promoters have been described above, but in one embodiment may be the maize ubiquitin 1 promoter.

Suitable methods for producing CRISPR nucleic acids and vector systems are known, for example, from Molecular Plant (Ma et al 2015, Molecular Plant, DOI: 10.1016/j.mol.2015.04.007), which is incorporated herein by reference.

In an alternative aspect of the invention, the nucleic acid construct comprises at least one nucleic acid sequence encoding a TAL effector, wherein the effector targets an NRT2.3 promoter sequence selected from SEQ ID NOs 12 to 15. Methods for designing TAL effectors, given a target sequence, will be well known to the skilled artisan. Examples of suitable methods are given by Sanjana et al and Cermak T et al, both of which are incorporated herein by reference. Preferably, the nucleic acid construct comprises two nucleic acid sequences encoding TAL effectors to produce a TALEN pair. In a further embodiment, the nucleic acid construct further comprises a Sequence Specific Nuclease (SSN). Preferably, such SSN is an endonuclease, such as FokI. In a further embodiment, the TALENs are assembled in a single plasmid or nucleic acid construct by the gold Gate (Golden Gate) cloning method.

In another aspect of the invention, sgRNA molecules are provided, wherein the sgRNA molecule comprises a crRNA sequence and a tracrRNA sequence, and wherein the crRNA sequence may bind to at least one sequence selected from SEQ ID NOs 12 to 15 or a variant thereof. "variants" are as defined herein. In one embodiment, the sgRNA molecule may comprise at least one chemical modification, e.g., to enhance its stability and/or binding affinity to the target sequence or crRNA sequence to the tracrRNA sequence. Such modifications are well known to those skilled in the art and include, for example and without limitation, the modifications described in Rahdar et al, 2015, which is incorporated herein by reference. In this example, the crRNA may comprise phosphorothioate backbone modifications, such as 2 '-fluoro (2' -F), 2 '-O-methyl (2' -O-Me), and S-constrained ethyl (cET) substitutions.

In a further aspect of the invention there is provided an isolated nucleic acid sequence encoding a protospacer element (as defined in any one of SEQ ID NOs 16 to 23).

In another aspect of the invention, there is provided a plant or part thereof or at least one isolated plant cell transfected with at least one nucleic acid construct as described herein. Cas9 and sgrnas can be combined or in separate expression vectors (or nucleic acid constructs, such terms being used interchangeably). In other words, in one embodiment, an isolated plant cell is transfected with a single nucleic acid construct comprising a sgRNA and Cas9 as described in detail above. In an alternative embodiment, the 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 homologue thereof. The second nucleic acid construct may be transfected below, after or simultaneously with the first nucleic acid construct. An 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 is thus not limited to a single cas function (as is the case when both cas and sgRNA are encoded on the same nucleic acid construct).

In one embodiment, the nucleic acid construct comprising the cas protein is first transfected and stably integrated into the genome prior to the second transfection with the nucleic acid construct comprising the at least one sgRNA nucleic acid. In an alternative embodiment, the 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. In one example, the expression vector comprises a nucleic acid sequence as defined in SEQ ID No.30, or a functional variant or homologue thereof, wherein said nucleic acid sequence is operably linked to a suitable promoter. Examples of suitable promoters include the actin, CaMV35S, U6, U3 or maize ubiquitin (e.g., Ubi1) promoter.

The scope of the present invention also includes the use of the above-described nucleic acid construct (CRISPR construct) or sgRNA molecule in any of the above-described methods. For example, there is provided the use of the above CRISPR constructs or sgRNA molecules in modulating NRT2.3 promoter activity and/or NRT2.3 gene splicing as described herein.

Thus, in a further aspect of the invention, there is provided a method of modulating NRT2.3 promoter activity and/or NRT2.3 gene splicing and/or increasing NRT2.3b expression, the method comprising introducing and expressing a CRISPR construct as described above or introducing a sgRNA molecule as also described above into a plant. In other words, also provided are methods of modulating NRT2.3 promoter activity and/or NRT2.3 gene splicing and/or increasing NRT2.3b expression, as described herein, wherein the method comprises introducing at least one mutation into an endogenous NRT2.3 promoter using a CRISPR/Cas9, in particular a CRISPR construct as described herein.

In an alternative aspect of the invention, there is provided an isolated plant cell transfected with at least one sgRNA molecule as described herein.

In a further aspect of the invention, there is provided a genetically modified or edited plant comprising a transfected cell as described herein. In one embodiment, one or more nucleic acid constructs may be integrated in a stable form. In alternative embodiments, one or more nucleic acid constructs are not integrated (i.e., are transiently expressed). Thus, in a preferred embodiment, the genetically modified plant does not contain any sgRNA and/or Cas protein nucleic acids. In other words, the plant does not contain a transgene.

The terms "introduction", "transfection" or "transformation" as referred to herein include the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for transfer. Plant tissue capable of subsequent clonal propagation by organogenesis or embryogenesis may be transformed with a genetic construct of the present invention and whole plants regenerated therefrom. The particular tissue selected will vary depending on the clonal propagation systems available and best suited to the particular species being transformed. Exemplary tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, gametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristematic tissue (e.g., cotyledon meristem and hypocotyl meristem). The resulting transformed plant cells can then be used to regenerate transformed plants in a manner known to those skilled in the art. The transfer of foreign genes into the genome of a plant is called transformation. Plant transformation is now a routine technique for many species. Any of several transformation methods known to the skilled artisan can be used to introduce the nucleic acid construct of interest or the sgRNA molecule into a suitable progenitor cell. The described methods for transforming and regenerating plants from plant tissues or plant cells can be used for transient or stable transformation.

Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, direct injection of DNA into plants (microinjection), gene gun (or biolistic particle delivery system as described in the examples (biology)), lipofection, transformation using viruses or pollen and microprojectiles. The method may be selected from calcium/polyethylene glycol methods for protoplasts, ultrasound-mediated gene transfection, optical or laser transfection, transfection using silicon carbide fibers, electroporation of protoplasts, microinjection into plant material, bombardment with DNA or RNA-coated particles, infection with viruses, etc. (non-integration). Transgenic plants may also be produced by Agrobacterium tumefaciens (Agrobacterium tumefaciens) mediated transformation, including but not limited to the use of floral dip/Agrobacterium vacuum infiltration methods as described in Clough & Bent (1998) and incorporated herein by reference.

Thus, in one embodiment, at least one nucleic acid construct or sgRNA molecule as described herein can be introduced into at least one plant cell using any of the above methods. In alternative embodiments, any of the nucleic acid constructs described herein can be first transcribed to form a pre-assembled Cas9-sgRNA ribonucleoprotein and then delivered to at least one plant cell using any of the methods described above, such as lipofection, electroporation, or microinjection.

Optionally, for the selection of transformed plants, the plant material obtained in the transformation is usually subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner may be planted and suitably selected by spraying after the initial growth period. Another possibility is, if appropriate after sterilization, to plant the seeds on agar plates using suitable selection agents, so that only the transformed seeds can grow into plants. As described in the examples, a suitable marker may be bar-glufosinate or PPT. Alternatively, transformed plants are screened for the presence of a selectable marker such as, but not limited to, GFP, GUS (β -glucuronidase). Other examples will be readily apparent to the skilled person. Alternatively, without selection, seeds obtained in the manner described above were planted and grown and nrt2.3b expression or protein levels were measured at appropriate times using standard techniques in the art. This alternative to avoid the introduction of transgenes is more suitable for the production of plants without transgenes.

Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for example using PCR to detect the presence, copy number and/or genomic organization of the gene of interest. Alternatively or additionally, Southern, Northern, and/or Western analysis may be used to monitor the integration and expression levels of the newly introduced DNA, both techniques being well known to those of ordinary skill in the art.

The resulting transformed plants can be propagated in a variety of ways, for example, by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants can be selfed and homozygous second generation (or T2) transformants selected, and the T2 plants can then be further propagated by classical breeding techniques.

The skilled person is familiar with specific protocols for using the above CRISPR constructs. As an example, a suitable protocol is described in Ma & Liu ("CRISPR/Casp based multiplex genome editing in monocots and dicots"), incorporated herein by reference.

In a further related aspect of the invention, there is also provided a method of obtaining a genetically modified plant as described herein, the method comprising

a. Selecting a part of a plant;

b. transfecting at least one cell of the plant part of paragraph (a) with at least one nucleic acid construct as described herein or at least one sgRNA molecule as described herein using the above transfection or transformation techniques;

c. regenerating at least one plant derived from the transfected cells;

d. selecting one or more plants obtained according to paragraph (c) that show increased expression of nrt2.3b or increased relative expression of nrt2.3b (relative to nrt 2.3a).

In a further embodiment, the method further comprises the step of screening the genetically modified plant for SSN (preferably CRISPR) -induced mutations in the NRT2.3 promoter sequence. In one embodiment, the method comprises obtaining a DNA sample from the transformed plant and performing DNA amplification to detect mutations in at least one NRT2.3 promoter sequence.

In a further embodiment, the method comprises producing a stable T2 plant, preferably homozygous for the mutation (i.e. the mutation in the at least one NRT2.3 promoter sequence).

A plant having a mutation in at least one NRT2.3 promoter sequence may also be crossed with another plant also comprising at least one mutation in at least one NRT2.3 promoter sequence to obtain a plant having an additional mutation in the NRT2.3 promoter sequence. These combinations will be apparent to the skilled person. Thus, this method can be used to generate T2 plants having mutations in all or an increased number of partial homologues when compared to the number of partial homologous mutations in a single T1 plant transformed as described above.

