Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the invention relates to methods for simultaneously increasing plant yield and abiotic resistance as well as plants expressing the improved trait and methods of making such plants.
• Enhancement of grain production under either normal or adverse conditions is one of the long-standing pursuits of crop research. With a continuous increase in the world’s population yet decrease in the amount of arable land, the grain yield per unit area must be continuously increased to match food demands. One option is to exploit inferior soils from salinized or deserted land. However, both solutions demand the development of superior crop cultivars that have a simultaneously high yield and high salt resistance. The deterioration and the unsteadiness of the environment or climate conditions reinforces the need for dependable crops with a stable high yield. However, this is particularly challenging for crop breeding because a high yield and stable yield phenotype are thought to be mutually exclusive as a result of an internal compensation mechanism in plants. In addition, the genetic basis linking grain yield and stress tolerance remains largely unclear, which has so far, impeded the molecular maker assisted breeding process that is commonly used nowadays.
• Seed size or weight is one of the main determinants of seed yield.
• a number of seed size associated genes including many quantitative trait locus (QTLs) have been identified and subsequently cloned. Many of these genes were suggested to be related to the regulation of phytohormones, such as brassinosteroid (BR), auxin, and cytokinin. Despite the regulation of plant growth and development under normal conditions, hormones also play an important role in regulating stress responses. In addition to the well-known prominent role of ABA in a number of stresses, a number of studies have suggested that other hormones, such as cytokinins and BRs, also play an important role in regulating resistance to various stresses, including salt stress.
• QTLs quantitative trait locus
• yield of rice is usually determined directly by grain weight, number of effective tillers, and grains per spike. Grain weight or grain size is regulated by multiple genes and a number of quantitative trait loci (QTL) affecting the grain size of rice have been cloned. At the same time, rice yield is also affected by the environment, and harsh environments such as saline-alkali, drought, and diseases can lead to severe reductions in yield. Under adverse conditions, plants can make the corresponding adjustments at the molecular, cellular and overall level to minimize the damage caused by the environment and survive. Many genes are expressed by stress, and the products of these genes cannot only directly participate in the stress response of plants, but can also regulate the expression of other related genes or participate in signal transduction pathways, so that plants can avoid or reduce the damage and enhance the resistance to the stress environment.
• QTL quantitative trait loci
• AG02 Argonaute member protein
• DLT as a GRAS family protein is a putative transcriptional factor, whereas AG02 belonging to ARGONAUTE family protein is generally involved in small RNA function.
• AG02 can modulate DLT activity to regulate gene expression.
• AG02 alone can also activate these promoters. When DLT and AG02 were co-expressed, the activities of the promoters were greatly enhanced.
• a method of increasing at least one of yield and/or abiotic stress tolerance in a plant comprising increasing the expression or activity of AG02 (argonaute member protein) in said plant.
• AG02 argonaute member protein
• the said increase is compared to a control or wild-type plant.
• the yield is grain yield. In another embodiment, the abiotic stress tolerance is salt tolerance.
• the method comprises introducing and expressing in said plant a nucleic acid construct, wherein said construct comprises a nucleic acid sequence encoding an AG02 polypeptide as defined in SEQ ID NO: 1 or a functional variant or homologue thereof, wherein said nucleic acid sequence is operably linked to a regulatory sequence.
• the nucleic acid sequence comprises SEQ ID NO: 2 or 3 or a functional variant or homologue thereof.
• the regulatory sequence is a promoter, preferably a CaMV 35S promoter.
• the AG02 polypeptide comprises at least one modification that affects protein function.
• the AG02 polypeptide comprises at least one peptide tag.
• the plant is a crop plant.
• the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.
• a method for making a transgenic plant having increased yield and/or abiotic stress tolerance comprising introducing and expressing in said plant at least one nucleic acid construct, wherein said construct comprises a nucleic acid sequence encoding an AG02 polypeptide as defined in SEQ ID NO: 1 or a functional variant or homologue thereof, wherein said nucleic acid sequence is operably linked to a regulatory sequence.
• a method for making a transgenic plant having increased yield and/or abiotic stress tolerance comprising introducing a mutation into the plant genome, wherein said mutation is the insertion of at least one or more additional copy of a nucleic acid encoding an AG02 polypeptide as defined in SEQ ID NO: 1 or functional variant or homologue thereof such that said sequence is operably linked to a regulatory sequence, and wherein such mutation is introduced using targeted genome editing (preferably wherein the mutation is introduced using ZFNs, TALENs or CRISPR/Cas9).
