CN113924367B - Method for improving rice grain yield - Google Patents

Abstract

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

Description

Methods for increasing rice grain yield

Technical Field

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

Background technique

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

Rice is one of the most important staple foods in the world, consumed by more than 50% of the world's population, especially in Asia (FAO, 2015). It is also one of the most important food crops nutritionally. However, in the field, rice can be affected by many adverse conditions, which can lead to huge losses in yield. One of the main limiting factors affecting yield is low nitrogen availability.

Nitrogen (N) is fundamental to crop development as it is a building block of many organic molecules, nucleic acids, and proteins. Nitrogen nutrition affects all levels of plant function, from metabolism to resource allocation, growth, and development. The most abundant source of N available to plant roots is nitrate (NO3-) in natural aerobic soils due to the intense nitrification of applied organic and fertilizer N. In contrast, ammonium (NH4+) is the major form of N available in flooded rice fields due to anaerobic soil conditions (Sasakawa and Yamamoto, 1978).

Therefore, soil inorganic nitrogen (N) is available to plants mainly in the form of nitrate in aerobic uplands and well-drained soils, and in the form of ammonium in poorly drained soils and flooded anaerobic rice fields. In many plants, nitrate acquired by 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 stem in organic form. In response to 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-affinity transport system (LATS), responsible for nitrate acquisition. Constitutive HATS (cHATS) and nitrate-inducible HATS (iHATS) take up nitrate at low nitrate concentrations in the external medium, with a saturation range of 0.2-0.5 mM. In contrast, LATS functions in nitrate acquisition at higher external nitrate concentrations. Uptake by 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 status of the plant (Miller et al., 2007).

Despite the ability of higher plants to utilize organic nitrogen, the main sources of nitrogen acquisition by roots are thought to be ^NO3- and NH4. The relative adaptability of plants to these two N sources varies greatly. Although NH4 should be the preferred N source because its metabolism requires less energy than ^NO3- , only a few species actually perform well when NH4 is provided as the sole N source. The latter are boreal conifers, Ericaceae plants, some vegetable crops and rice (Oryza sativa L.). In contrast to these species, most agricultural species sometimes show severe toxicity symptoms on NH4, and therefore, it can be seen that these species grow faster on ^NO3- . However, when both N sources are provided simultaneously, growth and yield are usually significantly improved compared to growth on NH4 or ^NO3- alone (Kronzucker et al., 1999).

Rice differs from other crops in its ability to grow completely on NH4 as the sole nitrogen source. Rice is traditionally grown under flooded anaerobic soil conditions, where ammonium is the major nitrogen source. However, specialized aerenchyma cells in rice roots can transfer oxygen from the stem to the roots and release it into the rhizosphere, where bacterial conversion of ammonium to nitrate (nitrification) occurs. Nitrification in the flooded rice rhizosphere can result in 25-40% of the total crop N being absorbed as nitrate, primarily via the high affinity transport system (HATS). Nitrate uptake is mediated by co-transport with protons (H ⁺ ), ^which can be expelled from the cell by plasma membrane H ⁺ -ATPases. The molecular mechanisms of nitrate uptake and transport in rice are not fully understood. Since the nitrate concentration in the rhizosphere of rice fields is estimated to be less than 10 μM (Kirk and Kronzucker, 2005), NRT2 family members play a major role in nitrate uptake by rice (Araki and Hasegawa, 2006; Yan et al., 2011). Therefore, up to 40% of the total N uptake by rice roots grown under wetland conditions may be in the form of nitrate, and the uptake rate is 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 different 5'- and 3'-untranscribed regions (UTRs), showing high similarity to NRT2 genes from other monocots, while OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 genes. OsNRT2.3 mRNA is actually spliced into two gene products, OsNRT2.3a (AK109776) and OsNRT2.3b (AK072215), whose deduced amino acid sequences have 94.2% similarity (Feng et al., 2011; Yan et al., 2011). OsNRT2.3a is mainly expressed in roots, and this pattern is enhanced by nitrate supply, while OsNRT2.3b is weakly expressed in roots and relatively abundant in shoots, with no effect of N form and concentration on transcript numbers (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 security and to reduce the cost of mineral fertilizer inputs and negative environmental impacts, such as air and water quality and biodiversity loss. The present invention is intended to address this need.

Summary of the invention

We describe the generation of rice OsNRT2.3 mutant lines using targeted induced localized lesions in the genome (TILLING). We identified the resulting mutant lines as sharing 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 to NRT2.3a and, moreover, significantly increased 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 showed that the TATA-box is a key cis-regulatory element for the transcription of OsNRT2.3 into 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 showed that the TATA-box mutant in the 5'UTR of OsNRT2.3b and the binding protein OsTBP2.1 together increased 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 start codon of the OsNRT2.3 gene is important for the differential transcription of OsNRT2.3a and OsNRT2.3b and ultimately crop growth.

In one aspect of the invention, a method for increasing at least one of the yield, biomass, nitrogen utilization efficiency (NUE), nitrogen transport and/or nitrogen content of a plant is provided, the method comprising introducing at least one mutation into a nucleic acid sequence encoding a 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, mutations are introduced using mutagenesis. In a preferred embodiment, mutations are introduced using TILLING or T-DNA insertion. In an alternative embodiment, mutations are introduced using targeted genome modification, preferably ZFN, TALEN or CRISPR/Cas9.

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 5' nucleotides of SEQ ID NO: 1. In another embodiment, the mutation is a deletion of at least 90, more preferably 100 5' nucleotides 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, a genetically altered plant, or a part of a plant cell thereof, is provided, wherein the plant comprises at least one mutation in at least one nucleic acid sequence encoding a 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, a mutation is introduced into SEQ ID NO: 1, preferably into SEQ ID NO: 9. Preferably, the mutation is an insertion, a deletion and/or a 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 substitution from T to C.

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 5' nucleotides of SEQ ID NO: 1.

Preferably, the genetically altered plant is rice.

In another aspect of the invention, a method is provided for identifying and/or selecting plants having or which will have increased yield, biomass, nitrogen utilization efficiency (NUE), nitrogen transport and/or nitrogen content, preferably compared to control or wild-type plants, the method comprising detecting at least one polymorphism in the NRT2.3 promoter gene sequence in a plant or plant germplasm and selecting the plant or its progeny.

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 at 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 a deletion of at least the first 60 5' nucleotides of SEQ ID NO: 1.

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

In another aspect of the present invention, a method for increasing at least one of the yield, biomass, nitrogen utilization efficiency (NUE), nitrogen transport and/or nitrogen content of a plant is provided, the method comprising introducing and expressing a nucleic acid construct in the plant, the nucleic acid construct comprising a NRT2.3 promoter sequence operably linked to a NRT2.3 gene sequence, wherein the NRT2.3 promoter sequence is selected from the group comprising SEQ ID NO: 2, 3, 4 or 5 or a functional variant thereof.

On the other hand, a method for preparing a plant with increased yield, biomass, nitrogen utilization efficiency (NUE), nitrogen transport and/or nitrogen content is provided, the method comprising introducing and expressing a nucleic acid construct in a plant or plant cell, the nucleic acid construct comprising a NRT2.3 promoter sequence operably linked to a NRT2.3 gene sequence, wherein the NRT2.3 promoter sequence is selected from the group comprising SEQ ID NO: 2, 3, 4 or 5 or a functional variant thereof.

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

In another aspect, a vector is provided, the vector comprising the above nucleic acid construct. A host cell is also provided, the host cell comprising the vector or nucleic acid construct. Finally, a transgenic plant is also provided, the transgenic plant expressing the vector or nucleic acid construct. Preferably, the plant is rice.

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

In another aspect of the present invention, a nucleic acid construct is provided, 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 a sequence that is at least 90% identical to SEQ ID NOs 16 to 23.

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

Preferably, the construct further comprises a nucleic acid sequence encoding a trans-activating 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 an alternative embodiment, 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 Fok1.

In another aspect of the present invention, an isolated plant cell is provided, which is transfected with at least one nucleic acid construct as described above. In an alternative aspect, an isolated plant cell is provided, which 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 present invention, a genetically modified plant is provided, wherein the plant comprises the above-mentioned transfected cell. In one embodiment, the nucleic acid encoding the sgRNA and/or the nucleic acid encoding the Cas protein is integrated in a stable form.

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

In another aspect, there is provided 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 for obtaining a genetically modified plant as defined above, the method comprising:

a. Select a part of the plant;

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

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

One or more plants obtained according to paragraph (c) showing increased expression of NRT2.3b are selected.

In any of the above aspects of the invention, the increase is relative to control or wild-type plants.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1 shows the biomass, NUE and nitrogen content of -83bp mutant lines at maturity in the field. (a) Characterization of -83bp mutant lines of OsNRT2.3 mutation in field trials. Wild type, Zhonghua 11 (WT), mutant lines of T8, T11, T12 and T20. (Bar = 30 cm); (b) Nucleotide sequence of -83bp mutant lines; (c) Grain yield and dry weight per plant of mutant lines and WT plants grown in the field. The dry weight mean represents the aboveground biomass, excluding grain yield; (d) NUE of mutant lines; (e) Total N concentration in stems and roots in OsNRT2.3 TILLING lines and (f) Total N content in stems and roots in OsNRT2.3 TILLING lines. Error bars: SE (n = 5). Significant differences between mutant lines and WT are indicated by different letters. (P < 0.05, Student's t-test).

Figure 2 shows the characterization and identification of the -83bp mutant line in hydroponics. (a) Phenotypes of WT and -83bp mutant lines. (Bar = 10 cm); (b) Dry weight of -83bp mutant line; (c) Total N concentration in stem and root; (d) Total nitrogen content in stem and root. Error bars: SE (n = 6). Seedlings of wild-type Zhonghua 11 (WT) and mutant lines were grown in a solution of IRRI containing 1.25 mm NH4NO3 for 2 weeks. RNA was then extracted to analyze OsNRT2.3a/b expression; (e) qRT-PCR analysis of the expression ratio of OsNRT2.3b to OsNRT2.3a. Error bars: SE (n = 3). Different letters indicate significant differences between transgenic lines and WT (P < 0.05, Student's t-test).

Figure 3 shows the effect of OsNRT2.3 promoter mutation on OsNRT2.3a/b expression; (a, c) Schematic diagram of OsNRT2.3 promoter and different mutation sites that change the expression of OsNRT2.3a and OsNRT2.3b. P is the WT promoter of OsNRT2.3, P1 is the -665bp mutation in the OsNRT2.3 promoter, P2 is the -44bp mutation in the OsNRT2.3 promoter, and mP is the -83bp mutation in the OsNRT2.3 promoter; (b) The expression of OsNRT2.3b in different promoter lines promotes OsNRT2.3b; (d) The expression of OsNRT2.3b and OsNRT2.3a in the mp::OsNRT2.3 line; (e) The ratio of OsNRT2.3b to OsNRT2.3a in the mp::OsNRT2.3a line.

Figure 4 shows that the -83bp mutation alters the translation pattern of OsNRT2.3 in backcross lines; (a, b) Characterization of the B1F2 generation of OsNRT2.3 mutant lines in field trials. B1F2 of two independent lines T11 and T12 confirmed the hyperphenotype of the -83bp mutation. (Bar = 30 cm) AA is the control, -83bp without mutation; aa is the homozygous -83bp mutation; Aa is the heterozygous -83bp mutation; (c) Western blot analysis of OsNRT2.3b and HSP expression in leaves and roots. B1F3 T11 and B1F3 T12 are the B1F3 generation of OsNRT2.3 mutant backcross lines. WT and mutant backcross homozygous lines of 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 15NO3–, 1.25mM NH415NO3 and 1.25mM 15NH4NO3 were measured within 5 minutes; (d) 15N influx rate in the stem; (e) 15N influx rate in the root; (f) 15N ratio in the stem and root. Error bars: SE (n=5). Different letters indicate significant differences between transgenic lines and WT. (P<0.05, Student's t-test).

Figure 5 shows the effect of -83bp mutation of promoters of different lengths on transcription of rice OsNRT2.3; (a) Schematic diagram of OsNRT2.3 promoter fragments of different lengths and -83bp mutant fragments, which drive the expression of 437bp ORF and ZIIIB reporter gene of OsNRT2.3. 141bp and 697bp are the original OsNRT2.3 promoters; 141M and 697M carry -83bp mutation; (b-d) Transgenic rice seedlings of control lines (no mutation) and mutant lines were grown in a solution containing 1.25mm NH4NO3IRRI for 2 weeks. RNA was then extracted to analyze the expression of OsNRT2.3a/b. (b) shows the expression of OsNRT2.3a in transgenic lines; (c) shows the expression of OsNRT2.3b in transgenic lines; (d) shows the expression ratio of OsNRT2.3b/OsNRT2.3a in transgenic lines. Error bars: SE (n=3). Different letters indicate significant differences between transgenic lines and WT (P<0.05, Student's t-test).

Figure 6 shows that OsTBP2.1 binds to the OsNRT2.3 promoter fragment and activates OsNRT2.3 expression. (a) OsTBP2.1 binds to the TATA-box of the OsNRT2.3b 5'UTR. Yeast cells were co-transformed with pTATA-box::AbAi and OsTBP2/2.1/2.2::pGADT7. Cells were grown on medium to screen for interactions (SD, -Ura, -Leu) and (800nM). AbA was used to inhibit background growth. (b) Constructs for transient assays in rice protoplasts. The OsNRT2.3 promoter or the -83bp mutant promoter was used to drive expression of the reporter. pNRT2.3::Luc, pmNRT2.3::Luc. (c) OsTBP2.1 activates the OsNRT2.3 promoter. Expression of OsTBP2.1 is driven by the Ubi promoter. The reporter and effector were co-transformed into rice protoplasts. (d) Stable rice genetic vector construction framework. The reporter proteins-eGFP and mCherry were constructed in one vector, and the 141bp and 697bp promoters promoted the expression of eGFP and mCherry. (e) The level of eGFP. (f) The level of mCherry. Error bars: SE (n=3). (P<0.05, Student's t test).