Plants obtained or obtainable by the above methods are also within the scope of the present invention.

Genetically altered plants of the invention may also be obtained by transferring any of the sequences of the invention by crossing, for example by pollinating wild type or control plants with pollen of the genetically altered plants described herein, or pollinating pistils of the plants described herein with other pollen which does not comprise a mutation in at least one NRT2.3 promoter sequence. The methods of obtaining the plants of the present invention are not limited to those described in this paragraph; for example, genetic transformation of germ cells can be performed as described above, but it is not necessary to regenerate the plant thereafter.

Method for screening plant for a naturally increased grain yield phenotype

In a further aspect of the invention, there is provided a method for screening a population of plants and identifying and/or selecting plants carrying or expressing at least one mutation in an NRT2.3 promoter, preferably at least one mutation in an NRT2.3b 5' UTR or NRT2.3a promoter. More preferably at least one of SEQ ID NO 1 or 9 or a variant thereof. Alternatively, methods for screening a population of plants and identifying and/or selecting plants having increased yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content are provided. In any aspect, the method comprises detecting at least one polymorphism in the NRT2.3 promoter and preferably at least one mutation in SEQ ID No.1 or 9 or variants thereof in a plant or plant germplasm. Preferably, the screening comprises determining the presence of at least one polymorphism, wherein the polymorphism is at least one insertion and/or at least one deletion and/or substitution.

In one embodiment, 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. In a preferred embodiment, the polymorphism is a deletion of at least one nucleotide of the 5' end of SEQ ID NO. 1. More preferably, the polymorphism is a deletion of at least the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 nucleotides of the 5' end of SEQ ID NO. 1. In a more preferred embodiment, the polymorphism is a deletion of at least the first 50, more preferably the first 60, even more preferably the first 62 nucleotides of the 5' end of SEQ ID NO. 1. In an alternative embodiment, the polymorphism is a deletion of the first 90, more preferably of the first 100, even more preferably of the first 101 nucleotides of the 5' end of SEQ ID NO. 1. In an alternative embodiment, the polymorphism is the deletion of

SEQ ID NO

6 or 7 from SEQ ID NO 1.

In an alternative embodiment, the polymorphism is a substitution of at least one nucleotide. In a preferred embodiment, the polymorphism is a substitution of at least one nucleotide at position 160 of SEQ ID NO:1 (which may also be referred to herein as the polymorphism at position-83 of the ATG start codon for the NRT2.3 gene), position 201 of SEQ ID NO:1 (which may also be referred to herein as position-42), and position 222 of SEQ ID NO:1 (which may also be referred to herein as position-21). In a preferred embodiment, the polymorphism is a substitution at position 160 of SEQ ID NO. 1. In a further preferred embodiment, the substitutions may be as follows:

position 160 (of SEQ ID NO: 1): t to C;

position 201 (of SEQ ID NO: 1): a to C;

position 222 (of SEQ ID NO: 1): c to T.

In a preferred embodiment, 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. In one embodiment, the TATA-box is defined in SEQ ID NO 9 or a variant thereof. Thus, in one embodiment, the polymorphism affects the binding of a transcription factor or histone to the NRT2.3 promoter and thus affects the transcription of the NRT2.3 gene. In another preferred embodiment, the at least one polymorphism affects the binding capacity of a TATA binding protein, such as TBP2.1 (thereby increasing the expression level of nrt 2.3b).

In a preferred embodiment, 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 polymorphisms are well known to the skilled artisan and include, but are not limited to, isozyme electrophoresis, Restriction Fragment Length Polymorphism (RFLP), randomly amplified polymorphic DNA (rapd), any primed polymerase chain reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), sequence feature amplification region (scarr), Amplified Fragment Length Polymorphism (AFLP), simple sequence repeat (SSR-also known as microsatellite), and Single Nucleotide Polymorphism (SNP). In one embodiment, competitive allele specific pcr (kasp) genotyping is used.

In one embodiment, the method comprises

a) Obtaining a nucleic acid sample from a plant, and

b) nucleic acid amplification of the NRT2.3 promoter sequence was performed using one or more pairs of primers.

In a further embodiment, the method may further comprise introgressing the chromosomal region comprising the NRT2.3 promoter sequence polymorphism into a second plant or plant germplasm to produce an introgressed plant or plant germplasm. Preferably, said second plant will exhibit an increase in yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content.

While the foregoing disclosure provides a general description of the subject matter contained within the scope of the invention, including the methods of making and using the invention and the best mode thereof, the following examples are provided to further enable those skilled in the art to practice the invention and to provide a full written description thereof. However, those skilled in the art will appreciate that the details of these examples are not to be construed as limitations of the present invention, the scope of which is to be understood from the claims appended to this disclosure and their equivalents. Various further aspects and embodiments of the invention will be apparent to those skilled in the art in view of this disclosure.

As used herein, "and/or" is to be taken as a specific disclosure of each of the two specific features or components, with or without the other. For example, "a and/or B" will be considered a specific disclosure of each of (i) a, (ii) B, and (iii) a and B, as if each were individually listed herein.

Unless the context indicates otherwise, the description and definition of the features described above is not limited to any particular aspect or embodiment of the invention, and applies equally to all aspects and embodiments described.

The above applications, as well as all documents and sequence accession numbers ("application-cited documents") cited therein or during prosecution thereof and all documents cited or referenced in the application-cited documents, as well as all documents cited or referenced herein ("herein-cited documents") and all documents cited or referenced in the herein-cited documents, along with any manufacturer's specifications, descriptions, product specifications, and product sheets for any products mentioned herein or any documents cited herein, are incorporated herein by reference, and may be used in the practice of the present invention. More specifically, all references are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The invention will now be described in the following non-limiting examples.

Examples

Materials and methods

Plant material and growth conditions

OsNRT2.3TIILLING lines, backcross lines and transgenic lines (in this study) were surface sterilized with 30% (v/v) NaClO for 30 minutes and then rinsed thoroughly with water. Seedlings were grown in a climatic chamber, illuminated for 16 hours (30 ℃) and dark for 8 hours (26 ℃). The plants used for the field trials were grown at Nanjing university of agriculture, Nanjing, Jiangsu. The chemistry of the test field soil is described by Chen et al. (Chen et al, 2016)

Identification of OsNRT2.3TILLING lines

When seedlings were grown in IRRI for 3 weeks, DNA samples were collected and stored at-80 ℃. DNA samples were extracted by CTAB method. We used two pairs of primers pOsNRT2.3-F/R and OsNRT2.3-F/R to amplify the promoter and gene of OsNRT2.3; each DNA sample was repeated 4 times. Product sequence by company name

And (4) determining. The amplified sequence of TILLING line was aligned with the sequence of ZHONGHUO11(WT) using DNAMAN.

Production of transgenic plants

To investigate the effect of OsNRT2.3 promoter fragments of different lengths on OsNRT2.3a and OsNRT2.3b expression, we amplified rice OsNRT2.3 promoters of different lengths, such as 697bp and 141 bp. To confirm that the-83 bp mutation affects the expression of OsNRT2.3a and OsNRT2.3b, we prepared-83 bp mutants in both the 141bp and 697bp promoters. Different promoter lengths were used to drive the 437bp Open Reading Frame (ORF) of osnrt 2.3. To better distinguish OsNRT2.3a from OsNRT2.3b, the 437bp ORF was linked to the sequence of ZIIIB (cct gca ggt cgc cac att agc aat gcc aca tta gca atg ccg act cta gag gat ccc) (SEQ ID NO: 31).

To demonstrate the effect of the-83 bp mutation on OsNRT2.3a and OsNRT2.3b transcription and translation, we incorporated OsNRT2.3a and OsNRT2.3b into one vector. In one vector, a 141bp fragment of the OsNRT2.3 promoter was used to drive expression of OsNRT2.3a and eGFP, as well as OsNRT2.3b and mCherry reporter genes. We then performed a-83 bp mutation in the 141bp promoter fragment. In another vector, we used a 697bp promoter fragment with a-83 bp mutation to drive expression of OsNRT2.3a and eGFP, and OsNRT2.3b and mCherry, just like the 141 promoter vector. The expression vector was transferred into Agrobacterium (EHA105) by electroporation and transformed into rice (Toki, 1997; Toki et al, 2006).

In order to explore the functions of the genes OsTBP2.1 and OsNRT2.3, we constructed an OsTBP2.1 overexpression vector (pUbi:: OsTBP2.1) and obtained a T-DNA mutant line.

Yeast single hybrid assay

Yeast single-hybridization experiments were performed according to the manufacturer's protocol "Matchmaker Gold Yeast One-Hybrid Screening System User Manual" (Clontech). Briefly, the pTATA-cassette-pAbAi vector was transferred to a yeast strain and grown on medium lacking uracil (Ura). We tested the bait strain on medium lacking Ura using different concentrations of Aureobasidin a (AbA). The vectors of pGADT7-TBP2, pGADT7-TBP2.1 and pGADT7-TBP2.2 were then transferred into the strain carrying the pTATA-cassette-pAbAi. The strain was then grown in a medium containing 800ng ml^-1AbA^rIs grown on a medium lacking leucine.

Quantitative real-time PCR

Total RNA was extracted from the roots and shoots of seedlings using Trizol reagent, respectively. The total RNA was reverse transcribed into cDNA using a reverse transcription kit, and the synthesized cDNA was used as a template for real-time PCR reaction. For real-time PCR reactions, the templates were reacted 3 times with AceQ qPCR SYBR Green Master Mix kit on an Applied Biosystems (ABI) Plus real-time PCR system.