• the mutation may be introduced into a regulatory sequence which is operably linked to a nucleic acid encoding an AG02 polypeptide, wherein said mutation results in increased AG02 expression or activity levels and wherein such mutation is introduced using targeted genome editing (preferably wherein the mutation is introduced using ZFNs, TALENs or CRISPR/Cas9).
• the nucleic acid sequence encoding an AG02 polypeptide comprises SEQ ID NO: 2 or 3 or a functional variant or homologue thereof.
• the regulatory sequence is a promoter, preferably a CaMV 35S promoter.
• the AG02 polypeptide comprises at least one modification.
• the at least one modification leads to moderate overexpression of AG02 (for example as defined herein).
• the at least one modification affects protein function.
• the AG02 polypeptide comprises at least one peptide tag.
• the plant is a crop plant.
• the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.
• transgenic plant part thereof or plant cell expressing at least one nucleic acid construct, wherein said construct comprises a nucleic acid sequence encoding an AG02 polypeptide as defined in SEQ ID NO: 1 or a functional variant or homologue thereof wherein said nucleic acid sequence is operably linked to a regulatory sequence.
• a transgenic plant, part thereof or plant cell expressing a mutation wherein the mutation is the insertion of at least one copy of an AG02 polypeptide as defined in SEQ ID NO: 1 or a functional variant or homologue thereof such that said sequence is operably linked to a regulatory sequence, wherein such mutation is introduced using targeted genome editing (preferably wherein the mutation is introduced using ZFNs, TALENs or CRISPR/Cas9).
• the mutation may be introduced into a regulatory sequence which is operably linked to a nucleic acid encoding an AGQ2 polypeptide, wherein said mutation results in increased AG02 expression or activity levels, and wherein such mutation is introduced using targeted genome editing (preferably wherein the mutation is introduced using ZFNs, TALENs or CRISPR/Cas9).
• the plant is characterised by an increase in yield and/or abiotic stress tolerance compared to a control plant.
• the plant is characterised by an increase in grain yield. In another embodiment, the plant is characterised by an increase in salt tolerance.
• the regulatory sequence is a promoter, preferably a CaMV 35S promoter.
• the plant is a crop plant.
• the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.
• nucleic acid construct comprising a nucleic acid sequence encoding an AG02 polypeptide as defined in SEQ ID NO: 1 or a functional variant or homologue thereof wherein said nucleic acid sequence is operably linked to a regulatory sequence, wherein the regulatory sequence is a a promoter, preferably a CaMV 35S promoter.
• a host cell comprising the nucleic acid construct or the vector described above.
• the host cell is a bacterial or plant cell.
• transgenic plant expressing the nucleic acid construct described above.
• the plant is a crop plant, and more preferably it is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet
• nucleic acid construct or the vector described above to increase yield and/or abiotic stress tolerance in a plant.
• a protein which is the following (a) or (b):
• the gene is a DNA molecule according to any one of the following (1)-(3):
• the protein or gene described above to regulate yield and/or tolerance in a plant, preferably abiotic stress tolerance.
• the plant is a dicotyledonous or monocotyledonous plant.
• a method for cultivating a transgenic plant comprising introducing the gene or recombinant vector described above into a target plant to obtain a transgenic plant; the transgenic plant having the following phenotypes: a yield higher than the target plant (or control plant) and/or stress tolerance higher than the target plant (or control plant).
• the recombinant expression vector can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium-mediated and the like.
• a method for culturing a transgenic plant comprising increasing the expression and/or activity of the protein described above in a target plant (or control plant) to obtain a transgenic plant; the transgenic plant having the following phenotype: yield higher than the target plant (or control plant) and/or stress tolerance higher than the target plant (or control plant).
• the use of the protein, gene or method described above in plant breeding is to breed plants having high yield and/or high stress tolerance and/or increased resistance to bacterial leaf blight and/or black streak dwarf.
• Figure 1 shows that AG02 is a potential DLT interacting protein.
• Figure 2 shows that the overexpression of AG02 improves grain size (grain length and weight of 100 grains (g)) as well as plant growth.
• Figure 3 shows that moderating the level of AG02 overexpression using tag-fused AG02 improves plant yields.
• FIG. 4 shows that overexpression of AG02 enhances salinity tolerance.
• Figure 5 shows that DLT and AG02 are involved in the ABA response.
• Figure 6 shows the effect of salt stress on the plants of the invention for up to 9 days.