Figure 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 OsTBP2.1 T-DNA mutant lines in field trials. Wild type, Huangyang (WT-HY). (Bar = 20 cm). (c) Expression of OsTBP2.1 in OEOsTBP2.1 and OsTBP2.1 lines. (d) Ratio of OsNRT2.3b to OsNRT2.3a in OsTBP2.1 overexpression and T-DNA mutant lines.

Figure 8 shows a schematic diagram of the transcription model of OsNRT2.3 upon TATA-box mutation. 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 leading to higher levels of OsNRT2.3b translation.

Figure 9 shows the characteristics of the TILLING lines in the field.

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

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

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

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

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

Figure 15 shows the influx of ¹⁵ NO3 ^- and ¹⁵ NH4 ⁺ into the OsNRT2.3 mutant line within 5 minutes. WT and OsNRT2.3 mutant seedlings were grown in 1.25 mM NH ₄ NO ₃ for 3 weeks and nitrogen starved for 1 week. Then the ¹⁵ N influx rates of 2.5 mM ¹⁵ NO3 ^- , 1.25 mM NH ₄ ¹⁵ NO ₃ and 1.25 mM NH ₄ ¹⁵ NO ₃ were measured within 5 minutes. (a) Root ¹⁵ N influx rate. (b) Stem ¹⁵ N influx rate. Error bars: SE (n = 5). Different letters indicate significant differences between transgenic lines and WT (P < 0.05, one-way ANOVA).

Figure 16 shows the influx of ¹⁵ NO3 ^- and ¹⁵ NH4 ⁺ into OsNRT2.3 mutant backcross lines over a 5-minute period. WT and mutant backcross homozygous lines of T11 and T12 were grown in 1.25 mM NH ₄ NO ₃ for 3 weeks and nitrogen starved for 1 week. The ¹⁵ N influx rates of 2.5 mM ¹⁵ NO3 ^- , 1.25 mM NH ₄ ¹⁵ NO ₃ , and 1.25 mM NH ₄ ¹⁵ NO ₃ were then measured over a 5-minute period. (a) Root ¹⁵ N influx rate. (b) Stem ¹⁵ N influx rate. Error bars: SE (n=5). Different letters indicate significant differences between transgenic lines and WT (P<0.05, one-way ANOVA).

Figure 17 shows nitrogen content in -83bp mutant backcross lines at maturity. Nitrogen content of leaves, sheaths, stems and ears of mutant backcross lines and controls.

DETAILED DESCRIPTION OF THE INVENTION

The rice transporter OsNRT2.3 has two splice forms, OsNRT2.3a and OsNRT2.3b. Some nitrate transporters require two genes to function; the second, smaller component (OsNAR21) is required for the transporter to be properly targeted to the plasma membrane. One of the two splice forms, OsNRT2.3a, requires this second component for function, while the other form, OsNRT2.3b, does not. We have previously shown that overexpressing OsNRT2.3b in rice improves growth and NUE.

We have now unexpectedly demonstrated that mutations 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, affect the splicing of the gene, resulting in an increased expression ratio of OsNRT2.3b to OsNRT2.3a. In addition, by targeting local lesions induced in the genome, we obtained many lines carrying mutations at the -83bp position of the OsNRT2.3 gene (relative to the ATG start codon of the NRT2.3 gene). Mutations in the sequence upstream of this position altered the transcription of the OsNRT2.3 gene and increased the ratio of OsNRT2.3b to OsNRT2.3a compared to the wild type. In addition, the backcross mutant lines increased the total biomass by about 28% and the NUE by about 75% compared to the field control plants. The weight per ear of the backcross mutant lines in the field also increased by about 60%. At the same time, the backcross mutant lines further improved the nitrogen absorption in the field compared to the control.

Therefore, we concluded that the mutation in the upstream sequence of the OsNRT2.3 gene at position -83 is key to controlling the splicing of the OsNRT2.3 gene, resulting in an increase in the relative expression of OsNRT2.3b and a positive effect on rice growth, yield and NUE.

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

Unless otherwise indicated, the practice of the present invention will employ conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are within the skill of the art and are fully explained 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, mutated, synthetic DNA or RNA molecules, and analogs of DNA or RNA produced using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, antisense sequences, and non-coding regulatory sequences that do not encode mRNA or protein products. These terms also encompass genes. The terms "gene" or "gene sequence" are widely used to refer to DNA nucleic acids associated with biological functions. Therefore, a gene may include introns and exons in a genomic sequence, or may only include a cDNA coding sequence, and/or may include a cDNA combined with a regulatory sequence.

Aspects of the present invention relate to recombinant DNA techniques and exclude embodiments based solely on the production of plants by traditional breeding methods and plants obtained 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, a method for increasing plant yield, biomass, nitrogen utilization efficiency (NUE), nitrogen transport and/or nitrogen content is provided, 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 the 224 nucleotides upstream (eg in the 5' direction) of the ATG start 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" 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, such as up to or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to control plants.

The term "yield" generally refers to a measurable product of economic value, usually associated with a specific crop, region and period of time. Individual plant parts directly contribute to yield by their number, size and/or weight. Actual yield is the yield per square meter for a crop and year, and is determined by dividing the total yield (including harvested and assessed yield) by the cultivated square meters.

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, above ground (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. The increased biomass may be expressed as g/plant or kg/hectare (b) increased seed yield per plant, which may include one or more of an increase in seed biomass (weight) on a per plant or individual basis, (c) increased seed filling rate, (d) increased number of filled seeds, (e) increased harvest index, which may be expressed as a ratio of 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 composition (i.e. lipids (also referred to herein as oil)), protein The results of changes in the total content and composition of carbohydrates, (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, such as 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 may be the result of increased seed size and/or seed weight, (p) reduced number of sterile tillers per plant and (q) sturdier or stronger culms 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 increases by at least or up to 40%, more preferably 50%, even more preferably 60%, compared to control plants.

In one example, the yield increase is at least or up to 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, 25%, 30%, 35%, 40%, 45% or 50% compared to a control or wild-type plant. Alternatively, the yield may alternatively increase by 20-70%, more preferably 25-75%, compared to a control plant.

The term "nitrogen use 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 use efficiency of a plant includes uptake and utilization efficiency and can be calculated as UpE. In one embodiment, NUE is increased by 5%-80% or more compared to control plants, such as by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% compared to control plants. In a preferred embodiment, NUE is increased by at least 60%, more preferably 70%, even more preferably 75% 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 can refer to the absorption of ammonium, nitrate and/or ammonium nitrate. In one embodiment, nitrogen influx in the stem and/or roots of a 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 a further embodiment, the nitrogen content in at least one of the leaves, leaf sheaths, stems and spikes is increased. In a further embodiment, the total nitrogen content in the plant is not increased.

"At least one mutation" means that when the NRT2.3 promoter gene exists in more than one copy or homologues (having the same or slightly different sequences), at least one mutation is present in at least one gene. Preferably, all genes are mutated.

As shown in FIG5 a , the NRT2.3a gene contains a 43 bp 5′UTR (untranslated region), while the NRT2.3b gene contains a 223 bp 5′UTR.

In one embodiment, the method includes introducing at least one mutation into a preferably endogenous NRT2.3 promoter. As used herein, "promoter" encompasses a nucleic acid sequence (start or start codon) of 1.5 kbp upstream of the ATG. As used herein, the term "promoter" includes the 5'UTR (untranslated region) of 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 the splicing of the NRT2.3 gene, in particular the mutation increases the relative expression of NRT2.3b to NRT2.3a. Compared to wild-type plants, the increase in relative expression is at least 2 times, more preferably at least 4 times, more preferably at least 6 times, even more preferably at least 8 times. In an alternative embodiment, the mutation increases the expression of NRT2.3b by at least 5 times, more preferably 6 times, even more preferably 7 times, 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, the expression of NRT2.3b in the stem of a plant is increased and/or the expression of NRT2.3a in the root of a plant is reduced.

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 natural sequence in a plant genome. 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 NO: 1 to 8 refers to a variant gene sequence or a portion of the gene sequence that retains the biological function of the complete 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, such as in non-conservative residues. Also included are variants that are substantially identical to the wild-type sequence shown herein, i.e., have only some sequence variations, such as in non-conservative residues, and have biological activity. Each modification proposed is within the routine technical scope of 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% or more of a similar nucleic acid or amino acid sequence to a non-variant nucleic acid or amino acid sequence. 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity.

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

In a further embodiment, the variant used herein may comprise a nucleic acid sequence encoding a NRT2.3 promoter as defined herein, which is capable of hybridizing to a nucleic acid sequence 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 higher degree than hybridization to other sequences (e.g., at least 2-fold above background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of hybridization and/or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probing).

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

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

1. "Insertion mutation" of one or more nucleotides;

2. "Deletion mutation" of one or more nucleotides;

3. "Substitution mutation", resulting in the replacement of at least one nucleotide by at least one different nucleotide.

4. "Inversion" mutation, which is a 180-degree rotation of the nucleic acid sequence.

In one embodiment, the mutation is the deletion of at least one nucleotide in the NRT2.3 promoter, wherein preferably, the NRT2.3 promoter comprises SEQ ID NO:1 or its functional variant or consists of it. In a preferred embodiment, the mutation is the deletion of at least one nucleotide at the 5' end of SEQ ID NO:1. More preferably, the mutation is the deletion of at least the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 nucleotides at the 5' end of SEQ ID NO:1. In a more preferred embodiment, the mutation is the deletion of at least the first 50, more preferably the first 60, and even more preferably the first 62 nucleotides at the 5' end of SEQ ID NO:1. In an alternative embodiment, the mutation is the deletion of the first 90, more preferably the first 100, and even more preferably the first 101 nucleotides at the 5' end of SEQ ID NO:1. In an alternative embodiment, the mutation is the 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 (again, may be referred to herein as position -21). At position -83 (relative to the ATG start codon), there are two putative motifs, designated OSE2ROOTNODULE (-82 bp to -86 bp) (SEQ ID NO: 10) and ASF1MOTIFCAMV (-76 bp to -83 bp) (SEQ ID NO: 11). We believe that the OSE2ROOTNODULE motif may control the tillering process and that the ASF1MOTIFCAMV motif is a repressor binding motif. It is possible that the loss of the gene repressor binding function of this motif in the mutant lines leads to the changes in the expression ratios observed in these lines.

In a further preferred embodiment, the substitution 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): 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, 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 changes the binding ability 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 OSE2ROOTNODULE motif and/or the ASF1MOTIFCAMV motif. Preferably, the mutation results in a loss of function or partial loss of one or both motifs. As described above, we believe that the OSE2ROOTNODULE 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 repressor bind. More preferably, the OSE2ROOTNODULE motif is defined in SEQ ID NO: 10 or a variant thereof and the ASF1MOTIFCAMV motif is defined in SEQ ID NO: 11 or a variant thereof. 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 deletions that remove promoter or enhancer functional regions, as these can affect the splicing of the NRT2 gene. For example, a mutation may result in the deletion of the TATA-box. In other words, the deletion of SEQ ID NO:9.

In one embodiment, the 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-mentioned genetic engineering methods, and does not cover naturally occurring varieties.

Targeted genome modification or targeted genome editing is a genome engineering technology 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 major classes of customizable DNA-binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcription factors, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and RNA-guided DNA endonucleases Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats). Meganucleases, ZFs, and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. While meganucleases integrate nuclease and DNA binding domains, ZF and TALE proteins consist of separate modules that target 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of FokI to direct nucleic acid hydrolysis activity to specific genomic sites.

After delivery to host cells via 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 a central domain of 33-35 amino acid repeats in tandem. This is followed by a single truncated repeat of 20 amino acids. Most naturally occurring TAL effectors examined have 12 to 27 complete repeats.

These repeats are distinguished only by two adjacent amino acids, their repeat variable diresidues (RVDs). The RVD determines which single nucleotide the TAL effector will recognize: one RVD corresponds to one nucleotide, and the four most common RVDs each preferentially bind to one of the four bases. The naturally occurring recognition sites are uniformly preceded by a T required for TAL effector activity. TAL effectors can be fused to the catalytic domain of the FokI nuclease to create TAL effector nucleases (TALENs), thereby generating targeted DNA double-strand breaks (DSBs) in vivo for genome editing. The use of this technology in genome editing is well described in the art, for example in US 8,440,431, US 8,440,432, and US 8,450,471. Cermak 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 GoldenGate method uses a type IIS restriction endonuclease that cuts outside its recognition site to produce a unique 4bp overhang. Cloning can be accelerated by digestion and ligation in the same reaction mixture because correct assembly eliminates enzyme recognition sites. The assembly of a custom TALEN or TAL effector construct includes two steps: (i) assembling the repeat modules into an intermediate array of 1-10 repeats and (ii) ligating the intermediate array into a backbone to prepare the final construct. Thus, TAL effectors targeting the NRT2.3 promoter sequence 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 present invention is CRISPR. The use of this technology in genome editing is also well described in the art, such as in US8,697,359 and the references cited herein. In short, CRISPR is a microbial nuclease system that participates in the defense against invading phages and plasmids. The CRISPR locus in the microbial host contains a combination of CRISPR-related (Cas) genes and non-coding RNA elements that can program CRISPR-mediated nucleic acid cleavage (sgRNA) specificity. Three types (I-III) of CRISPR systems have been identified in a wide range of bacterial hosts. A key feature of each CRISPR locus is the presence of a series of repetitive sequences (direct repeats), separated by a short non-repetitive sequence (spacer). The non-coding CRISPR array is transcribed and cut into short crRNAs containing a single spacer sequence in the direct repeat sequence, which guide the Cas nuclease to the target site (protospacer). Type II CRISPR is one of the most fully characterized systems, which performs targeted DNA double-strand breaks in four consecutive steps. First, two non-coding RNAs, pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Secondly, tracrRNA hybridizes with the repeat region of pre-crRNA and mediates the processing of pre-crRNA into mature crRNA containing a single spacer sequence. Third, the mature crRNA:tracrRNA complex guides Cas9 to the target DNA through Watson-Crick base pairing between the spacer on the crRNA and the original spacer adjacent to the original spacer motif (PAM) on the target DNA, which is an additional requirement for target identification. Finally, Cas9 mediates the cutting of the target DNA, producing double-strand breaks in the original spacer.