Western blot

Yan et al (2011) describe specific osnrt2.3b antibodies. Total protein was extracted from the-83 bp mutant backcross line for SDS-PAGE. The protein was then transferred to PVDF membrane and incubated with OsHSP (1:5000) and OsNRT2.3b (1:2000) overnight at 4 ℃. The PVDF membrane was then incubated with a secondary antibody (1: 20000; Pierce). Bound antibodies are detected by chemiluminescence (Yan et al, 2011; Tang et al, 2012).

Measurement of Nitrogen uptake Using 15N

WT, T11/WT (aa), and T12/WT (aa) lines were grown in IRRI nutrient solution for 3 weeks, then grown under N starvation for 3 days. Plants were initially transferred to 0.1mM CaSO₄For 1 minute, and then they were individually transferred to a medium containing 2.5mM 15NO₃And 1.25mM NH₄15NO₃For 5 minutes, and then again transferred to 0.1mM CaSO₄For 1 minute. All plants were placed at 105 ℃ for 30 minutes to inactivate the enzyme. The roots and stems are separated. They were then dried in an oven at 70 ℃ for 7 days and the dried samples were ground. Approximately 1mg of powder was analyzed for each sample using an isotope ratio mass spectrometer system (Thermo Fisher Scientific). Calculation of 15NO from the 15N concentration of roots and shoots₃-inflow of water.

Analysis of nitrate content

The strain was left at 105 ℃ for 30 minutes to inactivate the enzyme. The samples were oven dried at 75 ℃ for 3 days and the dry weight was recorded as a biomass value. The sample was ground to powder form. By means of H₂SO₄-H₂O₂Digestion of the sample is completed. In a continuous flow automatic analyzer (model Auto analyzer3, Bran) as described by Leleu, 2004&Lueebbe, germany) was performed.

Promoter Activity assay

The-83 bp mutation on the 1.5-kb promoter and 1.5-kb promoter fragment of OsNRT2.3 was amplified from rice and inserted into the luciferase reporter. The plasmid was transferred to rice protoplasts together with pUbi:OsTBP2.1 and harvested at 24 hours. Protoplasts were analyzed by using the kit dual-luciferase reporter assay system (Promega) to calculate the ratio of firefly Luciferase (LUC) to Renilla (Ren) luciferase.

RNA-sequence analysis

Total RNA was extracted from the shoots and roots of wild type (WT-27, WT-HY), OsTBP2.1 overexpression line (OE) and OsTBP2.1T-DNA mutation line (Mu) with three biological repeats. The samples were tested by Genepioneer corporation.

Results

The-83 bp region at the upstream of the translation initiation codon OsNRT2.3 is important for rice development

The mutant lines T8, T11, T12 and T20 were obtained using TILLING (targeting a localised lesion induced in the genome) (Tsai et al, 2011). (FIG. 9, FIG. 1 a). We extracted DNA from these strains to determine the location of the mutation. The results showed that T8, T11 and T12 all carried a mutation at-83 bp, upstream of the translation initiation codon OsNRT2.3. (FIG. 1 b). However, strain T20 carries no mutation upstream of the translation start codon of the gene OsNRT2.3. (FIG. 1 b). In the field, grain yield, dry weight and NUE were increased compared to WT and T20. (FIG. 1c, d). Although there was no difference in total N concentration between leaves, leaf sheaths, stems and ears, total nitrogen content of stems and ears was increased compared to WT and T20 lines (fig. 1e, f). Therefore, the mutation of the OsNRT2.3ATG upstream site improves the nitrogen transport efficiency, thereby promoting the growth of rice.

We then cultured 4 lines and WT in IRRI nutrient solution. The data show that T8, T11 and T12 strains showed significant growth and biomass compared to wild type, but that T20 strain was not different (fig. 2a, b). The total nitrogen concentration of the shoots and roots in the-83 bp mutant line and the T20 strain was not different from that of WT (FIG. 2c), but the total nitrogen content of the shoots and roots in the-83 bp mutant line was increased compared with that of WT and the T20 strain (FIG. 2 d). Expression data for osnrt2.3a and osnrt2.3b showed that osnrt2.3a was down-regulated but osnrt2.3b was up-regulated in T8, T11 and T12 lines compared to WT and T20 (fig. 10a, b). Thus, the mutant at-83 bp upstream of ATG of OsNRT2.3 increased the ratio of OsNRT2.3b to OsNRT2.3a (FIG. 2 e).

Furthermore, as shown in FIG. 15, for all three types studied¹⁵N-Source, accumulated in roots (FIG. 15a) and shoots (FIG. 15b) in strains T8, T11 and T12, compared to W¹⁵The N content is significantly higher. The T20 strain did not show any significant differences compared to WT in roots or shoots (fig. 15a, b).

FIG. 17 also shows nitrogen content in mature-83 bp mutant backcross lines. Mutant backcross lines and controls were compared to WT for nitrogen content in leaves, leaf sheaths, stems and ears of T8, T8/WT (aa) (loss of-83 bp mutation) lines. Compared to WT, WT and T8/WT (aa) did not differ significantly between all parts of the plant.

The-83 bp region upstream of ATG of OsNRT2.3 can increase the ratio of OsNRT2.3b to OsNRT2.3a

The results show that-83 bp site mutation in the osnrt2.3 promoter significantly promoted osnrt2.3b expression compared to no mutation or other site mutation (fig. 3a, b). When we driven osnrt2.3a expression using osnrt2.3 promoter with-83 bp site mutation, osnrt2.3a expression was down-regulated compared to the non-mutated line, however, osnrt2.3b expression was significantly up-regulated (fig. 3c, d). A-83 bp mutation upstream of OsNRT2.3ATG also increased the ratio of OsNRT2.3b to OsNRT2.3a (FIG. 3 e). Therefore, we concluded that the-83 bp region upstream of OsNRT2.3ATG is important for translation of OsNRT2.3 into OsNRT2.3a and OsNRT2.3b, and thus important for rice development.

The 83bp mutation increases the OsNRT2.3b protein level and nitrogen transport efficiency

To further verify that a-83 bp mutation site upstream of osnrt2.3 can affect transcription of osnrt2.3, we crossed T11 and T12 lines with WT. We obtained homozygous (Aa), heterozygous (Aa) and non-mutant (Aa) lines backcrossed at T11 and T12 (fig. 4a, b and fig. 11). The sequence results show that the OsNRT2.3 gene has only 1 mutation at the upstream-83 bp of ATG (FIGS. 12a and b). In contrast to WT, expression of OsNRT2.3a was down-regulated in T11/WT (aa) and T12/WT (aa) lines, whereas OsNRT2.3b was up-regulated. (FIG. 12 c). The homozygous (AA) lines T11/wt (AA) and T12/wt (AA) showed a significant increase in yield, dry weight and nitrogen use efficiency compared to the mature stage non-mutant (AA) lines. (FIG. 13)

We next analyzed OsNRT2.3b protein levels in T11/WT (aa) and T12/WT (aa) leaves and roots by Western blotting, and found that OsNRT2.3b protein levels in T11/WT (aa) and T12/WT (aa) leaves were elevated compared to WT, but not in roots. (FIG. 4 c). Previous research results indicate that OsNRT2.3a is primarily responsible for the long-distance transport of nitrate from root to shoot, whereas OsNRT2.3b is mainly responsible for the stems. (Tang et al, 2012; Yan et al, 2011) knock-out OsNRT2.3a increases the accumulation of root nitrate, while overexpression of OsNRT2.3b increases nitrate uptake and transport. (Tang et al, 2012; Fan et al, 2016). To determine the effect of a-83 bp site mutant upstream of the translation initiation codon of OsNRT2.3 on the different forms of nitrogen application, the-83 bp mutation on 15NO was measured within 5 min at pH5.5₃-and NH₄15NO₃Influence of absorption (fig. 4d, e). Compared to WT, the backcross showed more 15N uptake into the shoots in the 5 min treatment experiment (fig. 4 d). However, the uptake of 15N in roots is only upon administration of 15NO₃-and not NH₄15NO₃Is significantly increased in (fig. 4 e). Using 15NO compared with wild type₃-and NH₄15NO₃The treatment also significantly increased shoots in the backcross line¹⁵Concentration of N (fig. 4 f). In summary, we conclude that a-83 bp mutation in the 5' UTR of osnrt2.3b improves N uptake efficiency and transport efficiency.

Furthermore, FIG. 16 shows OsNRT2.3 mutant backcross lines¹⁵NO₃-and¹⁵NH₄+ inflow in 5 minutes. Mutant backcross homozygous lines for WT and T11 and T12 at 1.25mM NH₄NO₃Medium growth for 3 weeks, nitrogen starvation for 1 week. Then 2.5mM was measured in 5 minutes¹⁵NO₃-、1.25mM NH₄ ¹⁵NO₃And 1.25mM¹⁵NH₄NO₃Is/are as follows¹⁵And (N) inflow rate. (a) Root of herbaceous plant¹⁵And (N) inflow rate. (b) Stem¹⁵And (N) inflow rate. Error line: different letters of SE (n ═ 5) indicate significant differences (P) between transgenic lines and WT<0.05, one-way analysis of variance).