• Figure 7 shows AG02 expression levels (A) and AG02-Flag fusion protein levels (B) in AG02-Flag overexpression lines.
• nucleic acid As used herein, the words “nucleic acid”, “nucleic acid sequence”, “nucleotide”, “nucleic acid molecule” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products.
• genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.
• polypeptide and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
• promoter typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in the binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid.
• transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue- specific manner.
• a transcriptional regulatory sequence of a classical prokaryotic gene in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences.
• the aspects of the invention involve recombination DNA technology and exclude embodiments that are solely based on generating plants by traditional breeding methods.
• a“genetically altered plant” or“mutant plant” is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant.
• a mutant plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant using a mutagenesis method, such as any of the mutagenesis methods described herein.
• the mutagenesis method is targeted genome modification or genome editing.
• the plant genome has been altered compared to wild type sequences using a mutagenesis method.
• Such plants have an altered phenotype as described herein, such as increased grain size, grain yield and abiotic stress tolerance.
• these traits are conferred by the presence of an altered plant genome, for example, a mutated endogenous AG02 gene or promoter.
• the endogenous promoter or gene sequence is specifically targeted using targeted genome modification and the presence of a mutated gene or promoter sequence is not conferred by the presence of transgenes expressed in the plant.
• the genetically altered plant can be described as transgene-free.
• transgenic means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
• genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or
• (c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues.
• the natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library.
• the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part.
• the environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp.
• a naturally occurring expression cassette for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above - becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815 both incorporated by reference.
• a plant according to all aspects of the invention described herein may be a monocot or a dicot plant.
• the plant is a crop plant.
• crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use.
• the plant is a cereal.
• the plant is Arabidopsis.
• the monocotyledonous plant may be a plant of the Poales.
• the Poales plant may be a gramineous plant.
• the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.
• the plant is from the genus Oryza, preferably rice, and in one example Zhonghua 11 rice.
• the plant is soybean.
• plant encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned comprise the nucleic acid construct as described herein or carry the herein described mutations (such as those introduced by genome editing techniques to introduce at least one additional copy of AG02).
• plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the nucleic acid construct or mutations as described herein.
• the invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs.
• the aspects of the invention also extend to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
• Another product that may be derived from the harvestable parts of the plant of the invention is biodiesel.
• the invention also relates to food products and food supplements comprising the plant of the invention or parts thereof. In one embodiment, the food products may be animal feed.
• a product derived from a plant as described herein or from a part thereof there is provided.
• the plant part or harvestable product is a seed or grain. Therefore, in a further aspect of the invention, there is provided a seed produced from a genetically altered plant as described herein.
• the plant part is pollen, a propagule or progeny of the genetically altered plant described herein. Accordingly, in a further aspect of the invention there is provided pollen, a propagule or progeny produced from a genetically altered plant as described herein.
• a control plant as used herein according to all of the aspects of the invention is a plant which has not been modified according to the methods of the invention. Accordingly, in one embodiment, the control plant does not have increased expression of an AG02 nucleic acid and/or altered activity of an AG02 polypeptide, as described above. In an alternative embodiment, the plant has not been genetically modified, as described above. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.
• a method of increasing yield preferably grain yield and/or abiotic stress tolerance in a plant, the method comprising increasing the expression or activity of AG02 (argonaute member protein 2). Preferably said increase is compared to a control or wild-type plant.
• a method of increasing both yield and abiotic stress tolerance meaning that yield can be increased under conditions of abiotic stress tolerance.
• abiotic stress tolerance may be selected from drought, salinity, wind, high or low temperature or high light.
• the abiotic stress tolerance is salt tolerance, and there is provided a method of increasing yield under high salt stress (e.g. when the plant is grown under salt stress conditions).
• high salt stress can be considered to be at least 100-250mM NaCI.
• yield in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight. The actual yield is the yield per square meter for a crop per year, which is determined by dividing total production per year (includes both harvested and appraised production) by planted square metres.
• the term“increased yield” as defined herein can be taken to comprise any or at least one of the following and can be measured by assessing one or more of (a) increased biomass (weight) of one or more parts of a plant, aboveground (harvestable parts), or increased root biomass, increased root volume, increased root length, increased root diameter or increased root length or increased biomass of any other harvestable part.