A major advantage of the CRISPR-Cas9 system over traditional gene targeting and other programmable endonucleases is the ease of multiplexing, which allows multiple genes to be mutated simultaneously by using multiple sgRNAs, each targeting a different gene. In addition, if two sgRNAs are used to flank a genomic region, the middle portion can be deleted or inverted.

Cas9 is thus the hallmark protein of the type II CRISPR-Cas system and is a large monomeric DNA nuclease that is guided to a DNA target sequence adjacent to a PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand, while the RuvC-like domain cleaves the non-complementary strand, thus, introducing blunt cuts in the target DNA. Heterologous expression of Cas9 with sgRNA can introduce site-specific double-strand breaks (DSBs) into genomic DNA of living cells from various organisms. For applications 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 the Cas9 nuclease. sgRNA is a synthetic RNA chimera produced by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, sgRNAs with different target specificities can be created. The standard length of the guide sequence is 20bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3. Therefore, sgRNA molecules targeting the NRT2.3 promoter sequence as described herein can be designed using techniques known in the art.

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

Alternatively, more conventional mutagenesis methods can be used to introduce at least one mutation into the NRT2.3 promoter sequence. These methods include physical mutagenesis and chemical mutagenesis. The skilled person will know that other methods can be used to produce such mutants, and methods for mutagenesis and polynucleotide changes are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al., (1987) Methods in Enzymol. 154: 367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, ed. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein.

In one embodiment, insertion mutagenesis is used, for example, T-DNA mutagenesis (which inserts a T-DNA fragment from the T-plasmid of Agrobacterium tumefaciens into DNA, resulting in loss of gene function or acquisition of gene function mutation), site-directed nuclease (SDN) or transposon as a mutagen. Insertion mutagenesis is another method of destroying gene function, which is based on inserting exogenous DNA into the target gene (see Krysan et al., The Plant Cell, Vol. 11, 2283-2290, December 1999). Therefore, in one embodiment, T-DNA is used as an insertion mutagen to destroy the activity of the NRT2.3 promoter. In an example, T-DNA can be inserted into SEQ ID NO:1 or its functional variants. T-DNA not only destroys the expression of the gene inserted into it, but also can be used as a marker for subsequent identification of mutations. Since the sequence of the inserted element is known, various clones or PCR-based strategies can be used to restore the gene inserted. Inserting a length of 5 to 25kb level T-DNA usually destroys gene function. If enough T-DNA transformed lines are generated, there is a high probability of finding transgenic plants carrying T-DNA insertions in any gene of interest. Transformation of spores with T-DNA is achieved by the Agrobacterium-mediated method, which involves exposing plant cells and tissues to a suspension of Agrobacterium cells.

The details of this method are well known to technicians. In short, the transformation of plants by Agrobacterium results in the integration of a sequence known as T-DNA into the nuclear genome, which is carried by a bacterial plasmid. 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 quickly characterized by direct sequencing and analysis of the DNA inserted flanking. The gene expression of NRT2.3b in the mutant or the relative expression of NRT2.3a and NRT2.3b is compared with that in wild-type plants. Phenotypic analysis was also performed.

In another embodiment, the mutagenesis is physical mutagenesis, such as the 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 a variant thereof.

In another embodiment of the various aspects of the invention, the method comprises mutagenizing the plant population with a mutagen. The mutagen can be 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 (EM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimethylbenz (a) anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, disulfane, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), etc.), 2-methoxy-6-chloro-9 [3- (ethyl-2-chloroethyl) aminopropylamino] acridine dihydrochloride (ICR-170) or formaldehyde. Again, the target population can then be screened to identify NRT2.3 promoter mutants.

In another embodiment, the method for generating and analyzing mutations is targeted local damage induced in the genome (TILLING), which is reviewed in Henikoff et al., 2004. In this method, seeds are induced with chemical mutagens such as EMS. The resulting M1 plants are self-fertilized, and the M2 generation individuals are used to prepare DNA samples for mutation screening. The DNA samples are collected and arranged on microtiter plates, and gene-specific PCR is performed. Any method for identifying heteroduplexes between wild-type and mutant genes can be used to screen NRT2.3 promoter mutations of PCR amplification products. For example, but not limited to, denaturing high pressure liquid chromatography (dHPLC), constant denaturing capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (TGCE), or by using chemical cutting fragmentation. Preferably, the PCR amplification product is incubated with a nuclease that preferentially cuts the mismatch in the heteroduplex between the wild-type and mutant sequences. The cleavage product is electrophoresed using an automatic sequencing gel device, and the gel image is analyzed with the help of a standard commercial image processing program. Any primer specific for the NRT2.3 promoter sequence can be used to amplify the NRT2.3 promoter sequence in the combined DNA sample. Preferably, the primers are designed to amplify the NRT2.3 promoter region where useful mutations are most likely to occur, particularly in the NRT2.3 promoter region that is highly conserved and/or confers activity, as described elsewhere. To facilitate detection of PCR products on gels, the PCR primers can be labeled using any conventional labeling method. In an alternative embodiment, the method for generating and analyzing mutations is EcoTILLING. EcoTILLING is a molecular technique similar to TILLING, except that its goal is to reveal the natural variation in a given population rather than inducing mutations. The first disclosure of the EcoTILLING method was described in Comai et al. 2004.

Rapid high throughput screening procedures allow analysis of amplification products to identify mutations in the NRT2.3 promoter, particularly mutations in SEQ ID NO: 1 compared to corresponding non-mutagenized wild type plants. Once a mutation is identified, seeds from M2 plants carrying the mutation are grown into adult M3 plants and screened for phenotypic characteristics associated with the mutations in the NRT2.3 promoter described herein.

Plants carrying mutations in the endogenous NRT2.3 promoter, in particular in SEQ ID NO: 1 or 9 or variants thereof or obtainable by such a method are also within the scope of the present invention.

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

In another aspect of the present invention, a method for altering NRT2.3 gene splicing and/or increasing the relative expression of NRT2.3b and NRT2.3a and/or increasing the expression of NRT2.3b and/or decreasing the expression of NRT2.3a is provided, 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, a method for increasing the yield, biomass, nitrogen utilization efficiency (NUE), nitrogen transport and/or nitrogen content of a plant is provided, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding a TATA binding protein (TBP), preferably TBP2.1. In one embodiment, the nucleic acid sequence encodes a 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 TBP. The present invention also includes a transgenic plant characterized in that the expression level of NRT2.3b is increased compared to a wild-type plant, wherein the plant expresses the above-mentioned nucleic acid construct.

Genetically altered or modified plants and methods for producing such plants

In another aspect of the present invention, a genetically altered plant, part thereof or plant cell is provided, characterized in that the plant has increased relative expression of NRT2.3b to NRT2.3a compared to a wild-type or control plant. In an alternative embodiment, the plant is characterized in that NRT2.3b expression increases compared to a wild-type or control plant. Alternatively, the plant is characterized in that NRT2.3a expression decreases compared to a wild-type or control plant. Preferably, the increase or decrease may be 1 times, 2 times, 3 times, times, 5 times, 6 times, 7 times, 8 times or 9 times higher than the expression level in the wild-type or control plant. Alternatively, the increase or decrease may be up to or higher than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the wild-type or control plant level.

In another aspect of the invention, a genetically altered plant, part thereof or plant cell is provided, wherein the 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 mutations, preferably deletions, insertions and/or substitutions, in the NRT2.3b 5'UTR or NRT2.3a promoter sequence by any of the above methods. Preferably, the mutation is introduced into at least one plant cell and a plant regenerated from 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 of preferably at least one plant cell using any mutagenesis technique 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, the selected plants comprise at least one mutation in the NRT2.3b 5'UTR or the NRT2.3a promoter. In one embodiment, as described herein, the plant is characterized by an increase in the relative expression of NRT2.3b to NRT2.3a, an increase in the level of NRT2.3b, and/or a decrease in the expression of NRT2.3a, as described herein. The expression level of the NRT2.3b 5'UTR or the 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, such as by clonal propagation or classical breeding techniques. For example, the first generation (or T1) transformed plants can be selfed and homozygous second generation (or T2) transformants can be selected, and then the T2 plants can be further propagated by classical breeding techniques. The transformed organisms produced can take a variety of forms. For example, they can be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells are transformed to contain expression cassettes); grafts of transformed and untransformed tissues (e.g., in plants, transformed stock is grafted onto untransformed young branches).

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, "genetically altered plants" or "mutant plants" are plants that have been genetically altered compared to naturally occurring wild-type (WT) plants. In one embodiment, mutant plants are plants that have been altered compared to naturally occurring wild-type (WT) plants using a mutagenesis method, such as any mutagenesis method 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 a mutagenesis method compared to the wild-type sequence. Such plants have a phenotype of changes as described herein, such as increased yield, biomass, NUE, nitrogen transport and/or nitrogen content. Therefore, in this example, the increased yield, biomass, NUE, nitrogen transport and/or nitrogen content are conferred by the presence of the altered plant genome, for example, a mutated NRT2.3b 5'UTR or NRT2.3a promoter sequence. In one embodiment, an endogenous promoter sequence is specifically targeted using targeted genome modification, and the presence of a mutant gene or promoter sequence is not conferred by the presence of a transgene expressed in the plant. In other words, genetically altered plants can be described as being free of transgenes.

Transgenic plants

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

Therefore, in wild-type or control plants, overexpression of a mutant promoter operably linked to a NRT2.3 gene will also increase yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or nitrogen content.

Therefore, in another aspect of the present invention, a nucleic acid construct is provided, comprising a NRT2.3 promoter sequence operably linked to a NRT2.3 gene sequence, wherein the NRT2.3 promoter sequence is selected from the group comprising SEQ ID NO: 2, 3, 4 or 5 or a functional variant thereof. 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 connection between a promoter sequence and a target gene, such that the promoter sequence is able to initiate transcription of the target gene.

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

In another aspect of the present invention, a host cell comprising a nucleic acid construct is provided. The host cell can be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell. The present 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, a transgenic plant expressing the above nucleic acid construct is 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 can be used to introduce the target gene into a suitable ancestral cell. The method described for transforming and regenerating plants from plant tissue or plant cells can be used for instantaneous or stable transformation. The transformation method includes using liposomes, electroporation, increasing chemicals absorbed by free DNA, injecting DNA directly into plants, particle gun bombardment, using viruses or pollen to transform and micro-injection. The method can be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection to plant materials, DNA or RNA coated particle bombardment, infection with (non-integrated) viruses, etc. Transgenic plants, including transgenic crop plants, are preferably produced by transformation mediated by Agrobacterium tumefaciens.

In order to select transformed plants, the plant material obtained in the conversion is usually subjected to selective condition treatment, so that transformed plants are distinguished from unconverted plants. For example, the seeds obtained in the above manner can be planted, and suitable selection is carried out by spraying after the initial growth period. Another possibility is that, if appropriate, after sterilization, a suitable selection agent is used to grow seeds on agar plates, so that only the seeds converted can grow into plants. Or, screening is performed to determine whether there is a selection marker, such as one of the above-mentioned markers, in the transformed plants. After DNA transfer and regeneration, the plant presumed to be converted can also be assessed, such as using Southern analysis, to determine whether there is a target gene, copy number and/or genomic organization. Or or in addition, Northern and/or Western analysis can be used to monitor the expression level of the newly introduced DNA, and these two technologies are well known to those of ordinary skill in the art.

The transformed plants produced can be propagated in a variety of ways, such as by clonal propagation or classical breeding techniques. For example, the first generation (or T1) transformed plants can be selfed and homozygous second generation (or T2) transformants can be selected, and then the T2 plants can be further propagated by classical breeding techniques. The transformed organisms produced can take a variety of forms. For example, they can be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells are transformed to contain expression cassettes); grafts of transformed and untransformed tissues (e.g., in plants, transformed stock is grafted onto untransformed young branches).

Unless otherwise stated, 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, and all plant parts and propagules thereof. The invention further extends to include progeny of primary transformed or transfected cells, tissues, organs or whole plants 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 parent in the method according to the invention.

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

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

In another aspect of the present invention, a method for producing plants having at least one of increased yield, biomass, nitrogen utilization efficiency (NUE), nitrogen transport and/or nitrogen content is provided, the method comprising introducing and expressing a nucleic acid construct as described herein in the plant.

The increase is relative to control or wild-type plants.

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

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

The present invention also extends to harvestable parts of the plants of the present invention as described herein, such as grains. Aspects of the present invention also extend to products derived from, preferably directly derived from, harvestable parts of such plants, i.e., cereals, such as, but not limited to, rice bran, rice bran oil, rice flour, rice husks, rice starch, husk ash, broken rice, and brewer's rice. In another aspect of the present invention, products derived from plants as described herein or parts thereof are provided.

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

According to all aspects of the present invention, the control plant used herein is a plant that is not modified according to the method of the present invention. Therefore, in one embodiment, the control plant does not have at least one mutation in the NRT2.3 promoter. In an alternative embodiment, the plant does not have genetic modification as described above. In one embodiment, the control plant is a wild-type plant. The control plant is typically the same plant species, preferably has the same genetic background as the modified plant.

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

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

"tracrRNA" (trans-activating RNA) means an RNA sequence that hybridizes to crRNA and binds to a CRISPR enzyme, such as Cas9, thereby activating the nuclease complex to introduce a double-stranded break at a specific site within the genomic sequence of at least one NRT2.3 promoter nucleic acid or promoter sequence.

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

"sgRNA" (single guide RNA) refers to a combination of tracrRNA and crRNA in a single RNA molecule, preferably also including a linker loop (which connects tracrRNA and crRNA into a single molecule). "sgRNA" may also be referred to as "gRNA" and in this context, these terms are interchangeable. sgRNA or gRNA provides targeting specificity and scaffold/binding ability for Cas nuclease. gRNA may refer to a dual RNA molecule comprising a crRNA molecule and a tracrRNA molecule.

"TAL effector" (transcription activator-like (TAL) effector) or TALE means a protein that can bind to a genomic DNA target sequence (a sequence within the NRT2.3 promoter gene or a promoter sequence) and can be fused to the cleavage domain of an endonuclease such as FokI to produce a TAL effector nuclease or TALENS or a large range of nucleases to produce a megaTAL. The TALE protein consists of a central domain responsible for DNA binding, a nuclear localization signal, and a domain that activates transcription of the 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, of which the two amino acids at positions 12 and 13 are highly variable (repeat variable diresidues, RVD). The RVD is responsible for recognizing a single specific nucleotide. HD targets cytosine; NI targets adenine, NG targets thymine, and NN targets guanine (although NN can also bind to adenine, the specificity is lower).