OsNRT2.3 different promoter lengths alter transcription of OsNRT2.3a and OsNRT2.3b

We designed expression vectors to study the effect of different promoter lengths of osnrt2.3 on osnrt2.3a and osnrt2.3b transcription (fig. 14a) and obtained transgenic seedlings. Expression of OsNRT2.3a is upregulated by longer promoter lengths. When the 141bp and 180bp promoters were used to drive expression of the 437bp ORF of OsNRT2.3a, its expression was down-regulated in seedlings. However, the longer promoter sequences, 243bp, 697bp and 1505bp promoters, increased the expression of OsNRT2.3a (FIG. 14 b). In contrast, in the case of OsNRT2.3b, there were only shorter promoters-141 bp and 180 bp-compared to the other lines, significantly increasing the expression of OsNRT2.3b (FIG. 14 c). The ratio of OsNRT2.3b to OsNRT2.3a was also increased in the 141bp and 180bp promoters compared to the other lines (FIG. 14 d). In conclusion, the expression ratio of OsNRT2.3b to OsNRT2.3a strongly correlates with the promoter length.

To confirm that the-83 bp site mutation affects the expression of OsNRT2.3a and OsNRT2.3b in different promoter lengths, we performed-83 bp site mutation on the 141bp and 697bp promoters. We then designed a vector using either a non-mutated or-83 bp site-mutated promoter to drive the expression of the 437bp ORF of osnrt2.3a and the reporter gene ZIIIB (fig. 5 a). Statistics of real-time PCR show that expression of osnrt2.3a is down-regulated when expression is driven by a mutant promoter (141bp or 697bp promoter) compared to the expression level of osnrt2.3a driven by an equivalent non-mutant promoter (fig. 5 b). However, when the mutation occurred on a short promoter (141bp promoter), the expression of OsNRT2.3b was reduced compared to the expression level of the non-mutated 141bp promoter. In contrast, when the mutation occurred on the long promoter (697bp), the expression of osnrt2.3b was up-regulated compared to the expression level on the equivalent (697bp) non-mutated promoter (fig. 5 c). Overall, the data indicate that whether the mutation is on a short or long promoter, it promotes transcription of osnrt2.3b but not osnrt2.3a (fig. 5 d). In conclusion, the-83 bp change can change the transcriptional regulation of OsNRT2.3 through promoters with different lengths.

OsTBP2.1 binds to the cis-element TATA-box of OsNRT2.3

Website using Softberry (http://linux1.softberry.com/berry.phtml) We analyzed the pre-ATG sequence of OsNRT2.3 and identified the-83 bp mutant within the TATA-box motif. We therefore searched the NCBI website (https:// www.ncbi.nlm.nih.gov) for three TATA-box binding proteins: OsTBP2, OsTBP2.1 and OsTBP2.2. To identify proteins that interact with the 83bp TATA-box upstream of OsNRT2.3, we performed a yeast single-hybrid assay. The results show that the binding protein OsTBP2.1 can be combined with TATA box-pAbAi reporter gene in 800ng ml^-1Gold basidin A (AbA)^r) SD/-Leu medium. Other effectors failed to grow. (FIG. 6a)

We used a dual luciferase assay to investigate whether the TATA-box binding protein OsTBP2.1 affects the transcriptional activity of the OsNRT2.3 promoter in the-83 bp mutant line. Reporter and effector vectors as shown in FIG. 6b were constructed and co-transformed into rice protoplasts. As shown in FIG. 6c, the greatest activation was observed when pmNRT2.3:: Luc and pUbi:: TBP2.1 were co-transformed into rice protoplasts. Notably, luciferase expression levels were lower in PnrT2.3:: Luc and pUbi:: TBP2.1 lines compared to pmNRT2.3:: Luc and pUbi:: TBP2.1 lines (FIG. 6 c). The lowest level of luciferase expression was observed in the individual transformation lines pNRT2.3:: Luc and pmNRT2.3:: Luc (FIG. 6 c). We next constructed the expression vector shown in FIG. 6d and obtained transgenic seedlings. OsNRT2.3a and OsNRT2.3b protein content was increased compared to non-mutant lines of different promoter lengths (FIG. 6e, f). Taken together, these results indicate that the transcription factor OsTBP2.1 can increase the transcription of OsNRT2.3 into OsNRT2.3b.

OsTBP2.1 increases the ratio of OsNRT2.3b to OsNRT2.3a and influences rice growth

To investigate the regulation of OsNRT2.3 by OsTBP2.1, we obtained over-expression and T-DNA mutant lines. (FIGS. 7a, b). As shown in FIG. 7c, the grain weight and dry biomass of the over-expressed lines were increased, while the grain weight and biomass of the T-DNA mutant lines were decreased, compared to WT-W27. This correlates with an increase in OsTBP2.1 and OsNRT2.3b expression in OE198 and OE200 overexpression lines compared to WT (WT-W27) and a decrease in OsTBP2.1 and OsNRT2.3b expression in T-DNA mutation lines (1A-19324) compared to WT Huangyant (WT-HY) (FIG. 7 d). Overexpression of OsTBP2.1 also increased the ratio of OsNRT2.3b to OsNRT2.3a (the opposite effect was observed in the T-DNA mutant line (FIG. 7 e)).

Discussion of the related Art

During growth and development, precise expression of functional genes is required for plants to respond to various environmental and developmental signals. In these processes, several transcription factors play important roles. For example, NLP transcription factors play an important role in the regulation of higher plant nitrates. (Konishi et al, 2019). Our studies have shown that the TATA-box binding factor ostbp2.1 regulates a variety of pathways, including the nitrate pathway. Our experiments show that cotransfection of OsTBP2.1 with OsNRT2.3 promoter can increase protein levels. (FIG. 6b, c) the ratio of OsNRT2.3b to OsNRT2.3a increased in the OsTBP2.1 overexpression line, but decreased in the ostbp2.1T-DNA line. (FIG. 7 d). In conclusion, OsTBP2.1 can enhance the expression of OsNRT2.3b and reduce the expression of OsNRT2.3a so as to influence rice development. When the TATA box is mutated to TACA, the binding capacity of ostbp2.1 is increased, resulting in higher expression levels of osnrt2.3b.

The key cis-acting elements play an important role in the transcriptional regulation of each gene. The TATA-box at the 5'UTR of OsNRT2.3b, but upstream of the OsNRT2.3a 5' UTR, controls transcription of OsNRT2.3 by binding to OsTBP2.1. However, when the TATA-box is mutated, the ability of OsTBP2.1 to bind to the TATA box is altered, resulting in an increased ratio of OsNRT2.3b to OsNRT2.3a. (FIG. 9). Thus, we concluded that transcription of OsNRT2.3 into OsNRT2.3a and OsNRT2.3b is mainly regulated by the TATA-box on the OsNRT2.3 promoter, and that the TATA-box plays an important regulatory role in the splicing of OsNRT2.3 into OsNRT2.3a and OsNRT2.3b.

The 5' UTR plays a regulatory role in RNA translation, RNA stability and RNA transcription. The number and length of introns at the 5'UTR will affect gene expression (Chung et al, 2006) as well as key cis-acting elements on the promoter and on the 5' UTR containing the first intron. (Hernandez-Garcia and Finer, 2014; Gallegos and Rose, 2015). In our study, TATA-box mutants in the 5' UTR of OsNRT2.3b increased the ratio of OsNRT2.3b to OsNRT2.3a, resulting in improved yield and growth.

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Sequence listing

1 NRT2.3 (partial) promoter of SEQ ID NO

1 GACGCGAGCG CGGAGACGGC AGCGCCGGCC TCCCACCGGT CGCGTAAGAT CACGCCCGAA

61 ATCTTTATTC ATTTTCTCTC CACCGGTTGC CCTCTCGCCG CACCCAACCA TCGCGCCACG

121 CCGCGCCGCG CTGCCGGAGC CGCGCTTTCC GCTATGCTAT AAGAGCTGAC GCGCAGGGCA

181 CAGCGGATGT ACGTACACAC AGTCACTAGC TAAGCTGCTA GCCTTGCTAC CACGTGTTGG

241 AG

2 NRT2.3 promoter with polymorphism (underlined) upstream-81 of ATG

1 GACGCGAGCG CGGAGACGGC AGCGCCGGCC TCCCACCGGT CGCGTAAGAT CACGCCCGAA

61 ATCTTTATTC ATTTTCTCTC CACCGGTTGC CCTCTCGCCG CACCCAACCA TCGCGCCACG

121 CCGCGCCGCG CTGCCGGAGC CGCGCTTTCC GCTATGCTAC AAGAGCTGAC GCGCAGGGCA

181 CAGCGGATGT ACGTACACAC AGTCACTAGC TAAGCTGCTA GCCTTGCTAC CACGTGTTGG

241 AG

3 NRT2.3 promoter with polymorphisms at-81, -42 and-21 upstream of ATG

1 GACGCGAGCG CGGAGACGGC AGCGCCGGCC TCCCACCGGT CGCGTAAGAT CACGCCCGAA

61 ATCTTTATTC ATTTTCTCTC CACCGGTTGC CCTCTCGCCG CACCCAACCA TCGCGCCACG

121 CCGCGCCGCG CTGCCGGAGC CGCGCTTTCC GCTATGCTAC AAGAGCTGAC GCGCAGGGCA

181 CAGCGGATGT ACGTACACAC CGTCACTAGC TAAGCTGCTA GTCTTGCTAC CACGTGTTGG

241 AG

180C-terminal residues of the promoter of SEQ ID NO 4 NRT2.3

CTTTATTC ATTTTCTCTC CACCGGTTGC CCTCTCGCCG CACCCAACCA TCGCGCCACG

CCGCGCCGCG CTGCCGGAGC CGCGCTTTCC GCTATGCTAC AAGAGCTGAC GCGCAGGGCA

CAGCGGATGT ACGTACACAC CGTCACTAGC TAAGCTGCTA GTCTTGCTAC CACGTGTTGG

AG

141C-terminal residues of the promoter of SEQ ID NO 5NRT 2.3

ACCCAACCA TCGCGCCACG CCGCGCCGCG CTGCCGGAGC CGCGCTTTCC GCTATGCTACAAGAGCTGAC GCGCAGGGCA CAGCGGATGT ACGTACACAC CGTCACTAGC TAAGCTGCTA GTCTTGCTAC CACGTGTTGG AG