• Increased biomass may be expressed as g/plant or kg/hectare, (b) increased grain yield per plant, which may comprise one or more of an increase in grain biomass (weight) per plant or on an individual basis, (c) increased grain filling rate, (d) increased number of filled grains, (e) increased harvest index, which may be expressed as a ratio of the yield of harvestable parts such as grains over the total biomass, (f) increased viability/germination efficiency, (g) increased number or size or weight of seeds or pods or beans or grain (h) increased seed or grain volume (which may be a result of a change in the composition (i.e.
• lipid also referred to herein as oil
• protein protein
• carbohydrate total content and composition (i) increased (individual or average) grain area, (j) increased (individual or average) grain length, (k) increased (individual or average) seed perimeter, (I) increased growth or increased branching, for example inflorescences on more branches, (m) increased fresh weight or grain fill (n) increased ear weight (o) increased thousand kernel weight (TKW) or 100 grain weight, which may be taken from the number of filled seeds counted and their total weight and may be as a result of an increase in seed size and/or seed weight (p) decreased number of barren tillers per plant and (q) sturdier or stronger culms or stems.
• branching for example inflorescences on more branches
• n increased fresh weight or grain fill
• o increased ear weight
• TKW thousand kernel weight
• TKW thousand kernel weight
• an increase in yield comprises an increase in at least one of the following, grain size, grain weight, grain yield and thousand kernel weight (TKW) or 100 grain weight.
• Yield is increased relative to a control or wild-type plant.
• the yield is increased by up to or at least 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%, 50%, 60%, 70%, 80%, 90%, 95% or 99% compared to a control or wild-type plant.
• yield is increased by between 5 and 15%, more preferably between 8 and 10% compared to a control plant.
• the increase in yield comprises an increase in grain yield by up to or at least 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.
• abiotic stress tolerance refers to outside, non-living, factors which can cause harmful effects to plants and are major limiting factors for plant growth and crop yield.
• abiotic stress arises from an excess or deficit in the physical or chemical environment, such as drought, salinity, wind, high or low temperature or high light.
• An increase is abiotic stress tolerance may be understood to mean that abiotic stress responses are increased, enhanced or improved, compared to a control or wild- type plant.
• said stress is salt stress.
• the method increases salt tolerance to high salt concentrations. Soil salinity is a severely limiting factor for plant growth and as such, there is a need to improve salt tolerance in crops.
• overexpressing AG02 increases salt tolerance ability.
• “salt tolerance” can be considered to be the ability of a plant to grow under saline conditions which comparatively would inhibit the growth of at least 95% of a control or wild-type plant.
• the growth rate of salt tolerant plants of the invention will be inhibited by less than 50%, preferably less than 30%, and most preferably will have a growth rate which is not significantly inhibited by a growth medium containing water soluble inorganic salts which inhibits growth of at least 95% of a comparative, non-salt tolerant plants.
• an increase in salt tolerance can be measured by looking at survival rate of the plant when grown under high salt concentration conditions (as defined herein).
• survival can be considered to be the ability to produce new green leaves after treatment.
• survival rate of a plant overexpressing AG02 is increased by at least 1- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold,
• the survival rate in a control plant More preferably the survival rate is increased by between 5 and 15-fold compared to the level in a control plant or wild-type plant.
• ABA abcisic acid
• ABA sensitivity is associated with various types of abiotic stress resistance such as salt, drought or cold.
• ABA sensitivity is increased by at least 0.5 fold, 1-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold-
• a method of increasing at least one of plant height and leaf length, preferably in the roots comprising increasing the expression or activity of AG02 in a plant.
• the method comprises introducing and expressing a nucleic acid construct comprising a nucleic acid sequence encoding an AG02 polypeptide as defined in SEQ ID NO: 1 or a functional variant or homolog thereof, wherein the nucleic acid sequence is operably linked to a regulatory sequence.
• the nucleic acid sequence encoding an AG02 polypeptide comprises or consists of SEQ ID NO: 2 or 3 or a functional variant or homolog thereof.
• the method comprises introducing a mutation into the plant genome, wherein said mutation is the insertion of at least one or more additional copies of a nucleic acid encoding an AG02 polypeptide as defined herein or a functional variant or homolog thereof such that said sequence is operably linked to a regulatory sequence, or wherein said mutation is the introduction, deletion or substitution of one or more nucleic acids (bases) of an AG02 regulatory sequence, such that said mutation increases expression of AG02, preferably to a moderate level, as described herein.
• such mutation is introduced using targeted genome editing.
• the mutation is introduced using ZFNs, TALENs or CRISPR/Cas9.
• An AG02 regulatory sequence is described elsewhere.
• Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events.