In another aspect of the present invention, a nucleic acid construct is provided, wherein the nucleic acid construct encodes at least one DNA binding domain, wherein the DNA binding domain can bind to a sequence in the NRT2.3 promoter gene, wherein the sequence is selected from SEQ ID NO 12 to 15 or a variant thereof, as defined herein. In one embodiment, the construct further comprises a nucleic acid encoding a (SSN) sequence-specific nuclease such as a 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 technician will know the appropriate sequence of additional nucleotides, as these are defined by the selection of the Cas protein.

In another embodiment, the nucleic acid construct further comprises a tracrRNA sequence. Likewise, suitable tracrRNA sequences are known to the skilled person because 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 an sgRNA (or gRNA). Again, as already discussed, the sgRNA typically comprises a crRNA sequence, a tracrRNA sequence and preferably comprises a sequence for a linker loop. In a preferred embodiment, the nucleic acid construct comprises at least one nucleic acid sequence encoding an sgRNA sequence as defined herein.

In a further embodiment, the nucleic acid construct may further include at least one nucleic acid sequence encoding an endoribonuclease cleavage site. Preferably, the endoribonuclease is Csy4 (also referred to as Cas6f). When the nucleic acid construct comprises multiple sgRNA nucleic acid sequences, the construct may comprise the same number of endoribonuclease cleavage sites. In another embodiment, the cleavage site is 5' of the sgRNA nucleic acid sequence. Therefore, the flank of each sgRNA nucleic acid sequence is an endoribonuclease cleavage site.

The term "variant" refers to a nucleotide sequence in which nucleotides are substantially identical to one of the above-mentioned sequences. Variant can be achieved by modification such as insertion, substitution or deletion of one or more nucleotides. In a preferred embodiment, 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 with any one of the above-mentioned sequences. In one embodiment, the sequence identity is at least 90%. In another embodiment, the sequence identity is 100%. The sequence identity can be determined by any sequence alignment program known in the art.

The present invention also relates to nucleic acid constructs, the nucleic acid constructs comprising a nucleic acid sequence operably connected to a suitable plant promoter. Suitable plant promoters can be constitutive or strong promoters, or can be tissue-specific promoters. In one embodiment, suitable plant promoters are selected from, but not limited to, tuberose yellow leaf curl virus (CmYLCV) promoter or switchgrass ubiquitin 1 promoter (PvUbil) U6 RNA polymerase III (TaU6) CaMV35S, U6 or corn ubiquitin (e.g. Ubil) promoter. Alternatively, expression can be specifically directed to a specific tissue of rice seeds by a gene expression regulatory sequence. In one embodiment, promoters are selected from U3 promoters (SEQ ID NO:25), U6a promoters (SEQ ID NO:26), U6b promoters (SEQ ID NO:27), U3b promoters (SEQ ID NO:28) and U6-1 promoters (SEQ ID NO:29) in dicotyledons.

The nucleic acid construct of the present invention may further comprise a nucleic acid sequence encoding a CRISPR enzyme. "CRISPR enzyme" means an RNA-guided DNA endonuclease that can be combined with a CRISPR system. Specifically, this enzyme is combined with a tracrRNA sequence. In one embodiment, the CRISPR enzyme is a Cas protein ("CRISPR associated protein"), preferably Cas 9 or Cpf1, more preferably Cas9. In a specific embodiment, Cas9 is a codon-optimized Cas9, and more preferably, has a sequence described 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, such as C2c1, C2C2 and/or C2c3. In one embodiment, the Cas protein is from Streptococcus pyogenes. In alternative embodiments, the Cas protein may be from any of Staphylococcus aureus, Neisseria meningitides, Streptococcus thermophiles, or Treponema denticola.

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, such as acting as a DNA endonuclease, or recognizing and/or binding to DNA. Functional variants also include variants of the target gene that have sequence changes that do not affect function, such as non-conservative residues. Also included are variants that are substantially the same as the wild-type sequence shown herein, i.e., have only some sequence variations, such as in non-conservative residues, and have biological activity. 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 with the amino acid represented by SEQ ID NO:30. In a further embodiment, the Cas9 protein has been modified to increase activity.

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

In a further embodiment, the Cas9 protein has been modified to increase activity. For example, in one embodiment, the Cas9 protein may include a D10A amino acid substitution, and the nickase only cuts the DNA chain that is complementary to the gRNA and recognized by the gRNA. In an alternative embodiment, the Cas9 protein may alternatively or additionally include an H840A amino acid substitution, and the nickase only cuts the DNA chain that does not interact with the sRNA. In this embodiment, Cas9 can be used with a pair (i.e., two) of sgRNA molecules (or a construct expressing such a pair), so that the target region on the opposite DNA chain can be cut, and it is possible to increase the specificity by 100-1500 times. In a further embodiment, the cas9 protein may include a D1135E substitution. The Cas 9 protein may also be a VQR variant. Alternatively, the Cas protein may include mutations in the two nuclease domains of HNH and RuvC, and is therefore catalytically inactive. This catalytically inactive Cas protein can be used to prevent the transcription extension process, rather than cutting the target chain, and when co-expressed with the sgRNA molecule, it will result in the loss of protein function that is not fully translated. An example of a catalytically inactive protein is dead Cas9 (dCas9) caused by point mutations in the RuvC and/or HNH nuclease domains (Komor et al., 2016 and Nishida et al., 2016).

In further embodiments, Cas proteins, such as Cas9, can be further fused to inhibitory effectors, such as histone modification/DNA methylases or cytidine deaminases (Komor et al., 2016) to achieve site-directed mutagenesis. In the latter, cytidine deaminases do not induce double-stranded DNA breaks, but mediate the conversion of cytidine to uridine, thereby achieving C to T (or G to A) substitutions.

In a further embodiment, the nucleic acid construct comprises an endoribonuclease. Preferably, the endoribonuclease is Csy4 (also referred to as Cas6f), more preferably rice codon-optimized csy4. In one embodiment, when the nucleic acid construct comprises a cas protein, the nucleic acid construct may comprise a sequence for expressing an endoribonuclease, such as Csy4, expressed as a 5' end P2A fusion (used as a self-cleaving peptide) to a cas protein, such as Cas9.

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

Suitable methods for producing CRISPR nucleic acids and vector systems are known, for example, as published in Molecular Plant (Ma et al., 2015, Molecular Plant, DOI: 10.1016/j.molp.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. Given a target sequence, methods for designing TAL effectors will be well known to the skilled person. 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, this SSN is an endonuclease, such as FokI. In a further embodiment, TALEN is assembled in a single plasmid or nucleic acid construct by the GoldenGate cloning method.

In another aspect of the invention, an sgRNA molecule is provided, wherein the sgRNA molecule comprises a crRNA sequence and a tracrRNA sequence, and wherein the crRNA sequence can bind to at least one sequence selected from SEQ ID NOs 12 to 15 or a variant thereof. "Variant" is as defined herein. In one embodiment, the sgRNA molecule may comprise at least one chemical modification, such as to enhance its stability and/or binding affinity to the target sequence or the crRNA sequence and the tracrRNA sequence. Such modifications are well known to those skilled in the art and include, for example, but not limited to, modifications described in Rahdar et al., 2015, which are incorporated herein by reference. In this example, the crRNA may comprise a phosphorothioate backbone modification, such as 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), and S-constrained ethyl (cET) substitutions.

In another aspect of the present 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 present invention, a plant or part thereof or at least one plant cell isolated by transfection with at least one nucleic acid construct as described herein is provided. Cas9 and sgRNA can be combined or in a separate expression vector (or nucleic acid construct, such terms are interchangeable). In other words, in one embodiment, the plant cell isolated is transfected with a single nucleic acid construct comprising sgRNA and Cas9 as described in detail above. In an alternative embodiment, the plant cell isolated is transfected with two nucleic acid constructs, the first nucleic acid construct comprising at least one sgRNA as defined above, and the second nucleic acid construct comprising Cas9 or its functional variant or homologue. The second nucleic acid construct can be transfected below, after or simultaneously with the first nucleic acid construct. The advantage of the separate second construct comprising 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 therefore not limited to a single cas function (such as when cas and sgRNA are encoded on the same nucleic acid construct, this is the case).

In one embodiment, a nucleic acid construct comprising a cas protein is first transfected and stably integrated into the genome before a second transfection with a nucleic acid construct comprising at least one sgRNA nucleic acid is performed. In an alternative embodiment, the plant or part thereof or at least one isolated plant cell is transfected with an mRNA encoding a cas protein and co-transfected with at least one nucleic acid construct as defined herein.

The Cas9 expression vector for 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 homolog thereof, wherein the nucleic acid sequence is operably linked to a suitable promoter. Examples of suitable promoters include actin, CaMV35S, U6, U3, or maize ubiquitin (e.g., Ubil) promoters.

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

Therefore, in a further aspect of the present invention, a method for regulating NRT2.3 promoter activity and/or NRT2.3 gene splicing and/or increasing NRT2.3b expression is provided, the method comprising introducing and expressing a CRISPR construct as described above or introducing an sgRNA molecule as described above into a plant. In other words, a method for regulating NRT2.3 promoter activity and/or NRT2.3 gene splicing and/or increasing NRT2.3b expression is also provided, as described herein, wherein the method comprises introducing at least one mutation into an endogenous NRT2.3 promoter using 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 present invention, there is provided a plant comprising a genetic modification or editing of a transfected cell as described herein. In one embodiment, one or more nucleic acid constructs can be integrated in a stable form. In an alternative embodiment, one or more nucleic acid constructs are not integrated (i.e., transiently expressed). Therefore, in a preferred embodiment, the genetically modified plant does not contain any sgRNA and/or Cas protein nucleic acid. In other words, the plant does not contain a transgenic.

The terms "introduction", "transfection" or "transformation" as referred to herein include the transfer of exogenous polynucleotides into a host cell, regardless of the method used for transfer. Plant tissues that can subsequently be cloned by organogenesis or embryogenesis can be transformed with the genetic construct of the present invention, and a complete plant is regenerated therefrom. The specific tissue selected will vary depending on the clonal propagation system that is available for and most suitable for the specific species to be transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, female gametophytes, callus, existing meristems (e.g., apical meristems, axillary buds, and root meristems) and induced meristems (e.g., cotyledon meristems and hypocotyl meristems). 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 exogenous genes into a plant genome is called transformation. Plant transformation is now a routine technique for many species. Any of several transformation methods known to technicians can be used to introduce a target nucleic acid construct or sgRNA molecule into a suitable ancestral 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 the plant (microinjection), gene guns (or biolistic particle delivery systems (biology) as described in the Examples), lipofection, transformation using viruses or pollen and microprojection. The method may be selected from the calcium/polyethylene glycol method for protoplasts, ultrasound-mediated gene transfection, optical or laser transfection, transfection using silicon carbide fibers, electroporation of protoplasts, microinjection into plant material, bombardment with DNA or RNA-coated particles, infection with viruses, etc. (non-integrating). Transgenic plants can also be produced by Agrobacterium tumefaciens-mediated transformation, including but not limited to the use of the floral dip/Agrobacterium vacuum infiltration method 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 nucleic acid construct described herein can be first transcribed to form a preassembled Cas9-sgRNA ribonucleoprotein and then delivered to at least one plant cell using any of the above methods, such as lipofection, electroporation, or microinjection.

Optionally, in order to select transformed plants, the plant material obtained in the transformation is usually subjected to selective condition treatment so that transformed plants can be distinguished from untransformed plants. For example, seeds obtained in the above manner can be planted, and suitable selection is carried out by spraying after the initial growth period. Another possibility is, if appropriate, after sterilization, seeds are planted on an agar plate using a suitable selection agent, so that only the transformed seeds can grow into plants. As described in the examples, suitable markers can be bar-phosphine or PPT. Alternatively, the presence of selective markers such as but not limited to GFP, GUS (β-glucuronidase) is screened for transformed plants. Technicians are easily aware of other examples. Alternatively, no selection is performed, the seeds obtained in the above manner are planted and grown, and NRT2.3b expression or protein levels are measured at the appropriate time using standard techniques in the art. This alternative scheme to avoid the introduction of transgenics is more suitable for producing plants that do not contain transgenics.

After DNA transfer and regeneration, the putative transformed plants can also be evaluated, for example, using PCR to detect the presence of the target gene, copy number and/or genomic organization. Alternatively or additionally, Southern, Northern and/or Western analysis can be used to monitor the integration and expression level of the newly introduced DNA, both of which techniques are well known to those of ordinary skill in the art.

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

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

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

a. Select a part of the 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-mentioned transfection or transformation techniques;

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

d. selecting one or more plants obtained according to paragraph (c) which show increased expression of NRT2.3b or increased relative expression of NRT2.3b (relative to NRT2.3a).

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

In a further embodiment, the method comprises generating stable T2 plants, preferably homozygous for the mutation (ie, a mutation in at least one NRT2.3 promoter sequence).

Plants with mutations in at least one NRT2.3 promoter sequence can also be crossed with another plant that also contains at least one mutation in at least one NRT2.3 promoter sequence to obtain plants with additional mutations in the NRT2.3 promoter sequence. These combinations are obvious to the skilled person. Thus, this method can be used to generate T2 plants with mutations in all homologs or an increased number of homologs when compared to the number of homologous mutations in a single T1 plant transformed as described above.

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

The genetically modified plants of the present invention can also be obtained by crossing to transfer any sequence of the present invention, for example, using pollen of the genetically modified plants described herein to pollinate wild-type or control plants, or using other pollen that does not contain a mutation in at least one NRT2.3 promoter sequence to pollinate the pistil of the plants described herein. The methods for obtaining the plants of the present invention are not limited to those described in this paragraph; for example, the genetic transformation of reproductive cells can be performed as described above, but there is no need to regenerate plants afterwards.