Deletion of 62 nucleotides of the promoter of SEQ ID NO 6 NRT2.3

GACGCGAGCG CGGAGACGGC AGCGCCGGCC TCCCACCGGT CGCGTAAGAT CACGCCCGAA AT

Deletion of 101 5' nucleotides of the promoter of SEQ ID NO 7 NRT2.3

GACGCGAGCG CGGAGACGGC AGCGCCGGCC TCCCACCGGT CGCGTAAGAT CACGCCCGAAATCTTTATTC ATTTTCTCTC CACCGGTTGC CCTCTCGCCG C

8 NRT2.3 genome sequence of SEQ ID NO

GAGCGCCGGCCTCCCACCGGTCGCGTAAGATCACGCCCGAAATCTTTATTCATTTTCTCTCCACCGGTTGCCCTCTCGCCGCACCCAACCATCGCGCCACGCCGCGCCGCGCTGCCGGAGCCGCGCTTTCCGCTATGCTATAAGAGCTGACGCGCAGGGCACAGCGGATGTACGTACACACAGTCACTAGCTAAGCTGCTAGCCTTGCTACCACGTGTTGGAGATGGAGGCTAAGCCGGTGGCGATGGAGGTGGAGGGGGTCGAGGCGGCGGGGGGCAAGCCGCGGTTCAGGATGCCGGTGGACTCCGACCTCAAGGCGACGGAGTTCTGGCTCTTCTCCTTCGCGAGGCCACACATGGCCTCCTTCCACATGGCGTGGTTCTCCTTCTTCTGCTGCTTCGTGTCCACGTTCGCCGCGCCGCCGCTGCTGCCGCTCATCCGCGACACCCTCGGGCTCACGGCCACGGACATCGGCAACGCCGGGATCGCGTCCGTGTCGGGCGCCGTGTTCGCGCGTCTGGCCATGGGCACGGCGTGCGACCTGGTCGGGCCCAGGCTGGCCTCCGCGTCTCTGATCCTCCTCACCACACCGGCGGTGTACTGCTCCTCCATCATCCAGTCCCCGTCGGGGTACCTCCTCGTGCGCTTCTTCACGGGCATCTCGCTGGCGTCGTTCGTGTCGGCGCAGTTCTGGATGAGCTCCATGTTCTCGGCCCCCAAAGTGGGGCTGGCCAACGGCGTGGCCGGCGGCTGGGGCAACCTCGGCGGCGGCGCCGTCCAGCTGCTCATGCCGCTCGTGTACGAGGCCATCCACAAGATCGGTAGCACGCCGTTCACGGCGTGGCGCATCGCCTTCTTCATCCCGGGCCTGATGCAGACGTTCTCGGCCATCGCCGTGCTGGCGTTCGGGCAGGACATGCCCGGCGGCAACTACGGGAAGCTCCACAAGACTGGCGACATGCACAAGGACAGCTTCGGCAACGTGCTGCGCCACGCCCTCACCAACTACCGCGGCTGGATCCTGGCGCTCACCTACGGCTACAGCTTCGGCGTCGAGCTCACCATCGACAACGTCGTGCACCAGTACTTCTACGACCGCTTCGACGTCAACCTCCAGACCGCCGGGCTCATCGCCGCCAGCTTCGGGATGGCCAACATCATCTCCCGCCCCGGCGGCGGGCTACTCTCCGACTGGCTCTCCAGCCGGTACGGCATGCGCGGCAGGCTGTGGGGGCTGTGGACTGTGCAGACCATCGGCGGCGTCCTCTGCGTGGTGCTCGGAATCGTCGACTTCTCCTTCGCCGCGTCCGTCGCCGTGATGGTGCTCTTCTCCTTCTTCGTCCAGGCCGCGTGCGGGCTCACCTTCGGCATCGTGCCGTTCGTGTCGCGGAGGTCGCTGGGGCTCATCTCCGGGATGACCGGCGGCGGGGGCAACGTGGGCGCCGTGCTGACGCAGTACATCTTCTTCCACGGCACAAAGTACAAGACGGAGACCGGGATCAAGTACATGGGGCTCATGATCATCGCGTGCACGCTGCCCGTCATGCTCATCTACTTCCCGCAGTGGGGCGGCATGCTCGTAGGCCCGAGGAAGGGGGCCACGGCGGAGGAGTACTACAGCCGGGAGTGGTCGGATCACGAGCGCGAGAAGGGTTTCAACGCGGCCAGCGTGCGGTTCGCGGAGAACAGCGTGCGCGAGGGCGGGAGGTCGTCGGCGAATGGCGGACAGCCCAGGCACACCGTCCCCGTCGACGCGTCGCCGGCCGGGGTGTGAAGAATGCCACGGACAATAAGGTCGCGGTTGTAGTACAACTGTACAAATTGATGGTACGTGTCGTTTGACCGCGCGCGCGCACAGTGTGGGTCGTGGCCTCGTGGGCTTAGTGGAGTACAGTGAGGGGTGTACGTGTGTCGTGGCGCGCGCGGTCACCTCGGTGGCCTTGGGATTGGGGGGGCACTATACGCTAGTACTCCAGATATATACGGGTTTGATTTACTTCTGTGGATCGGCGCTTGTTGGTGGTTTGCTCCCTGTGGTTTTTGTGATGGTAATCATACTCATACTCAAACAGTC

SEQ ID NO 9 NRT2.3 TATA Box

CC GCTATGCTAT AAGAGCTGAC

10 in SEQ ID NO; OSE2ROOTNODULE (-82bp to-86 bp)

CTAC

11 is SEQ ID NO; ASF1MOTIFCAMV (-76bp to-83 bp)

CGCGCAGG

12 CRISPR target sequence of SEQ ID NO

13 CRISPR target sequence of SEQ ID NO

14 CRISPR target sequence of SEQ ID NO

15 CRISPR target sequence of SEQ ID NO

16 Problocker sequence of SEQ ID NO

GCTATAAGAGCTGACGCGCA

17 Problocker sequence of SEQ ID NO

GAGCCGCGCTTTCCGCTATG

18 Problocker sequence of SEQ ID NO

GTTGCCCTCTCGCCGCACCC

19 Problocker sequence of SEQ ID NO

CGACGCGAGCGCGGAGACGG

20 Problocker sequence of SEQ ID NO

5’GGCAGCTATAAGAGCTGACGCGCA 3’

5’AAACTGCGCGTCAGCTCTTATAGC 3’(SEQ ID NO:34)

21 Protospacer sequence of SEQ ID NO

5’GGCACATAGCGGAAAGCGCGGCTC 3’

5’AAACGAGCCGCGCTTTCCGCTATG 3’(SEQ ID NO:35)

22 Problocker sequence of SEQ ID NO

5’GGCAGGGTGCGGCGAGAGGGCAAC3’

5’AAACGTTGCCCTCTCGCCGCACCC 3’(SEQ ID NO:36)

23 Problocker sequence of SEQ ID NO

5’GGCACCGTCTCCGCGCTCGCGTCG 3’

5’AAACCGACGCGAGCGCGGAGACGG 3’(SEQ ID NO:37)

24 is SEQ ID NO; tracrRNA nucleic acid sequences

ATGCTACTACTAAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGACTAGCCTTATTTTAACTTGCTATGCTTTTCAGCATAGCTCTAAAAC

25U 3 promoter sequence:

AAAGGAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCA

26U 6a promoter sequence:

TTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCG

27U 6b promoter sequence:

TGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCACATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATGGGGCTCAGACTTGAGCTACGAGGCCGCAGGCGAGAGAAGCCTAGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATACGGCCGACGAGCGTGTACTACCGCGCGGGATGCCGCTGGGCGCTGCGGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGGAGACAGGTTTAGTACCACCTCGCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTG

28U 3b in dicotyledonous plants

TTTACTTTAAATTTTTTCTTATGCAGCCTGTGATGGATAACTGAATCAAACAAATGGCGTCTGGGTTTAAGAAGATCTGTTTTGGCTATGTTGGACGAAACAAGTGAACTTTTAGGATCAACTTCAGTTTATATATGGAGCTTATATCGAGCAATAAGATAAGTGGGCTTTTTATGTAATTTAATGGGCTATCGTCCATAGATTCACTAATACCCATGCCCAGTACCCATGTATGCGTTTCATATAAGCTCCTAATTTCTCCCACATCGCTCAAATCTAAACAAATCTTGTTGTATATATAACACTGAGGGAGCAACATTGGTCA

U6-1 of SEQ ID NO 29 in dicotyledonous plants

AGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAAACGAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTATATATGGTTAGAGACGAAGTAGTGATTG