• DSBs DNA double-strand breaks
• HR homologous recombination
• customisable DNA binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcription factors, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats).
• ZF and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate nuclease and DNA-binding domains, ZF and TALE proteins consist of individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of Fokl to direct nucleolytic activity toward specific genomic loci.
• TAL effectors Upon delivery into host cells via the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in host gene promoters and activate transcription. Their targeting specificity is determined by a central domain of tandem, 33-35 amino acid repeats. This is followed by a single truncated repeat of 20 amino acids. The majority of naturally occurring TAL effectors examined have between 12 and 27 full repeats.
• RVD repeat- variable di-residue
• Naturally occurring recognition sites are uniformly preceded by a T that is required for TAL effector activity.
• TAL effectors can be fused to the catalytic domain of the Fokl nuclease to create a TAL effector nuclease (TALEN) which makes targeted DNA double-strand breaks (DSBs) in vivo for genome editing.
• TALEN TAL effector nuclease
• CRISPR Another genome editing method that can be used according to the various aspects of the invention is CRISPR.
• CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids.
• CRISPR loci in microbial hosts contain a combination of CRISPR- associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid
• each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers).
• the non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer).
• the Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus.
• tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences.
• the mature crRN A: tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition.
• Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.
• Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
• the Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases.
• the HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA.
• sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms.
• DSBs site-specific double strand breaks
• codon optimized versions of Cas9 which is originally from the bacterium Streptococcus pyogenes, have been used.
• the single guide RNA is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease.
• sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA.
• the sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities.
• the canonical length of the guide sequence is 20 bp.
• sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3.
• Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.
• aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and in a preferred embodiment exclude embodiments that are solely based on generating plants by traditional breeding methods.
• the term“functional variant of a nucleic acid sequence” as used herein with reference to any of SEQ ID NOs 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant sequence, for example confers increased yield and/or abiotic stress tolerance when expressed in a transgenic plant.
• a functional variant also comprises a variant of the gene of interest which has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non- conserved residues, compared to the wild type sequences as shown herein and is biologically active.
• nucleic acid sequence or amino acid sequence comprising or consisting of a sequence selected from any of SEQ ID NOs 1 to 45 but also functional variants or parts of any of SEQ ID NOs 1 to 45 that does not affect the biological activity and function of the resulting protein.
• Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site that do not affect the functional properties of the encoded polypeptide are well known in the art.
• a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
• a codon encoding another less hydrophobic residue such as glycine
• a more hydrophobic residue such as valine, leucine, or isoleucine.
• changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product.
• Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide.
• a functional variant has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%,
• nucleic acid construct encoding an AG02 polypeptide as defined in SEQ ID NO: 1 or comprising or consisting of a nucleic acid sequence as defined in SEQ ID NO: 2 or 3, but to homologs of any of SEQ ID NOs 1 , 2 and 3.
• homolog also designates an AG02 orthologue from other plant species.
• a homolog of AG02 has, in increasing order of preference, at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,
• overall sequence identity is at least 37%. In one embodiment, overall sequence identity is at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%,
• an AG02 homologue has an amino acid sequence selected from any of SEQ ID NOs 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 and 23 and a nucleotide sequence selected from any of SEQ ID NOs 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22.
• AG02 encodes a member of the argonaute protein family.
• Argonaute family members are characterised by at least one PAZ (preferably N-terminal) and PIWI domain (preferably C-terminal).
• PAZ preferably N-terminal
• PIWI domain preferably C-terminal
• AG02 may also be referred to as DIP2, and such terms can be used interchangeably.
• the PAZ domain comprises the following sequence: AGPVLDLVQKSVRYLDYRTTLNKHQLDTLKNELKGQRVTVNHRRTKQKYIVKGLTDKP ASQITFVDSESGQTKKLLDYYSQQYGKVI EYQM LPCLDLSKSKDKQNYVPI ELCDLLE GQRYPKASLNRNSDKTLKEMA (SEQ ID NO: 30)
• the PIWI domain comprises the following sequence:
• a homolog or variant comprises a PAZ and/or PIWI domain. Accordingly, in one embodiment, the homolog or variant encodes an AG02 polypeptide with 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%,
• nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below.
• the terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
• sequence identity When percentage of sequence identity is used in reference to proteins or peptides, it is recognised that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
• sequence comparison algorithm calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.