Methods for screening plants for naturally increased grain yield phenotypes

In a further aspect of the invention, a method is provided for screening a plant population and identifying and/or selecting plants carrying or expressing at least one mutation in the NRT2.3 promoter, preferably at least one mutation in the NRT2.3b 5'UTR or the NRT2.3a promoter. , More preferably at least one mutation in SEQ ID NO:1 or 9 or a variant thereof. Alternatively, a method is provided for screening a plant population and identifying and/or selecting plants with increased yield, biomass, nitrogen utilization efficiency (NUE), nitrogen transport and/or nitrogen content. In either 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 a variant 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 the deletion of at least one nucleotide in the NRT2.3 promoter, wherein preferably the NRT2.3 promoter comprises SEQ ID NO:1 or consists of it. In a preferred embodiment, the polymorphism is the deletion of at least one nucleotide at the 5' end of SEQ ID NO:1. More preferably, the polymorphism is the deletion of at least the first 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 nucleotides at the 5' end of SEQ ID NO:1. In a more preferred embodiment, the polymorphism is the deletion of at least the first 50, more preferably the first 60, and even more preferably the first 62 nucleotides at the 5' end of SEQ ID NO:1. In an alternative embodiment, the polymorphism is the deletion of the first 90, more preferably the first 100, and even more preferably the first 101 nucleotides at 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 alternative embodiments, the polymorphism is a replacement of at least one nucleotide. In a preferred embodiment, the polymorphism is a replacement of at least one nucleotide at position 160 of SEQ ID NO:1 (this may also be referred to herein as a polymorphism at position-83 of the ATG start codon about the NRT2.3 gene), position 201 of SEQ ID NO:1 (may also be referred to herein as position-42) and position 222 of SEQ ID NO:1 (may also be referred to herein as position-21). In a preferred embodiment, the polymorphism is a replacement at position 160 of SEQ ID NO:1. In a further preferred embodiment, the replacement 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, 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, at least one polymorphism affects the binding ability of a TATA binding protein such as TBP2.1 (thereby increasing the expression level of NRT2.3b).

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 person and include, but are not limited to, isozyme electrophoresis, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), arbitrarily initiated polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), sequence signature amplification region (SCAR), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR-also known as microsatellites) and single nucleotide polymorphisms (SNPs). In one embodiment, competitive allele-specific PCR (KASP) genotyping is used.

In one embodiment, the method comprises

a) obtaining a nucleic acid sample from the plant, and

b) using one or more pairs of primers to perform nucleic acid amplification on the NRT2.3 promoter sequence.

In a further embodiment, the method may also include introgressing a chromosomal region comprising a NRT2.3 promoter sequence polymorphism into a second plant or plant germplasm to produce an introgressed plant or plant germplasm. Preferably, the second plant will show an increase in yield, biomass, nitrogen use efficiency (NUE), nitrogen transport and/or nitrogen content.

Although the above disclosure provides a general description of the subject matter included within the scope of the present invention, including methods of making and using the present invention and the best mode thereof, the following examples are provided to further enable one skilled in the art to practice the present invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the details of these examples should not be construed as limitations on the present invention, and the scope of the present invention should be understood from the claims appended to the present disclosure and their equivalents. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in light of this disclosure.

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

Unless the context dictates otherwise, the descriptions and definitions of the features described above are not limited to any particular aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

The above-mentioned application, and all documents and serial accession numbers cited therein or during the prosecution thereof ("application-cited documents") and all documents cited or referenced in documents cited in the application, and all documents cited or referenced herein ("herein-cited documents") and all documents cited or referenced in documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any product mentioned herein or any document 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.

Example

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 min and then rinsed thoroughly with water. Seedlings were grown in an artificial climate chamber with 16 h of light (30°C) and 8 h of darkness (26°C). Plants for field trials were grown at Nanjing Agricultural University, Nanjing, Jiangsu. The chemical properties of the experimental field soil were described by Chen et al. (Chen et al., 2016)

Identification of OsNRT2.3 TILLING lines

When the seedlings were grown in IRRI for 3 weeks, DNA samples were collected and stored at -80°C. DNA samples were extracted using the 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 sequences are provided by company name The sequences of the amplified TILLING lines were aligned with the sequence of Zhonghuo11 (WT) using DNAMAN.

Generation of transgenic plants

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

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

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

Yeast one-hybrid assay

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

Quantitative real-time PCR

Total RNA was extracted from the root and stem of the seedlings using Trizol reagent. 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 reaction, the template was reacted 3 times using AceQ qPCR SYBR Green Master Mix kit on Applied Biosystems (ABI) Plus real-time PCR system.

Western blotting

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

Determination of nitrogen absorption using 15N

WT, T11/WT(aa) and T12/WT(aa) lines were grown in IRRI nutrient solution for 3 weeks and then grown under N starvation for 3 days. Plants were initially moved to 0.1 mM CaSO ₄ for 1 minute, then they were moved to nutrient solution containing 2.5 mM 15NO ₃ - and 1.25 mM NH ₄ 15NO ₃ for 5 minutes, respectively, and then moved to 0.1 mM CaSO ₄ again for 1 minute. All plants were placed at 105°C for 30 minutes to inactivate the enzyme. The roots and stems were separated. They were then dried in an oven at 70°C for 7 days, and the dried samples were ground. About 1 mg of powder from each sample was analyzed using an isotope ratio mass spectrometer system (Thermo Fisher Scientific). The influx of 15NO ₃ - was calculated based on the 15N concentrations of the roots and stems.

Nitrate content analysis

The strains were placed at 105°C for 30 minutes to inactivate the enzymes. The samples were dried in an oven at 75°C for 3 days and the dry weight was recorded as the biomass value. The samples were ground into powder form. The digestion of the samples was done using H ₂ SO ₄ -H ₂ O _2. The total nitrogen content was determined on a continuous flow autoanalyzer (model Auto analyzer 3, Bran & Luebbe, Germany) as described by Leleu, 2004.

Promoter activity analysis

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

RNA-seq analysis

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

result

The -83 bp region upstream of the translation start codon OsNRT2.3 is essential for rice development

Mutant lines T8, T11, T12, and T20 were obtained using TILLING (Targeted Local Lesions Induced in Genomes) (Tsai et al., 2011). (Fig. 9, Fig. 1a). We extracted DNA from these lines to determine the location of the mutation. The results showed that T8, T11, and T12 all carried mutations at -83 bp, upstream of the translation start codon OsNRT2.3. (Fig. 1b). However, the T20 line did not carry a mutation upstream of the translation start codon of the gene OsNRT2.3. (Fig. 1b). In the field, grain yield, dry weight, and NUE were increased compared with WT and T20. (Fig. 1c, d). Although there was no difference in total N concentration between leaves, sheaths, stems, and panicles, the total N content of stems and panicles increased compared with WT and T20 lines (Fig. 1e, f). Therefore, the mutation of the site upstream of OsNRT2.3 ATG improves the nitrogen transport efficiency, thereby promoting rice growth.

We then cultured the four strains and WT in IRRI nutrient solution. The data showed that the T8, T11, and T12 strains showed significant growth and biomass compared to the wild type, but there was no difference in the T20 strain (Figure 2a, b). The total nitrogen concentration in the stem and root of the -83bp mutant and T20 strains was not different from that of the WT (Figure 2c), but the total nitrogen content in the stem and root of the -83bp mutant was increased compared to the WT and T20 strains (Figure 2d). The expression data of OsNRT2.3a and OsNRT2.3b showed that OsNRT2.3a was downregulated in the T8, T11, and T12 strains compared to the WT and T20, but OsNRT2.3b was upregulated (Figure 10a, b). Therefore, the mutant at -83bp upstream of the ATG of OsNRT2.3 increased the ratio of OsNRT2.3b to OsNRT2.3a (Figure 2e).

Furthermore, as shown in Figure 15 , for all three types of ¹⁵ N sources studied, significantly higher ¹⁵ N contents were accumulated in the roots ( Figure 15 a) and stems ( Figure 15 b) in the T8, T11, and T12 lines compared to W. The T20 line did not show any significant differences compared to WT in either the roots or stems ( Figures 15 a, b).

Figure 17 also shows the nitrogen content in the -83bp mutant backcross lines at the mature stage. The nitrogen content in leaves, leaf sheaths, stems and ears of the mutant backcross lines and controls in T8, T8/WT (aa) (loss of -83bp mutation) lines was compared with WT. There were no significant differences between WT and T8/WT (aa) in all parts of the plant compared to WT.

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

The results showed that the -83bp site mutation in the OsNRT2.3 promoter significantly promoted the expression of OsNRT2.3b compared with no mutation or mutations at other sites (Fig. 3a, b). When we used the OsNRT2.3 promoter with the -83bp site mutation to drive the expression of OsNRT2.3a, the expression of OsNRT2.3a was downregulated compared with the non-mutant line, however, the expression of OsNRT2.3b was significantly upregulated (Fig. 3c, d). The -83bp mutation upstream of the OsNRT2.3 ATG could also increase the ratio of OsNRT2.3b to OsNRT2.3a (Fig. 3e). Therefore, we concluded that the -83bp region upstream of the OsNRT2.3 ATG is essential for the translation of OsNRT2.3 into OsNRT2.3a and OsNRT2.3b and is therefore important for rice development.

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

To further verify that the -83bp mutation site upstream of OsNRT2.3 can affect the transcription of OsNRT2.3, we crossed T11 and T12 lines with WT. We obtained homozygous (aa), heterozygous (Aa), and non-mutant (AA) lines of T11 and T12 backcrosses (Figures 4a, b and 11). Sequence results showed that there was only one mutation at -83bp upstream of the ATG of the OsNRT2.3 gene (Figures 12a, b). Compared with WT, the expression of OsNRT2.3a was downregulated in T11/WT (aa) and T12/WT (aa) lines, however, OsNRT2.3b was upregulated. (Figure 12c). Compared with the non-mutant (AA) line at the maturity stage, the yield, dry weight, and nitrogen use efficiency of the homozygous (aa) lines T11/WT (aa) and T12/WT (aa) were significantly increased. (Figure 13)

We next analyzed the OsNRT2.3b protein levels in leaves and roots of T11/WT(aa) and T12/WT(aa) by Western blotting and found that the OsNRT2.3b protein levels were increased in leaves of T11/WT(aa) and T12/WT(aa) compared with WT, but not in roots. (Fig. 4c). Previous results showed that OsNRT2.3a is mainly responsible for the long-distance transport of nitrate from roots to stems, while OsNRT2.3b is mainly responsible for stems. (Tang et al., 2012; Yan et al., 2011) Knockout of OsNRT2.3a increased nitrate accumulation in roots, while overexpression of OsNRT2.3b increased nitrate absorption and transport. (Tang et al., 2012; Fan et al., 2016). To determine the effect of the -83 bp site mutant upstream of the OsNRT2.3 translation start codon on different forms of nitrogen application, the effect of the -83 bp mutation on 15NO ₃ - and NH ₄ 15NO ₃ uptake within 5 min was measured at pH 5.5 (Figure 4d, e). Compared with WT, the backcross lines showed more 15N uptake into the stem in the 5 min treatment experiment (Figure 4d). However, the uptake of 15N in the roots was significantly increased only in the roots applied with 15NO ₃ - but not NH ₄ 15NO ₃ (Figure 4e). Treatment with 15NO ₃ - and NH ₄ 15NO ₃ also significantly increased the concentration of stem ¹⁵ N in the backcross lines compared with the wild type (Figure 4f). In summary, we conclude that the -83 bp mutation in the 5'UTR of OsNRT2.3b improves N uptake efficiency and transport efficiency.

In addition, Figure 16 shows the influx of ¹⁵ NO ₃ - and ¹⁵ NH ₄ + in the OsNRT2.3 mutant backcross lines within 5 minutes. WT and T11 and T12 mutant backcross homozygous lines were grown in 1.25 mM NH ₄ NO ₃ for 3 weeks and nitrogen starved for 1 week. Then, the ¹⁵ N influx rates of 2.5 mM ¹⁵ NO ₃ -, 1.25 mM NH ₄ ¹⁵ NO ₃ and 1.25 mM ¹⁵ NH ₄ NO ₃ were measured within 5 minutes. (a) Root ¹⁵ N influx rate. (b) Shoot ¹⁵ N influx rate. Error bars: SE (n=5) Different letters indicate significant differences between transgenic lines and WT (P<0.05, one-way ANOVA).

Different promoter lengths of OsNRT2.3 alter the transcription of OsNRT2.3a and OsNRT2.3b

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

To confirm that the -83bp site mutation affects the expression of OsNRT2.3a and OsNRT2.3b at different promoter lengths, we performed -83bp site mutation on 141bp and 697bp promoters. We then designed vectors to drive the expression of the 437bp ORF of OsNRT2.3a and the reporter gene ZIIIB using either the non-mutated or -83bp site mutant promoter (Figure 5a). Real-time PCR statistics showed that the expression of OsNRT2.3a was downregulated when the expression was driven by the mutant promoter (141bp or 697bp promoter) compared to the expression level of OsNRT2.3a driven by the equivalent non-mutated promoter (Figure 5b). However, when the mutation occurred on the short promoter (141bp promoter), the expression of OsNRT2.3b was reduced compared to the expression level of the unmutated 141bp promoter. In contrast, when the mutation occurred on the long promoter (697 bp), the expression of OsNRT2.3b was upregulated compared with the expression level on the equivalent (697 bp) non-mutated promoter (Fig. 5c). Overall, the data suggest that regardless of whether the mutation is on the short or long promoter, it can promote the transcription of OsNRT2.3b but not OsNRT2.3a (Fig. 5d). In conclusion, the -83 bp mutation can alter the transcriptional regulation of OsNRT2.3 through promoters of different lengths.