SEQ ID NO:30 Cas9

ATGGCCCCAAAGAAGAAGCGCAAGGTCGACAAGAAGTACTCCATCGGCCTCGACATCGGCACCAATTCTGTTGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCGTCCAAGAAGTTCAAGGTCCTCGGCAACACCGACCGCCACTCCATCAAGAAGAATCTCATCGGCGCCCTGCTGTTCGACTCTGGCGAGACAGCCGAGGCTACAAGGCTCAAGAGGACCGCTAGACGCAGGTACACCAGGCGCAAGAACCGCATCTGCTACCTCCAAGAGATCTTCTCCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGGCTCGAGGAGAGCTTCCTCGTCGAGGAGGACAAGAAGCACGAGCGCCATCCGATCTTCGGCAACATCGTGGATGAGGTGGCCTACCACGAGAAGTACCCGACCATCTACCACCTCCGCAAGAAGCTCGTCGACTCCACCGATAAGGCCGACCTCAGGCTCATCTACCTCGCCCTCGCCCACATGATCAAGTTCAGGGGCCACTTCCTCATCGAGGGCGACCTCAACCCGGACAACTCCGATGTGGACAAGCTGTTCATCCAGCTCGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCGATCAACGCCTCTGGCGTTGACGCCAAGGCTATTCTCTCTGCCAGGCTCTCTAAGTCCCGCAGGCTCGAGAATCTGATCGCCCAACTTCCGGGCGAGAAGAAGAATGGCCTCTTCGGCAACCTGATCGCCCTCTCTCTTGGCCTCACCCCGAACTTCAAGTCCAACTTCGACCTCGCCGAGGACGCCAAGCTCCAGCTTTCCAAGGACACCTACGACGACGACCTCGACAATCTCCTCGCCCAGATTGGCGATCAGTACGCCGATCTGTTCCTCGCCGCCAAGAATCTCTCCGACGCCATCCTCCTCAGCGACATCCTCAGGGTGAACACCGAGATCACCAAGGCCCCACTCTCCGCCTCCATGATCAAGAGGTACGACGAGCACCACCAGGACCTCACACTCCTCAAGGCCCTCGTGAGACAGCAGCTCCCAGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACATCGATGGCGGCGCTTCTCAAGAGGAGTTCTACAAGTTCATCAAGCCGATCCTCGAGAAGATGGACGGCACCGAGGAGCTGCTCGTGAAGCTCAATAGAGAGGACCTCCTCCGCAAGCAGCGCACCTTCGATAATGGCTCCATCCCGCACCAGATCCACCTCGGCGAGCTTCATGCTATCCTCCGCAGGCAAGAGGACTTCTACCCGTTCCTCAAGGACAACCGCGAGAAGATTGAGAAGATCCTCACCTTCCGCATCCCGTACTACGTGGGCCCGCTCGCCAGGGGCAACTCCAGGTTCGCCTGGATGACCAGAAAGTCCGAGGAGACAATCACCCCCTGGAACTTCGAGGAGGTGGTGGATAAGGGCGCCTCTGCCCAGTCTTTCATCGAGCGCATGACCAACTTCGACAAGAACCTCCCGAACGAGAAGGTGCTCCCGAAGCACTCACTCCTCTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGGATGAGGAAGCCAGCTTTCCTTAGCGGCGAGCAAAAGAAGGCCATCGTCGACCTGCTGTTCAAGACCAACCGCAAGGTGACCGTGAAGCAGCTCAAGGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTCGAGATCTCCGGCGTCGAGGATAGGTTCAATGCCTCCCTCGGGACCTACCACGACCTCCTCAAGATTATCAAGGACAAGGACTTCCTCGACAACGAGGAGAACGAGGACATCCTCGAGGACATCGTGCTCACCCTCACCCTCTTCGAGGACCGCGAGATGATCGAGGAGCGCCTCAAGACATACGCCCACCTCTTCGACGACAAGGTGATGAAGCAGCTGAAGCGCAGGCGCTATACCGGCTGGGGCAGGCTCTCTAGGAAGCTCATCAACGGCATCCGCGACAAGCAGTCCGGCAAGACGATCCTCGACTTCCTCAAGTCCGACGGCTTCGCCAACCGCAACTTCATGCAGCTCATCCACGACGACTCCCTCACCTTCAAGGAGGACATCCAAAAGGCCCAGGTGTCCGGCCAAGGCGATTCCCTCCATGAGCATATCGCCAATCTCGCCGGCTCCCCGGCTATCAAGAAGGGCATTCTCCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCAGGCACAAGCCAGAGAACATCGTGATCGAGATGGCCCGCGAGAACCAGACCACACAGAAGGGCCAAAAGAACTCCCGCGAGCGCATGAAGAGGATCGAGGAGGGCATTAAGGAGCTGGGCTCCCAGATCCTCAAGGAGCACCCAGTCGAGAACACCCAGCTCCAGAACGAGAAGCTCTACCTCTACTACCTCCAGAACGGCCGCGACATGTACGTGGACCAAGAGCTGGACATCAACCGCCTCTCCGACTACGACGTGGACCATATTGTGCCGCAGTCCTTCCTGAAGGACGACTCCATCGACAACAAGGTGCTCACCCGCTCCGACAAGAACAGGGGCAAGTCCGATAACGTGCCGTCCGAAGAGGTCGTCAAGAAGATGAAGAACTACTGGCGCCAGCTCCTCAACGCCAAGCTCATCACCCAGAGGAAGTTCGACAACCTCACCAAGGCCGAGAGAGGCGGCCTTTCCGAGCTTGATAAGGCCGGCTTCATCAAGCGCCAGCTCGTCGAGACACGCCAGATCACAAAGCACGTGGCCCAGATCCTCGACTCCCGCATGAACACCAAGTACGACGAGAACGACAAGCTCATCCGCGAGGTGAAGGTCATCACCCTCAAGTCCAAGCTCGTGTCCGACTTCCGCAAGGACTTCCAGTTCTACAAGGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTCAATGCCGTGGTGGGCACAGCCCTCATCAAGAAGTACCCAAAGCTCGAGTCCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGCAAGATGATCGCCAAGTCCGAGCAAGAGATCGGCAAGGCGACCGCCAAGTACTTCTTCTACTCCAACATCATGAATTTCTTCAAGACCGAGATCACGCTCGCCAACGGCGAGATTAGGAAGAGGCCGCTCATCGAGACAAACGGCGAGACAGGCGAGATCGTGTGGGACAAGGGCAGGGATTTCGCCACAGTGCGCAAGGTGCTCTCCATGCCGCAAGTGAACATCGTGAAGAAGACCGAGGTTCAGACCGGCGGCTTCTCCAAGGAGTCCATCCTCCCAAAGCGCAACTCCGACAAGCTGATCGCCCGCAAGAAGGACTGGGACCCGAAGAAGTATGGCGGCTTCGATTCTCCGACCGTGGCCTACTCTGTGCTCGTGGTTGCCAAGGTCGAGAAGGGCAAGAGCAAGAAGCTCAAGTCCGTCAAGGAGCTGCTGGGCATCACGATCATGGAGCGCAGCAGCTTCGAGAAGAACCCAATCGACTTCCTCGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTCATCATCAAGCTCCCGAAGTACAGCCTCTTCGAGCTTGAGAACGGCCGCAAGAGAATGCTCGCCTCTGCTGGCGAGCTTCAGAAGGGCAACGAGCTTGCTCTCCCGTCCAAGTACGTGAACTTCCTCTACCTCGCCTCCCACTACGAGAAGCTCAAGGGCTCCCCAGAGGACAACGAGCAAAAGCAGCTGTTCGTCGAGCAGCACAAGCACTACCTCGACGAGATCATCGAGCAGATCTCCGAGTTCTCCAAGCGCGTGATCCTCGCCGATGCCAACCTCGATAAGGTGCTCAGCGCCTACAACAAGCACCGCGATAAGCCAATTCGCGAGCAGGCCGAGAACATCATCCACCTCTTCACCCTCACCAACCTCGGCGCTCCAGCCGCCTTCAAGTACTTCGACACCACCATCGACCGCAAGCGCTACACCTCTACCAAGGAGGTTCTCGACGCCACCCTCATCCACCAGTCTATCACAGGCCTCTACGAGACACGCATCGACCTCTCACAACTCGGCGGCGATTGA

32: OsTBP2.1 amino acid SEQ ID NO

MAAAEAAAEAAAALEGSEPVDLVKHPSGIIPTLQNIVSTVNLDCKLDLKAIALQARNAEYNPKRFAAVIMRIREPKTTALIFASGKMVCTGAKSEQQSKLAARKYARIIQKLGFAAKFKDFKIQNIVGSCDVKFPIRLEGLAYSHGAFSSYEPELFPGLIYRMKQPKIVLLIFVSGKIVLTGAKVRDETYTAFENIYPVLTEFRKVQQ