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

Using Softberry's website ( http://linux1.softberry.com/berry.phtml ), we analyzed the sequence before the ATG of OsNRT2.3 and identified that the -83bp mutant sequence was 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 TATA-box 83bp upstream of OsNRT2.3, we performed a yeast one-hybrid assay. The results showed that the binding protein OsTBP2.1 could interact with the TATA box-pAbAi reporter gene grown on SD/-Leu medium containing 800ng ml ^-1 aurobasidin A (AbA ^r ). Other effectors failed to grow. (Figure 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 -83bp mutant line. The reporter and effector vectors shown in Figure 6b were constructed and co-transformed into rice protoplasts. As shown in Figure 6c, the greatest activation was observed when pmNRT2.3::Luc and pUbi::TBP2.1 were co-transformed into rice protoplasts. It is noteworthy that the luciferase expression levels in the Pnrt2.3::Luc and pUbi::TBP2.1 lines were lower than those in the pmNRT2.3::Luc and pUbi::TBP2.1 lines (Figure 6c). The lowest levels of luciferase expression were observed in the single transformed lines pNRT2.3::Luc and pmNRT2.3::Luc (Figure 6c). We next constructed the expression vectors shown in Figure 6d and obtained transgenic seedlings. Compared with the null mutant lines with different promoter lengths, the protein levels of OsNRT2.3a and OsNRT2.3b were increased (Fig. 6e, f). Taken together, these results suggest that the transcription factor OsTBP2.1 can increase the transcription of OsNRT2.3 to OsNRT2.3b.

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

To investigate the regulation of OsTBP2.1 on OsNRT2.3, we obtained overexpression and T-DNA mutant lines. (Fig. 7a, b). As shown in Fig. 7c, the grain weight and dry biomass of the overexpression lines increased compared with WT-W27, while the grain weight and biomass of the T-DNA mutant lines decreased. This was associated with the increase in OsTBP2.1 and OsNRT2.3b expression in the OE198 and OE200 overexpression lines compared with WT (WT-W27) and the decrease in OsTBP2.1 and OsNRT2.3b expression in the T-DNA mutant line (1A-19324) compared with WT Huangyant (WT-HY) (Fig. 7d). 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. 7e)).

discuss

During growth and development, the response of plants to various environmental and developmental signals requires the precise expression of functional genes. In these processes, some transcription factors play important roles. For example, NLP transcription factors play an important role in the regulation of nitrate in higher plants. (Konishi et al., 2019). Our study showed that the TATA-box binding factor OsTBP2.1 regulates multiple pathways, including the nitrate pathway. Our experiments showed that co-transfection of OsTBP2.1 with the OsNRT2.3 promoter could increase protein levels. (Figure 6b, c) In the OsTBP2.1 overexpression line, the ratio of OsNRT2.3b to OsNRT2.3a increased, but in the ostbp2.1T-DNA line, the ratio decreased. (Figure 7d). In summary, OsTBP2.1 can enhance the expression of OsNRT2.3b and reduce the expression of OsNRT2.3a to affect rice development. When the TATA box was mutated to TACA, the binding ability of OsTBP2.1 was increased, resulting in higher expression levels of OsNRT2.3b.

Key cis-acting elements play an important role in the transcriptional regulation of each gene. The TATA-box located in the 5'UTR of OsNRT2.3b but upstream of the 5'UTR of OsNRT2.3a controls the 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 increase in the ratio of OsNRT2.3b to OsNRT2.3a. (Figure 9). Thus, we conclude that the transcription of OsNRT2.3 to OsNRT2.3a and OsNRT2.3b is mainly regulated by the TATA-box on the OsNRT2.3 promoter, and therefore the TATA-box plays an important regulatory role in the splicing of OsNRT2.3 to 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 affect gene expression (Chung et al., 2006) as well as key cis-acting elements at the promoter and at the 5'UTR containing the first intron (Hernandez-Garcia and Finer, 2014; Gallegos and Rose, 2015). In our study, the TATA-box mutant in the 5'UTR of OsNRT2.3b increased the ratio of OsNRT2.3b to OsNRT2.3a, resulting in improved yield and growth.

references

Araki R, Hasegawa H. Expression of rice(Oryza sativa L.) genes involved in high-affinity nitrate transport during the period of nitrateinduction. Breed. Sci. 2006; 56:295–302

Cermak,T et al.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting.Nucleic acid Res.39(2011).

Chen J, Zhang Y, Tan Y, Zhang M, Zhu L, Xu G, Fan X. 2016. Agronomicnitrogen-use efficiency of rice can be increased by driving OsNRT2.1expression with the OsNAR2.1 promoter. Plant Biotechnology Journal 14:1705 –1715.

Chung B Y, Simons C, Firth A E, et al. Effect of 5’UTR introns on gene expression in Arabidopsis thaliana[J]. Bmc Genomics, 2006, 7(1):120-0.

Clough SJ, Bent AF. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6):735-43.

Comai L,Young K,Till BJ,Reynolds SH,Greene EA,Codoma CA,Enns LC,Johnson JE,Burtner C,Odden AR,Heinkoff.2004.Efficient discovery of DNApolymorphisms in natural populations by Ecotilling.Plant J.37(5 ):778-86.

Crawford NM, Glass ADM. 1998. Molecular and physiological aspects of nitrate uptake in plants. Trends Plant Sci 3:389–395.

Fan X,Feng H,Tan Y,Xu Y,Miao Q,Xu G.2016.A putative 6-transmembranenitrate transporter OsNRT1.1b plays a key role in rice under lownitrogen.Journal of Integrative Plant Biology 58:590–599.

Fan Sciences:201525184.

Fan –11.

FAO.2015.FAO Statistical Pocketbook 2015.

Feng H, Fan X, Yan M, Liu

Feng,Q.,Zhang,Y.,Hao,P.,Wang,S.,Fu,G.,Huang,Y.,Li,Y.etal.2002.Sequence and analysis of rice chromosome 4.Nature 420:316 –320.

Forde BG. 2000. Nitrate transporters in plants: structure, function and regulation. Biochim Biophys Acta 1465:219–235.

Gallegos JE, Rose AB. 2015. The enduring mystery of intron-mediatedenhancement. Plant Science 237:8–15.

Henikoff S, Till BJ, Comai L. 2004. TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol. 135(2):630-6.

Hernandez-Garcia CM, Finer JJ. 2014. Identification and validation of promoters and cis-acting regulatory elements. Plant Science 217–218:109–119.

Jingguang C,Yong Z,Yawen T.2016.The Effects of OsNRT2.1 Over-expression on Plant Growth and Nitrogen use Efficiency in Rice Nipponbare(Oryza sativa L.ssp.japonica).14.

Kirk GJ, Kronzucker HJ. 2005. The potential for nitrification and nitrate uptake in the rhizosphere of wetland plants: a modeling study. Annalsof Botany 96:639–646.

Komor,A.C.;Kim,Y.B.;Packer,M.S.;Zuris,J.A.;Liu,D.R.2-16.Programmable editing of a target base in genomic DNA without double-stranded DNAcleavage.Nature,533,420–424

Konishi M, Yanagisawa S. 2019. The role of protein-protein interactions mediated by the PB1 domain of NLP transcription factors in nitrate-induciblegene expression. BMC Plant Biology 19(1):90.

Kronzucker,H.J.,Siddiqi,M.Y.,Glass,A.D.,&Kirk,G.J.(1999).Nitrate-ammonium synergism in rice.A subcellular flux analysis.Plant physiology,119(3),1041–1046.

Krysan PJ,Young JC,Sussman MR.1999.T-DNA as an insertional mutagen in Arabidopsis.Plant Cell.11(12):2283-90.

Kunkel TA.1985.Rapid and efficient dite-specifc mutagenesis without phenotypic selection.PNAS.82(2):488-92.

Kunkel TA,Roberts JD,Zakour RA.1987.Rapid and efficient dite-specifcmutagenesis without phenotypic selection.Methods Enzmol.154.367-82.

Leleu O, Vuylsteker C. 2004. Unusual regulatory nitrate reductaseactivity in otyledons of Brassica napus seedlings:enhancement of nitratereductase activity by ammonium supply. J Exp Bot 55:815–823

Ma X,Zhang Q,Zhu Q,Liu W,Chen Y,Qiu R,Wang B,Yang Z,Li H,Lin Y,Xie Y,Shen R,Chen S,Wang Z,Chen Y,Guo J,Chen L ,Zhao

Miller AJ, Fan

Nishida K,et al.2016.Targeted nucleotide editing using hybridprokaryotic and vertebrate adaptive immune systems.Science 353,aaf8729

Orsel M,Krapp A,Daniel-Vedele F.2002.Analysis of the NRT2 nitratetransporter family in Arabidopsis Structure and gene expression.PlantPhysiology 129:886-896.

Orsel M.,Krapp A.,Daniel-Vedele F.2002.Analysis of the NRT2 nitratetransporter family in Arabidopsis.Structure and gene expression.Plant Physiol129:886–896

Okamoto M, Vidmar J J, Glass A D M. 2003. Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana: responses to nitrate provision. PlantCell Physiology 44:304-317.

Peng X, Wang H, Jang J, Xiao T, He H, Jiang D, Tang X. 2016. OsWRKY80-OsWRKY4Module as a Positive Regulatory Circuit in Rice Resistance Against Rhizoctonia solani.

Radhar M,McMahon MA,Prakash TP,Swayze EE,Bennett F,ClevelandDW.2015.Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.PNAS.112(51)E7110-E7117.

Sanjana NE, et al. A transcription activator-like effector toolbox forgenome engineering. Nat Protoc. 2012;7(1):171–192

Sasaki, T., Matsumoto, T., Yamamoto, K. et al. 2002. The genome sequence and structure of rice chromosome 1. Nature 420:312–316.

Sasakawa,H.and Yamamoto,Y.1978.Comparison of the uptake of nitrateand ammonium by rice seedlings.Plant Physiology 62:665–669.

Schortemeyer M, Feil B., Stamp P. 1993. Root morphology and nitrogenuptake of maize simultaneously supplied with ammonium and nitrate in a split-root system. Ann Bot 72:107–115

Tang Z, Fan

Tegeder M, Rentsch D. 2010. Uptake and partitioning of amino acids and peptides. Molecular Plant 3:997–1011.

Toki S.1997. Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Molecular Biology Reporter 15:16–21.

Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H. 2006. Earlyinfection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant Journal 47:969–976.

Tsai H,Howell T,Nitcher R,Missirian V,Watson B,Ngo KJ,Lieberman M,Fass J,Uauy C,Tran RK,et al.2011.Discovery of rare mutations in populations:tilling by sequencing.Plant Physiology 56( 3):1257-1268.

Upadhyaya NM, Surin B, Ramm K, Gaudron J, Schunmann PHD, Taylor W, Waterhouse PM, Wang MB. 2000. Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers. Aust J Plant Physiol 27:201– 210.

Xu G,Fan X,Miller AJ 2012.Plant nitrogen assimilation and useefficiency.Annu Rev Plant Biol 63:153–182.

Yan M, Fan Environment 34:1360-1372.

Sequence Listing

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

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

SEQ ID NO: 2 NRT2.3 promoter with polymorphism (underlined) at -81 upstream of ATG

1 GACGCGAGCG CGGAGACGGC AGCGCCGGCC TCCCACCGGT CGCGTAAGAT CACGCCCGAA

61 ATCTTTATTC ATTTTCTCTC CACCGGTTGC CCTCTCGCCG CACCCAACCA TCGCGCCACG

121 CCGCGCCGCG CTGCCGGAGC CGCGCTTTCC GCTATGCTA C AAGAGCTGAC GCGCAGGGCA

181 CAGCGGATGT ACGTACACAC AGTCACTAGC TAAGCTGCTA GCCTTGCTAC CACGTGTTGG

241 AG

SEQ ID NO: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 GCTATGCTA C AAGAGCTGAC GCGCAGGGCA

181 CAGCGGATGT ACGTACACAC C GTCACTAGC TAAGCTGCTA G T CTTGCTAC CACGTGTTGG

241 AG

SEQ ID NO:4 180 C-terminal residues of NRT 2.3 promoter

CTTTATTC ATTTTCTCTC CACCGGTTGC CCTCTCGCCG CACCCAACCA TCGCGCCACG

CCGCGCCGCG CTGCCGGAGC CGCGCTTTCC GCTATGCTA C AAGAGCTGAC GCGCAGGGCA

CAGCGGATGT ACGTACACAC C GTCACTAGC TAAGCTGCTA G T CTTGCTAC CACGTGTTGG

AG

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

ACCCAACCA TCGCGCCACG CCGCGCCGCG CTGCCGGAGC CGCGCTTTCC GCTATGCTA C AAGAGCTGAC GCGCAGGGCA CAGCGGATGT ACGTACACAC C GTCACTAGC TAAGCTGCTA G T CTTGCTACCACGTGTTGG AG

SEQ ID NO:6 Deletion of 62 5' nucleotides of NRT2.3 promoter

GACGCGAGCG CGGAGACGGC AGCGCCGGCC TCCCACCGGT CGCGTAAGAT CACGCCCGAA AT

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

GACGCGAGCG CGGAGACGGC AGCGCCGGCC TCCCACCGGT CGCGTAAGATCACGCCCGAAATCTTTATC ATTTTCTCTC CACCGGTTGC CCTCTCGCCG C

SEQ ID NO:8 NRT 2.3 genome sequence

GAGCGCCGGCCTCCCACCGGTCCGGCGTAAGATCACGCCCGAAATCTTTATTCATTTTCTCCCACCGGTTGCCCTCTCGCCGCACCCAACCATCGCGCCACGCCGCGCCGCGCTGCCGGAGCCGCGCTTTCCGCTATGCTATAAGAGCTGACGCGCAGGGCACAGCGGATGTACGTACACACAGTCACTAGCTAAGCTGCTAGCCTTGCTACCACGTGTTGGAGATGGAGGCTAAGCCGGTGGCGATGGA GGTGGAGGGG GTCGAGGCGGCGGGGGGCAAGCCCGGTTCAGGATGCCGGTGGACTCCGACCTCAAGGCGACGGAGTTCTGGCTCTTCTCCTTCGCGAGGCCACACATGGCCTCCTTCCACATGGCGTGGTTCTCCTTCTTCTGCTGCTTCGTGTCCACGTTCGCCGCGCCGCCGCTGCTGCCGCTCATCCGCGACACCCTCGGGCTCACGGCCACGGACATCGGCAACGCCGGGATCGCGTCCCGTGTCGGGCGCCGTGTT CGCGCGTC TGGCCATGGGCACGGCGTGCGACCTGGTCGGGCCCAGGCTGGCCTCCGCGTCTCTGATCCTCCTCACCACACCGGCGGTGTACTGCTCCTCCATCATCCAGTCCCCGTCGGGGTACCTCCTCGTGCGCTTCTTCACGGGCATCTCGCTGGCGTCGTTCGTGTCGGCGCAGTTCTGGATGAGCTCCATGTTCTCGGCCCCCAAAGTGGGGCTGGCCAACGGCGTGGCCGGCGGCTGGGGCAACCTCGGCGGCGGCG CCGT CCAGCTGCTCATGCCGCTCGTGTACGAGGCCATCCACAAGATCGGTAGCACGCCGTTCACGGCGTGGCGCATCGCCTTCTTCATCCCGGGCCTGATGCAGACGTTCTCGGCCATCGCCGTGCTGGCGTTCGGGCAGGACATGCCCGGCGGCAACTACGGGAAGCTCCACAAGACTGGCGACATGCACAAGGACAGCTTCGGCAACGTGCTGCGCCACGCCCTCACCAACTACCGCGGCTGGATCCTGGCGCTCACCTACG GCTACAGCTTCGGCGTCGAGCTCACCATCGACAACGTCGTGCACCAGTACTTCTACGACCGCTTCGACGTCAACCTCCAGACCGCCGGGCTCATCGCCGCCAGCTTCGGGATGGCCAACATCATCTCCCGCCCCGGCGGCGGGCTACTCTCCGACTGGCTCTCCAGCCGGTACGGCATGCGCGGCAGGCTGTGGGGGCTGTGGACTGTGCAGACCATCGGCGGCGTCCTCTGCGTGGTGCTCGGAATCGTCGACT TCTC CTTCGCCGCGTCCGTCCTTCTTCGTCCAGGCCGCGTGCGGGCTCACCTTCGGCATCGTGCCGTTCGTGTCGCGGAGGTCCGCTGGGGCTCATCTCCGGGATGACCGGCGGCGGGGCAACGTGGGCGCCGTGCTGACGCAGTACATCTTCTTCCACGGCACAAAGTACAAGACGGAGACCGGGATCAAGTACATGGGGCTCATGATCATCGCGTGCACGCTGCCCGTCATG CTCATCTAC TTCCCGCAGTGGGGCGGCATGCTCGTAGGCCCGAGGAAGGGGGCCACGGCGGAGGAGTACTACAGCCGGGAGTGGTCGGATCACGAGCGCGAGAAGGGTTTTCAACGCGGCCAGCGTGCGGTTCGCGGAGAACAGCGTGCGCGAGGGCGGGAGGTCGTCGGCGAATGGCGGACAGCCCAGGCACACCGTCCCCGTCGACGCGTCGCCGGCCGGGGTGTGAAGAATGCCACGGACAATAAGGTCGCGGTTGTA GTACAACT GTACAAATTGATGGTACGTGTCGTTTGACCGCGCGCGCGCACAGTGTGGGTCGTGGCCTCGTGGGCTTAGTGGAGTACAGTGAGGGGTGTACGTGTGTCGTGGCGCGCGGTCACCTCGGTGGCCTTGGGATTGGGGGGGCACTATACGCTAGTACTCCAGATATATACGGGTTTGATTTACTTCTGTGGATCGGCGCTTGTTGGTGGTTTGCTCCCTGTGGTTTTTGTGATGGTAATCATACTACTCAAACA GTC

SEQ ID NO:9: NRT2.3 TATA box

CC GCTATGCTAT AAGAGCTGAC

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

CTAC

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

CGCGCAGG

SEQ ID NO:12 CRISPR target sequence

SEQ ID NO:13 CRISPR target sequence

SEQ ID NO:14 CRISPR target sequence

SEQ ID NO:15 CRISPR target sequence

SEQ ID NO: 16 Protospacer sequence

GCTATAAGAGCTGACGCGCA

SEQ ID NO: 17 Protospacer sequence

GAGCCGCGCTTTCCGCTATG

SEQ ID NO: 18 Protospacer sequence

GTTGCCCTCTCGCCGCACCC

SEQ ID NO: 19 Protospacer sequence

CGACGCGAGCGCGGAGACGG

SEQ ID NO:20 Protospacer sequence

5' GGCA GCTATAAGAGCTGACGCGCA 3'

5' AAAC TGCGCGTCAGCTCTTATAGC 3' (SEQ ID NO:34)

SEQ ID NO:21 Protospacer sequence

5' GGCA CATAGCGGAAAGCGCGGCTC 3'

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

SEQ ID NO:22 Protospacer sequence

5' GGCA GGGTGCGGCGAGAGGGCAAC3'

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

SEQ ID NO:23 Protospacer sequence

5' GGCA CCGTCTCCGCGCTCGCGTCG 3'

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

SEQ ID NO: 24; tracrRNA nucleic acid sequence

ATGCTACTACTAAAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGACTAGCCTTATTTTAACTTGCTATGCTTTTCAGCATAGCTCTAAAAC

SEQ ID NO:25 U3 promoter sequence:

AAAGGAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAG GACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCA

SEQ ID NO:26 U6a promoter sequence:

TTTTTTCCTGTAGTTTTCCCACAACCATTTTTTACCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGG GAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTGGCTGCCG

SEQ ID NO:27 U6b promoter sequence:

TGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCACATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATGGGGCTCAGACTTGAGCTACGAGGCCGCAGGCGAGAAGCCTAGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATACGGCCGACGAGCGTGTACTACCGCGCGGGATGCCGCTGGGCGCTGCGGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGG AGACAGGTTTAGTACCACCTCGCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTG

SEQ ID NO:28 U3b in dicotyledons

TTTACTTTAAATTTTTTCTTATGCAGCCTGTGATGGATAACTGAATCAAACAAATGGCGTCTGGGTTTAAGAAGATCTGTTTTGGCTATGTTGGACGAAACAAGTGAACTTTTAGGATCAACTTCAGTTTTATATATGGAGCTTATATCGAGCAATAAGATAAGTGGGCTTTTTATGTAATTTAATGGGCTATCGTCCATAGATTCACTAATACCCATGCCCAGTACCCATGTATGCGTTTCATATAAGCTCCTAATTTCTC CCACATCGCTCAAATCTAAACAAATCTTGTTGTATATATAACACTGAGGGAGCAACATTGGTCA

SEQ ID NO:29 U6-1 in dicotyledons

AGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAAACGAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGAAAAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTTCTAAAATAGGCCCATTTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCT GAGTTTATATATGGTTAGAGACGAAGTAGTGATTG

SEQ ID NO:30 Cas9

ATGGCCCCAAAGAAGAAGCGCAAGGTCGACAAGAAGTACTCCATCGGCCTCGACATCGGCACCAATTCTGTTGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCGTCCAAGAAGTTCAAGGTCCTCGGCAACACCGACCGCCACTCCATCAAGAAGAATCTCATCGGCGCCCTGCTGTTCGACTCTGGCGAGACAGCCGAGGCTACAAGGCTCAAGAGGACCGCTAGACGCAGGTACACCAGGCCGCAAGAACCGC ATCTGCTACCTCCAAGAGATCTCTCCAACGAGATGGCCAAGGTGGACGACAGCTTTCTTCCACAGGCTCGAGGAGAGCTTCCTCGTCGAGGAGGACAAGAAGCACGAGCGCCATCCGATCTTCGGCAACATCGTGGATGAGGTGGCCTACCACGAGAAGTACCCGACCATCTACCACCTCCGCAAGAAGCTCGTCGACTCCACCGATAAGGCCGACCTCAGGCTCATCTACCTCGCCCTCGCCCACATGATCAAGTTC AGGGGCCACTTCCTCATCGAGGGCGACCTCAACCCGGACAACTCCGATGTGGACAAGCTGTTCATCCAGCTCGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCGATCAACGCCTCTGGCGTTGACGCCAAGGCTATTCTCTCTGCCAGGCTCTTCAAGTCCCGCAGGCTCGAGAATCTGATCGCCCAACTTCCGGGCGAGAAGAAGAATGGCCTCTTCGGCAACCTGATCGCCCTCTCTCTTGGCCTCACCCCG AACTTCAAGTCCAACTTCGACCTCGCCGAGGACGCCAAGCTCCAGCTTTCCAAGGACACCTACGACGACGACCTCGACAATCTCCTCGCCCAGATTGGCGATCAGTACGCCGATCTGTTCCTCGCCGCCAAGAATCTCTCCGACGCCATCCTCCTCAGCGACATCCTCAGGGTGAACACCGAGATCACCAAGGCCCCACTCTCCGCCTCCATGATCAAGAGGTACGACGAGCACCACCAGGACCTCACACTCCTCAAG GCCCTCGTGAGACAGCAGCTCCCAGAGAAGTACAAGGAGATCTTCTTCGACCAGTCCAAGAACGGCTACGCCGGCTACATCGATGGCGGCGCTTCTCAAGAGGAGTTCTACAAGTTCATCAAGCCGATCCTCGAGAAGATGGACGGCACCGAGGAGCTGCTCGTGAAGCTCAATAGAGAGGACCTCCTCCGCAAGCAGCGCACCTTCGATAATGGCTCCATCCCGCACCAGATCCACCTCGGCGAGCTTCATGCTATC CTCCCGCAGGCAAGAGGACTTCTACCCGTTCCTCAAGGACAACCGCGAGAAGATTGAGAAGATCCTCACCTTCCGCATCCCGTACTACGTGGGCCCGCTCGCCAGGGGCAACTCCAGGTTCGCCTGGATGACCAGAAAGTCCGAGGAGACAATCACCCCCTGGAACTTCGAGGAGGTGGTGGATAAGGGCGCCTCTGCCCAGTCTTTCATCGAGCGCATGACCAACTTCGACAAGAACCTCCCGAACGAGAAGGTGCTC CCGAAGCACTCACTCCTCTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGGATGAGGAAGCCAGCTTTCCTTAGCGGCGAGCAAAAGAAGGCCATCGTCGACCTGCTGTTCAAGACCAACCGCAAGGTGACCGTGAAGCAGCTCAAGGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTCGAGATCTCCGGCGTCGAGGATAGGTTCAATGCCTCCCTCGGGACCTA C CACGACCTCCTCAAGATTATCAAGGACAAGGACTTCCTCGACAACGAGGAGAACGAGGACATCCTCGAGGACATCGTGCTCACCCTCACCCTCTTCGAGGACCGCGAGATGATCGAGGAGCGCCTCAAGACATACGCCCACCTCTTCGACGACAAGGTGATGAAGCAGCTGAAGCGCAGGCGCTATACCGGCTGGGGCAGGCTTCTAGGAAGCTCATCAACGGCATCCGCGACAAGCAGTCCGGCAAGACGATCCTCG ACTTCCTCAAGTCCGACGGCTTCGCCAACCGCAACTTCATGCAGCTCATCCACGACGACTCCCTCACCTTCAAGGAGGACATCCAAAAGGCCCAGGTGTCCGGCCAAGGCGATTCCCTCCATGAGCATATCGCCAATCTCGCCGGCTCCCCGGCTATCAAGAAGGGCATTCCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCAGGCACAAGCCAGAGAACATCGTGATCGAGATGGCCCGCGAGA ACCAGACCACACAGAAGGGCCAAAAGAACTCCCGCGAGCGCATGAAGAGGATCGAGGAGGGCATTAAGGAGCTGGGCTCCCAGATCCTCAAGGAGCACCCAGTCGAGAACACCCAGCTCCAGAACGAGAAGCTCTACCTCTACTACCTCCAGAACGGCCGCGACATGTACGTGGACCAAGAGCTGGACATCAACCGCCTCTCCGACTACGACGTGGACCATATTGTGCCGCAGTCCTTCCTGAAGGACGACTCCATCG ACAACAAGGTGCTCACCCGCTCCGACAAGAACAGGGGCAAGTCCGATAACGTGCCGTCCGAAGAGGTCGTCAAGAAGATGAAGAACTACTGGCGCCAGCTCCTCAACGCCAAGCTCATCACCCAGAGGAAGTTCGACAACCTCACCAAGGCCGAGAGAGGCGGCCTTTCCGAGCTTGATAAGGCCGGCTTCATCAAGCGCCAGCTCGTCGAGACACGCCAGATCACAAAGCACGTGGCCCAGATCCTCGACTCCCGCA TGAACACCAAGTACGACGAGAACGACAAGCTCATCCGCGAGGTGAAGGTCATCACCCTCAAGTCCAAGCTCGTGTCCGACTTCCGCAAGGACTTCCAGTTCTACAAGGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTCAATGCCGTGGTGGGCACAGCCCTCATCAAGAAGTACCCAAAGCTCGAGTCCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGCAAGATGATCGCCAAGTC CGAGCAAGAGATCGGCAAGGCGACCGCCAAGTACTTCTTCTACTCCAACATCATGAATTTCTTCAAGACCGAGATCACGCTCGCCAACGGCGAGATTAGGAAGAGGCCGCTCATCGAGACAAACGGCGACAGGCGAGATCGTGTGGGACAAGGGCAGGGATTTCGCCACAGTGCGCAAGGTGCTCTCCATGCCGCAAGTGAACATCGTGAAGAAGACCGAGGTTCAGACCGGCGGCTTCTCCAAGGAGTCCATCCT CCCAAAGCGCAACTCCGACAAGCTGATCGCCCGCAAGAAGGACTGGGACCCGAAGAAGTATGGCGGCTTCGATTCTCCGACCGTGGCCTACTCTGTGCTCGTGGTTGCCAAGGTCGAGAAGGGCAAGAGCAAGAAGCTCAAGTCCGTCAAGGAGCTGCTGGGCATCACGATCATGGAGCGCAGCAGCTTCGAGAAGAACCCAATCGACTTCCTCGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTCATCATCAA GCTCCCGAAGTACAGCCTCTTCGAGCTTGAGAACGGCCGCAAGAGAATGCTCGCCTCTGCTGGCGAGCTTCAGAAGGGCAACGAGCTTGCTCTCCCGTCCAAGTACGTGAACTTCCTCTACCTCGCCTCCCACTACGAGAAGCTCAAGGGCTCCCCAGAGGACAACGAGCAAAAGCAGCTGTTCGTCGAGCAGCACAAGCACTACCTCGACGAGATCATCGAGCAGATCTCCGAGTTCTCCAAGCGCGTGATCCTCGC CGATGCCAACCTCGATAAGGTGCTCAGCGCCTACAACAAGCACCGCGATAAGCCAATTCGCGAGCAGGCCGAGAACATCATCCACCTCTTCACCCTCACCAACCTCGGCGCTCCAGCCGCCTTCAAGTACTTCGACACCACCATCGACCGCAAGCGCTACACCTCTACCAAGGAGGTTCTCGACGCCACCCTCATCCACCAGTCTATCACAGGCCTCTACGAGACACGCATCGACCTCTCACAACTCGGCGGCGATTGA