OsTBP2.1 nucleic acid (CDS) 33: SEQ ID NO

GATTTCCGATCGCTATATAAAAGACCTAGGATTTCGAAATTTTTCCCTCCCCCTCCCCCTTCGCGCGCGCGCTCTCTTCCCGCCCTTTTTTTTTCCTTCTTCTTCACCGGTGGGATTGATTCGTGGGGTGCGGATCTGGTTTTTGGGGGTGTGTATGGCGGCGGCGGAGGCGGCGGCGGAGGCGGCGGCGGCGCTGGAGGGGAGCGAGCCCGTGGACCTGGTCAAGCACCCCTCCGGCATCATCCCCACGCTCCAGTAAGTCCCCCCCGCCCCCGCCCGGATCTGTATGTGCGGGTTCATCGAGCTGGTTGAGTTTGAGGTGGAGCGGTTAACTCGTCGCGCGCTGTTGATTTGGTTTTGTTTTGGGGGCCGGGGATATTATTTGTTGTTGCAAATGATGTGATTGGTGGTAGGTTTAGCTTGGGGGGTTACTACATGTTTGAGGTTCTGATTTGCTTGATTGATGAAAAAGGGGGGCGATCTGAGATCCGCGAGTCCGGTACGAGAAGAAATAGAGGCAGCCGATGTGCTTGTGCTGTGACGGATCCATTGTCCGGTCTTCATAACAACTCTTTTTAGCTGGACAGATGTTTGTTTTCTCATGAATGAATTCATATGGTTGGAGTCTTGGAGAATCAGCTTGTCAGCTGTTCTTTTTTTTAAAAAAAAATTAAGGTTTATAAATAAATAAATAATCAATCAATCAATCATGCATCTAAATTGTAGGATTTAATTCTTTCCCCTCATCACGTATTTCAACCTTAGAGGGAGGTATTGTGTCTGTGGAGAGTTTATGGTGCTCATGTGATACATACAGTTGACAATGGGTCAATGCATGTTTTTAGACAGCATCAAGCTCTGAACAAGTGAACCACATATTTAGGCTTGATCCATGTATAACCCCACTAAGTTTTATACTTGTTTGTACGGGCCAATATGCCCAAACCTGGCTGTTTATTAGCATGCTGTACTTGCTGGTGTGCATCGAAACGTTTTTGGGGTTTGCCTACATTGCTACCAGGCATGTATATTTTGCATGCTGGTGTGCATTGCAGAAAAAGTCGAGCATGCTTATATTGCTGCTTGTCATAGTGTACCTGCTTCCATGTGTTCTCACAAGTGCGCATGCCTGTTTTGCTGCCTTTCTGGGTTGTTTATGTTTCAGCGTTGGGATCATAGCTGGTAGACTGCATGTGTTCCACTCTGTTTTGGCTTATCATGCTTCCTTTGGATACTCTCACCAAGGAAATTGAAGTCCTCATTATGTGTTCTCATCTATTTTGATGGCAGAAACATCGTGTCGACGGTCAATTTGGATTGCAAATTAGACCTCAAAGCTATAGCTTTGCAAGCACGCAATGCAGAATATAATCCAAAGGTATTATGATGGCTTTGGTGTGTTCTTGTGTCTTTGATTTTGCTCGAAAGGATATCTTCTTTGCATGTGAAAATTTTACCTATTTTAATCCTGCTAGCATGTAATATGTAGACAAGTCCATACAATCCTATGGTCTGTTCCTAGGATGATACTTCCTTTGTGTATAACGCAAGTGTGAGTGCAATTTTAGAGGTCGGTATGGATAAATGCATAGCTGGCAGTCCAGTTTTATATTGTAAATCTCAGTTAAGAGAGTGAATACATTCATCACGCTCAGCTTGTCCCATGGATAAGCAAATTGCTTTACAATTAAGATTCTAAAATTGCTCTATTTAGTGCTTATGCACTTTCTAATCTGTGCTGCACGTATCAGATACAGCATGATGTATTGGGGGATTTTGTAATTATATGTAATATCATAATCTAAATCTAGGCTATTATGTTTATTTTAATAATGGATACATTTTGAAAGAAACAGAGATAACTGCACACTGAGGTTATAGCATCTCTATCTACTCCATCCGTTCCAAAATATAAGCACTGTAATATATGTTTTAGTTTGCTAACTCCTCTTTTCACAAACAGCGTTTTGCTGCAGTTATCATGAGAATAAGAGAACCGAAAACTACAGCTCTGATATTTGCATCGGGTAAAATGGTATGGTTCAACCTCTTTGTATAGCTAAGACTGCAAATATTCTGTTTGTTCATCTTCTTATTGACTTGTGAATTGCTTTATCACCTGTCTTTGTTGCTCTTGTAGCTATTATAACGCATATTGATTGATGAACTCTTTTTGCTGATGTGTAACTGCTTTCTATTAGCCTACTATCTTTTTAGTTTTTCTTTGTTTTTTTAACAGCTATCACATCCATACCTGATCCATGCAGCCTGTAGTTCAATGCTGAATCTGTAAATTTAAGTTTGCTCTGATTTTCTGACATGTTTGCTGTTGCATTGCTGTTACATAATAACAGATAAGTAGTGCTCTTTCTGTAAATGTATAGTTTGTCCTTGTTAACTTTTTTATTGTCTTAAAGCATAATCAGAATTGACAATAAATTGAACCTGTCATATTTATTTGTTGATGGATAATCAGCAATGACACATCACATGGACTTCATGTCGCGTCAATATCTTAATTTTAAAAGGCTTGTTTATGAAAATTCACTTGATTTTCCTTTTGGATCAATGTCTTGATCCGAATAAGCTTGTTGAAGGATAATTGGTGGTCAGCATTTTTGGGATAAAATCTTCATTAAAAGTTGCTTTGCTGGTTCTCATCAGGAGGGATAATATCTGTGTTACAGACTTAAAGTAATACATTTTTCTGATAATAAATTCACATTTTAAGCATGAGAACGAAGGAGTTCACAGCCTAGGCTTACTAGTACGCTGTATGGGGCCTGACTATTAGGCAGTGGACTTAAGAAGGGGTTGTCTTGGGTTTGATGCACCGATTTGCTGCGTCTTTGCTTCTTTTTTATAAAAATCAAATTTGCTTGTTAACCAAAATTTCTCTGTGGGCACCATGACCTGCTAGCATTTGTGGTGGAATTAAAAGCATGTTTATAAATCTTTTGTTGTCCTTTACCAGGTATGTACTGGGGCAAAGAGCGAACAACAATCAAAGCTTGCAGCAAGAAAGGTATGGGGAGTATTTATTTTATTTCATTTTATTTTGCTACGTGAAGTCTAACACTTTTTTATTGCCAGTATGCTCGTATTATCCAAAAGCTTGGCTTTGCTGCTAAGTTTAAGGTAACTATTTCAGGCTTGATTAATTTTCATCTGTGCAAATGCCTACCATTTTCATTCTGTAAGATTGTACCTAAATGTTTAACTCATTTGCACTATACCAGGACTTCAAGATTCAGAACATTGTTGGTTCTTGTGATGTTAAATTTCCAATCAGGCTGGAGGGACTTGCATATTCTCATGGTGCTTTCTCAAGTGTAAGTTGAATCCTTGCATGTTTTTTTAGGATATTTCTTTCACATATGTTATGCTATTCTCATGTCTTGTGCCTTTTGTCTTCCAGTATGAGCCTGAACTCTTTCCTGGTCTGATATATCGGATGAAGCAACCGAAGATTGTTCTTCTGATTTTTGTTTCAGGCAAGATTGTTTTGACCGGAGCAAAGGTAAGCAGCCTTTCCTTTTGTATACCCTTGATTGTCTATTCCTTTTTGTATGTCTGAATGTACTTGTCTTTATAGGTGAGGGATGAGACGTATACCGCCTTTGAGAACATATACCCTGTGCTAACAGAGTTCAGAAAAGTCCAGCAATGGTACGTCTTTATTTTGTTGTTTAGGTATGCAGTGTGTGGTAGAAACATCGGAAGTTCGAAGTAAGAATTATACAGTGGATAATTGCACTTGTGTCTTTTGTTATACGTGGGAGTGAACTTTGAGCACACTCCTTACTAAATTGCAATATGTTGAATTTTTTAATATTACAAGCACCTATAAATCTGAACTTTCCAATTTGAATCCAGATGGTCAGCAGAAACTGAAGGAATAAATCATCTCAAGAAAGATTTTAATTTCCACACTACCTTGATCTTAGATAGGATTTGGATACTTGTTTCAGATATGATTCATCACTACCATGTGATGGACTGTTGGCTGCCCTTGCCTACTTACTGTGCTTGACTGCAGTTTACTTCCAATTTAGGAGAACACAATTTTAAATAATAATAATAATGATGGCTCCTTGCACCTTCAAATTTGCAGATAACCTATGGAGGTCACAACTACAACGCTTCCTTGAGGATTTTGCTGCCTTGTAACTGCTAATTTTAATCTGTACATATATGTAGTCTGGAGGGGCGTACAGCATCTTGTAATTTATGTGAGCCCCTGGATGAATGAGCACTGTAGACTTGTAGCTGGGTGAGTATGTTGTTAGTAGTCTCTTGTGGCATGGAGTTCAGTCCAACCGATCTGATGGAGTTTTCGTACGTTTGTAGCCCTTGCCGATCTTTTTCCCTTTTCTTCCCAATAGACATGTTGCTAAACTTTTACTAACTTGTTAACAGACAGACAGAATGATAACATGGACTGTGGATGCTTAGCGTTTGTGGCCG

Claims

1. A method for increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content of a plant, comprising introducing at least one mutation into a nucleic acid sequence encoding an NRT2.3 promoter.

2. The method of claim 1, wherein the plant is rice.

3. The method of any one of the preceding claims, wherein the nucleic acid sequence encoding the NRT2.3 promoter comprises SEQ ID NO 9 or a functional variant thereof.

4. The method of any one of the preceding claims, wherein the nucleic acid sequence encoding the NRT2.3 promoter comprises SEQ ID No.1 or a functional variant thereof.

5. The method of any one of the preceding claims, wherein the mutation is introduced using mutagenesis.

6. The method of claim 4, wherein the mutation is introduced using TILLING or T-DNA insertion.

7. The method of any one of claims 1 to 4, wherein the mutation is introduced using a targeted genome modification, preferably a ZFN, TALEN or CRISPR/Cas 9.

8. The method of any one of the preceding claims, wherein the mutation is introduced into SEQ ID No.1, preferably into SEQ ID No. 9.

9. The method of any one of the preceding claims, wherein the mutation is an insertion, deletion and/or substitution.

10. The method of claim 9, wherein the mutation is a substitution of at least one nucleotide.

11. The method of claim 10, wherein the substitution is at position 160, position 201, or position 222 of SEQ ID NO 1.

12. The method of claim 11, wherein the mutation is a substitution at position 160.

13. The method of claim 12, wherein the mutation is a T to C substitution.

14. The method of any one of claims 1 to 9, wherein the mutation is a deletion of at least one nucleotide.

15. The method of claim 14, wherein the mutation is a deletion of at least fifty, more preferably sixty 5' nucleotides of SEQ ID No. 1.

16. The method of claim 14, wherein the mutation is a deletion of at least ninety, more preferably one hundred 5' nucleotides of SEQ ID No. 1.

17. The method of any one of the preceding claims, wherein the increase is relative to a control or wild type plant.

18. The method of any one of claims 1-17, wherein the method further comprises regenerating a plant and screening the plant for an increase in at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content.

19. A plant obtained or obtainable by the method of any one of claims 1 to 18.

20. The plant of claim 19, wherein said plant is rice.

21. A genetically altered plant, a part of a plant cell thereof, wherein said plant comprises at least one mutation in at least one nucleic acid sequence encoding an NRT2.3 promoter.

22. The genetically altered plant of claim 21, wherein said nucleic acid sequence comprises SEQ ID NO 9 or a functional variant thereof.

23. The genetically altered plant of claim 22, wherein said nucleic acid sequence comprises SEQ ID No.1 or a functional variant thereof.

24. The genetically altered plant of claim 21, wherein the plant is characterized by an increase in at least one of yield, biomass, Nitrogen Utilization Efficiency (NUE), nitrogen transport, and/or an increase in at least one of nitrogen content.

25. The genetically altered plant of any one of claims 21 to 24, wherein said mutation is introduced into SEQ ID No.1, and preferably into SEQ ID No. 9.

26. The genetically altered plant of any one of claims 21 to 25, wherein said mutation is an insertion, deletion and/or substitution.

27. The genetically altered plant of claim 26, wherein the mutation is a substitution of at least one nucleotide.

28. The genetically altered plant of claim 27, wherein said substitution is at position 160, position 201, or position 222 of SEQ ID No. 1.

29. The genetically altered plant of claim 27, wherein the mutation is a substitution at position 160 of SEQ ID No. 1.

30. The genetically altered plant of claim 28, wherein the mutation is a T to C substitution.

31. The genetically altered plant of any one of claims 21 to 26, wherein said mutation is a deletion of at least one nucleotide.

32. The genetically altered plant of claim 31, wherein said mutation is a deletion of at least fifty, more preferably sixty 5' nucleotides of SEQ ID No. 1.

33. The genetically altered plant of claim 31, wherein said mutation is a deletion of at least ninety, more preferably one hundred 5' nucleotides of SEQ ID No. 1.

34. The genetically altered plant of any one of claims 21 to 33, wherein said increase is relative to a control or wild type plant.

35. The genetically altered plant of any one of claims 21 to 34, wherein said plant is rice.

36. A method for identifying and/or selecting for plants having or to have increased yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content, preferably as compared to a control or wild type plant, comprising detecting at least one polymorphism in the NRT2.3 promoter gene sequence in a plant or plant germplasm and screening said plant or its progeny.

37. The method of claim 36, wherein the NRT2.3 promoter gene sequence comprises SEQ ID No. 9, more preferably SEQ ID No.1 or a functional variant thereof.

38. The method of claim 36, wherein the polymorphism is at least one substitution at least position 160 of SEQ ID No. 1.

39. The method of claim 36, wherein the polymorphism is a deletion of at least one 5 'nucleotide of SEQ ID No.1, more preferably of at least the first sixty 5' nucleotides of SEQ ID No. 1.

40. The method of any one of claims 36 to 39, wherein said method further comprises introgressing the chromosomal region comprising the at least one polymorphism in the NRT2.3 promoter into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.

41. A method of increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content of a plant, the method comprising introducing and expressing in the plant a nucleic acid construct comprising an NRT2.3 promoter sequence operably linked to an 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.

42. A method for making a plant with increased yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content, said method comprising introducing into a plant or plant cell an expression nucleic acid construct comprising an NRT2.3 promoter sequence operably linked to an NRT2.3 gene sequence, wherein said NRT2.3 promoter sequence is selected from the group comprising SEQ ID NO:2, 3, 4 or 5 or a functional variant thereof.

43. The method of claim 41 or 42, wherein the NRT2.3 gene sequence comprises SEQ ID NO 8 or a functional variant thereof.

44. The method of any one of claims 41 to 43, wherein the plant is rice.

45. A plant obtained or obtainable by the method of claim 42.

46. A nucleic acid construct comprising an NRT2.3 promoter sequence operably linked to an NRT2.3 gene sequence, wherein said NRT2.3 promoter sequence is selected from the group comprising SEQ ID NO 2, 3, 4 or 5 or a functional variant thereof.

47. The nucleic acid construct of claim 46, wherein the NRT2.3 gene sequence comprises SEQ ID NO 8 or a functional variant thereof.

48. A vector comprising the nucleic acid construct of claim 46 or 47.

49. A host cell comprising the vector of claim 48 or the nucleic acid construct of claim 46 or 47.

50. A transgenic plant expressing the vector of claim 48 or the nucleic acid construct of claim 46 or 47.

51. The transgenic plant of claim 50, wherein the plant is rice.

52. Use of the vector of claim 48 or the nucleic acid construct of claim 46 or 47 to increase at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content in a plant.

53. A method of altering splicing of an NRT2.3 gene, said method comprising introducing at least one mutation into a nucleic acid sequence encoding an NRT2.3 promoter.

54. A nucleic acid construct comprising a nucleic acid sequence encoding at least one DNA binding domain capable of binding to at least one NRT2.3 promoter.

55. The nucleic acid construct of claim 54, wherein said nucleic acid sequence encodes at least one protospacer element, and wherein the sequence of said protospacer element is selected from SEQ ID Nos 16 to 23 or a sequence having at least 90% identity to SEQ ID Nos 16 to 23.

56. The nucleic acid construct of claim 54 or 55, wherein the construct further comprises a nucleic acid sequence encoding CRISPR RNA (crRNA) sequence, wherein the crRNA sequence comprises the protospacer sequence and additional nucleotides.

57. The nucleic acid construct of any one of claims 54 to 56, wherein 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.

58. The nucleic acid construct of any one of claims 54-57, wherein the construct encodes at least one single guide RNA (sgRNA), wherein the sgRNA comprises a tracrRNA sequence and a crRNA sequence.

59. The nucleic acid construct of any one of claims 54-58, wherein the construct is operably linked to a promoter.

60. The nucleic acid construct of claim 59, wherein the promoter is a constitutive promoter.

61. The nucleic acid construct of any one of claims 54-60, wherein the nucleic acid construct further comprises a nucleic acid sequence encoding a CRISPR enzyme.

62. The nucleic acid construct of claim 61, wherein the CRISPR enzyme is a Cas protein.

63. The nucleic acid construct of claim 62, wherein the Cas protein is Cas9 or a functional variant thereof.

64. The nucleic acid construct of claim 54, wherein the nucleic acid construct encodes a TAL effector.

65. The nucleic acid construct of claim 54 or 64, wherein the nucleic acid construct further comprises a sequence encoding an endonuclease or a DNA cleavage domain thereof.

66. The nucleic acid construct of claim 65, wherein the endonuclease is FokI.

67. An isolated plant cell transfected with at least one nucleic acid construct as defined in any of claims 54 to 66.

68. An isolated plant cell transfected with at least one nucleic acid construct according to any of claims 54 to 60 and a second nucleic acid construct, wherein said second nucleic acid construct comprises a nucleic acid sequence encoding a Cas protein, preferably a Cas9 protein or a functional variant thereof.

69. The isolated plant cell of claim 68, wherein the second nucleic acid construct is transfected before, after, or simultaneously with the nucleic acid construct of any of claims 54-60.

70. A genetically modified plant, wherein the plant comprises a transfected cell as defined in any of claims 67 to 69.

71. The genetically modified plant of claim 70, wherein a nucleic acid encoding the sgRNA and/or a nucleic acid encoding the Cas protein are integrated in a stable form.

72. A method of increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content of a plant, the method comprising introducing and expressing in a plant the nucleic acid construct of any one of claims 54 to 66, wherein preferably the increase is relative to a control or wild type plant.

73. A plant obtained or obtainable by the method of claim 72.

74. Use of a nucleic acid construct as defined in any of claims 54 to 66 for increasing at least one of yield, biomass, Nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content in a plant.

75. A method of obtaining a genetically modified plant as defined in any one of claims 70 to 71, said method comprising:

a. selecting a part of a plant;

b. transfecting at least one cell of the plant part of paragraph (a) with a nucleic acid construct as defined in any one of claims 54 to 66;

c. regenerating at least one plant derived from the transfected cells;