SEQ ID NO:32: OsTBP2.1 amino acid

MAAAEAAAEAAAALEGSEPVDLVKHPSGIIPTLQNIVSTVNLDCKLDLKAIALQARNAEYNPKRFAAVIMRIREPKTTALIFASGKMVCTGAKSEQQSKLAARKYARIIQKLGFAAKFKDFKIQNIVGSCDVKFPIRLEGLAYSHGAFSSYEPELFPGLIYRMKQPKIVLLIFVSGKIVLTGAKVRDETYTAFENIYPVLTEFRKVQQ

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

GATTTCCGATCGCTATAAAAGACCTAGGATTTCGAAATTTTTCCCTCCCCTCCCCCTTCGCGCGCGCGCTCTCTCTTCCCGCCCTTTTTTTTTCCTTCTTCTTCACCGGTGGGATTGATTCGTGGGGTGCGGATCTGGTTTTTGGGGGTGTATGGCGGCGGCGGAGGCGGCGGCGGAGGCGGCGGCGGCGCTGGAGGGGAGCGAGCCCGTGGACCTGGTCAAGCACCCTCCGGCATCATCCCCACGCTCCAGTA AGTCCCCCCCGCCCCCGCCCG GATCTGTATGTGCGGGTTCATCGAGCTGGTTGAGTTTGAGGTGGAGCGGTTAACTCGTCGCGCGCTGTTTGATTTGGTTTGTTTTGGGGGCCGGGGATATTATTTGTTGTTGCAAATGATGTGATTGGTGGTAGGTTTAGCTTGGGGGGTTACTACATGTTTGAGGTTCTGATTTGCTTGATTGATGAAAAAGGGGGGCGATCTGAGATCCGCGAGTCCGGTACGAGAAGAAATAGAGGCAGCCGATGTG CTTGTGCTGTGACGGATCCATTGTCCGGT CTTCATAACAACTCTTTTTAGCTGGACAGATGTTTGTTTTCTCATGAATGAATTCATATGGTTGGAGTCTTGGAGAATCAGCTTGTCAGCTGTTCTTTTTTTTAAAAAAAAATTAAAGGTTTATAAATAAATAAATAATCAATCAATCAATCATGCATCTAAATTGTAGGATTTAATTCTTTCCCCTCATCACGTATTTCAACCTTAGAGGGAGGTATTGTGTCTGTGGAGAGTTTATGGTGCTCATGTGATACATACAGTTG ACAATGGGTCAATGCAT GTTTTTAGACAGCATCAAGCTCTGAACAAGTGAACCACATATTTAGGCTTGATCCATGTATAACCCCACTAAGTTTTATACTTGTTTGTACGGGGCCAATATGCCCAAACCTGGCTGTTTATTAGCATGCTGTACTTGCTGGTGTGCATCGAAACGTTTTTTGGGGTTTGCCTACATTGCTACCAGGCATGTATATTTTGCATGCTGGTGTGCATTGCAGAAAAAGTCGAGCATGCTTATATTGCTGCTTGTCATAGTGTACCTGCT TCCATGTGTTCTCCA CAAGTGCGCATGCCTGTTTTGCTGCCTTTCTGGGTTGTTTATGTTTCAGCGTTGGGATCATAGCTGGTAGACTGCATGTGTTCCACTCTGTTTTGGCTTATCATGCTTCCTTTGGATACTCTCACCAAGGAAATTGAAGTCCTCATTATGTGTTCTCATCTATTTTGATGGCAGAAACATCGTGTCGACGGTCAATTTGGATTGCAAATTAGACCTCAAAGCTATAGCTTTGCAAGCACGCAATGCAGAATAATCCAAA GGTATTATGATGGCTTTG GTGTGTTCTTGTGTCTTTGATTTTGCTCGAAAGGATATCTTCTTTGCATGTGAAAATTTTACCTATTTTAATCCTGCTAGCATGTAATATGTAGACAAGTCCATACAATCCTATGGTCTGTTCCTAGGATGATACTTCCTTTGTGTATAACGCAAGTGTGAGTGCAATTTTAGAGGTCGGTATGGATAAATGCATAGCTGGCAGTCCAGTTTTATATTGTAAATCTCAGTTAAGAGAGTGAATACATTCATCACGCT CAGCTTGTCCCATGGATAAGCA AATTGCTTTACAATTAAGATTCTAAAATTGCTCTATTTAGTGCTTATGCACTTTCTAATCTGTGCTGCACGTATCAGATACAGCATGATGTATTGGGGGATTTTGTAATTATATGTAATATCATAATCTAAATCTAGGCTATTATGTTTATTTTAATAATGGATACATTTTGAAAGAAACAGAGATAACTGCACACTGAGGTTATAGCATCTCTATCTACTCCATCCGTTCCAAAATATAAGCACTGTAATATATGTTTTTA GTTTGCTAACTCCTCTTT TCACAAACAGCGTTTTGCTGCAGTTATCATGAGAATAAGAGAACCGAAAACTACAGCTCTGATATTTGCATCGGGTAAAATGGTATGGTTCAACCTCTTTGTATAGCTAAGACTGCAAATATTCTGTTTGTTCATCTTCTTATTGACTTGTGAATTGCTTTATCACCTGTCTTTGTTGCTCTTGTAGCTATTATAACGCATATTGATTGATGAACTCTTTTTGCTGATGTGTAACTGCTTTCTATTAGCCTACTATCTTT TTAGTTTTTCTTTGTTTTTT TAACAGCTATCACATCCATACCTGATCCATGCAGCCTGTAGTTCAATGCTGAATCTGTAAATTTAAGTTTGCTCTGATTTTCTGACATGTTTGCTGTTGCATTGCTGTTACATAACAGATAAGTAGTGCTCTTTCTGTAAATGTATAGTTTGTCCTTGTTAACTTTTTTATTGTCTTAAAGCATAATCAGAATTGACAATAAATTGAACCTGTCATATTTATTTGTTGATGGATAATCAGCAATGACACATCACATGGACT TCATGTCGCGTCAAT ATCTTAATTTTAAAAGGCTTGTTTATGAAAATTCACTTGATTTTCCTTTTGGATCAATGTCTTGATCCGAATAAGCTTGTTGAAGGATAATTGGTGGTCAGCATTTTTGGGATAAAATCTTCATTAAAAGTTGCTTTGCTGGTTCTCATCAGGAGGGATAATATCTGTGTTACAGACTTAAAGTAATACATTTTTCTGATAATAAATTCACATTTTAAGCATGAGAACGAAGGAGTTCACAGCCTAGGCTTACTAGTACGCTGTAT GGGGCCTGACTAT TAGGCAGTGGACTTAAGAAGGGGTTGTCTTGGGTTTGATGCACCGATTTGCTGCGTCTTTGCTTCTTTTTTATAAAAATCAAATTTGCTTTGTTAACCAAAATTTCTCTGTGGGCACCATGACCTGCTAGCATTTGTGGTGGAATTAAAAGCATGTTTATAAATCTTTTGTTGTCCTTTACCAGGTATGTACTGGGGCAAAGAGCGAACAACAATCAAAGCTTGCAGCAAGAAAGGTATGGGGAGTATTTATTTTATTTCATTTT ATTTTGCTACGTGAA GTCTAACACTTTTTTATTGCCAGTATGCTCGTATTATCCAAAAGCTTGGCTTTGCTGCTAAGTTTAAGGTAACTATTTCAGGCTTGATTAATTTTCATCTGTGCAAATGCCTAACCATTTTCATTCTGTAAGATTGTACCTAAATGTTTAACTCATTTGCACTATACCAGGACTTCAAGATTCAGAACATTGTTGGTTCTTGTGATGTTAAATTTCCAATCAGGCTGGAGGGACTTGCATATTCTCATGGTGCTTTCTCAAGTGT AAGTTGAATCCTTGCA TGTTTTTTTAGGATATTTCTTTCACATATGTTATGCTATTCTCATGTCTTGTGCCTTTTGTCTTCCAGTATGAGCCTGAACTCTTTCCTGGTCTGATATATCGGATGAAGCAACCGAAGATTGTTCTTCTGATTTTTGTTTCAGGCAAGATTGTTTTGACCGGAGCAAAGGTAAGCAGCCTTTCCTTTTGTATACCCTTGATTGTCTATTCCTTTTTGTATGTCTGAATGTACTTGTCTTTAGGTGAGGG ATGAGACGTATACCGCCTTTGAGAACA TATACCCTGTGCTAACAGAGTTCAGAAAAGTCCAGCAATGGTACGTCTTTATTTTGTTGTTTAGGTATGCAGTGTGTGGTAGAAACATCGGAAGTTCGAAGTAAGAATTATACAGTGGATAATTGCACTTGTGTCTTTTGTTATACGTGGGAGTGAACTTTGAGCACACTCCTTACTAAATTGCAATATGTTGAATTTTTTAATATTACAAGCACCTATAAATCTGAACTTTCCAATTTGAATCCAGATGGTCAGCAGAA ACTGAAGGAATAAATCATC TCAAGAAAGATTTTAATTTCCACACTACCTTGATCTTAGATAGGATTTGGATACTTGTTTCAGATATGATTCATCACTACCATGTGATGGACTGTTGGCTGCCCTTGCCTACTTACTGTGCTTGACTGCAGTTTTACTTCCAATTTAGGAGAACACAATTTTAAATAATAATAATAATGATGGCTCCTTGCACCTTCAAATTTGCAGATAACCTATGGAGGTCACAACTACAACGCTTCCTTGAGGATTTTGCTGCCTTGTAACTGCTA ATTTAATCTG TACATATATGTAGTCTGGAGGGGCGTACAGCATCTTGTAATTTATGTGAGCCCCTGGATGAATGAGCACTGTAGACTTGTAGCTGGGTGAGTATGTTGTTAGTAGTCTCTTGTGGCATGGAGTTCAGTCCAACCGATCTGATGGAGTTTTCGTACGTTTGTAGCCCTTGCCGATCTTTTTCCCTTTCTTCCCAATAGACATGTTGCTAAACTTTACTAACTTGTTAACAGACAGACAGAATGATAACATGGACT GTGGATGCTTAGCGTTTGTGGCCG

Claims (22)

1. A method of increasing at least one of yield, biomass, nitrogen Use Efficiency (NUE), nitrogen transport, and/or nitrogen content in a plant, said method comprising introducing at least one mutation into a nucleic acid sequence encoding an NRT2.3 promoter, wherein said NRT2.3 promoter comprises SEQ ID No.1, and wherein said mutation is selected from the group consisting of

At least one mutation of at least one nucleotide in the TATA box of the nrt2.3 promoter, wherein said at least one mutation is a substitution of T to C at position 160 of SEQ ID No. 1; and/or

Deletion of SEQ ID No. 6 or 7 in SEQ ID No. 1.

2. The method of claim 1, wherein the method further introduces a mutation from a to C at position 201 and a mutation from C to T at position 222.

3. The method of claim 1 or2, wherein the plant is rice.

4. The method of claim 1 or 2, wherein the mutation is introduced using mutagenesis.

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

6. The method of claim 4, wherein the mutation is introduced using targeted genomic modification.

7. The method of claim 1, wherein the increase is relative to a control or wild-type plant.

8. The method of claim 1, wherein the NRT2.3 promoter is 141bp, 180bp, or 697bp in length.

9. The method of claim 1, 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.

10. A method of identifying and/or selecting a plant having or to have increased yield, biomass, nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content as compared to a control or wild type plant, the method comprising detecting in the plant or plant germplasm at least one polymorphism in the NRT2.3 promoter gene sequence and screening for said plant or progeny thereof, wherein said NRT2.3 promoter comprises SEQ ID No. 1, and wherein said polymorphism is selected from at least one mutation of at least one nucleotide in the TATA box of the NRT2.3 promoter, wherein said at least one mutation is a T to C substitution at position 160 of SEQ ID No. 1; and/or the deletion of SEQ ID NO 6 or 7 in SEQ ID NO 1.

11. The method of claim 10, wherein the method further comprises introgressing a chromosomal region comprising at least one polymorphism in the NRT2.3 promoter into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.

12. 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 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 NOs 2,4, or 5.

13. A method of making 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 said NRT2.3 promoter sequence is selected from the group comprising SEQ ID NOs 2,4 or 5.

14. The method of claim 12 or 13, wherein the NRT2.3 gene sequence comprises SEQ ID No. 8.

15. The method of claim 10, 11, 12 or 13, wherein the plant is rice.

16. 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 NOs 2,4 or 5.

17. The nucleic acid construct of claim 16, wherein the NRT2.3 gene sequence comprises SEQ ID No. 8.

18. A vector comprising the nucleic acid construct of claim 17.

19. A host cell comprising the vector of claim 18 or the nucleic acid construct of claim 16.

20. Use of the vector of claim 18 or the nucleic acid construct of claim 16 for increasing at least one of yield, biomass, nitrogen Use Efficiency (NUE), nitrogen transport and/or nitrogen content in a plant.

21. 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, wherein said NRT2.3 promoter comprises SEQ ID No. 1, and wherein said mutation is selected from at least one mutation of at least one nucleotide in the TATA box of the NRT2.3 promoter, wherein said at least one mutation is a substitution of T to C at position 160 of SEQ ID No. 1; and/or the deletion of SEQ ID NO 6 or 7 in SEQ ID NO 1.

22. A method of obtaining a genetically modified plant, the method comprising:

a. selecting a portion of a plant;

b. Transfecting at least one cell of the plant part of paragraph (a) with 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 NOs 2,4 or 5;

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

Selecting one or more plants obtained according to paragraph (c) that show increased expression of nrt2.3b and/or increased ratio of osnrt2.3b to osnrt2.3 a.