• Suitable homologues can be identified by sequence comparisons and identification of conserved domains. For example, there are homologues provided in this application for ten further species: Setaria italica (SEQ ID NO: 4 and 5), Sorghum bicolor (SEQ ID NO: 6 and 7), Zea mays (SEQ ID NO: 8 and 9), Aegilops tauschii (SEQ ID NO: 10 and 11), Triticum aestivum (SEQ ID NO: 12 and 13), Hordeum vulgare (SEQ ID NO: 14 and 15), Brachypodium distachyon (SEQ ID NO: 16 and 17), Glycine max (SEQ ID NO: 18 and 19), Gossypium hirsutum (SEQ ID NO: 20 and 21), and Medicago truncatula (SEQ ID NO: 22 and 23). There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function
• nucleotide sequences of the invention and described herein can also be used to isolate corresponding sequences from other organisms, particularly other plants, for example crop plants.
• methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein.
• Topology of the sequences and the characteristic domains structure can also be considered when identifying and isolating homologs.
• Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof.
• hybridization techniques all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen plant.
• the hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker.
• Hybridization of such sequences may be carried out under stringent conditions.
• stringent conditions or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background).
• Stringent conditions are sequence dependent and will be different in different circumstances.
• target sequences that are 100% complementary to the probe can be identified (homologous probing).
• stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
• a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
• stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 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). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
• stringent conditions may be crossing and washing of the membrane in a DNA or RNA crossing experiment at 65°C using a solution of 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS.
• a variant as used herein can comprise a nucleic acid sequence encoding an AG02 polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to a nucleic acid sequence as defined in SEQ ID NO: 2 or 3.
• a variant can be considered to comprise one of the following:
• nucleic acid sequence encoding a polypeptide with at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the amino acid represented by SEQ ID NO: 1 , 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 or 23;
• nucleic acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the amino acid represented by SEQ ID NO: 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22;
• nucleic acid sequence encoding an AG02 polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to the nucleic acid sequence of any of (a) to (c).
• “increasing the expression” means an increase in the nucleotide levels and“increasing the levels” as used herein means an increase in the protein levels of at least one AG02 polypeptide.
• the expression or levels or activity of AG02 are increased by up to or more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% when compared to the level in a wild-type or control plant.
• Methods for determining AG02 nucleotide expression or protein levels would be well known to the skilled person. In particular increases can be measured by any standard technique known to the skilled person.
• an increase in the expression and/or protein levels of AG02 may comprise a measure of protein and/or nucleic acid levels and can be measured by any technique known to the skilled person, such as, but not limited to, any form of gel electrophoresis or chromatography (e.g. HPLC).
• the level of expression or the level of increase in activity is moderate.
• a moderate increase in expression or a moderate increase in activity may comprise increasing the levels of expression or activity by up to 5%, 10%, 20%, 30%, 40%, 50% or 60% when compared to the level of expression in a control or wild-type plant.
• moderate expression may comprise increasing levels of expression or activity by up to 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x,
• a moderate level of expression may be determined by varying the level of overexpression or activity of AG02 (for example, using different strength promoters or protein tags, as described herein or by any other means known to the skilled person), determining an effect on yield, and determining that the level of overexpression or activity increase is moderate if there is an increase in yield.
• An increase in yield is defined herein (such as an increase in grain yield).
• Moderate overexpression of AG02 may be particularly preferably to produce plants with an increase in yield (more so than an increase in abiotic stress tolerance).
• a moderate increase in activity of AG02 may be achieved by the addition of at least one synthetic tag to the AG02 polypeptide to moderate or decrease protein function by up to 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% compared to the activity of an AG02 polypeptide without a synthetic tag.
• a protein tag may be fused to the C-terminal of AG02 to impair protein function by affecting protein structure or protein-protein interactions.
• a plant expressing an AG02 with at least one synthetic tag has reduced grain size compared to a plant expressing an AG02 with no synthetic tag.
• tags include Flag, GFP, Myc, HA and His, as shown in Table 1 , although the skilled person would be aware that any synthetic tag that has the ability to affect protein function could be used.
• the tag is a FLAG-tag, and comprises the amino acid sequence: DYKDDDDK (SEQ ID NO: 32).
• a moderate increase in activity of AG02 may be achieved by the post-translational addition of at least one synthetic tag as described herein.
• a moderate increase in activity of AG02 may be achieved by expressing an AG02 polypeptide fused to at least one synthetic tag as described herein. This could be achieved by the introduction and expression of a construct comprising a sequence encoding at least one AG02 polypeptide fused to at least one synthetic tag. Alternatively this could be achieved by inserting and expressing at least one AG02 polypeptide fused to at least one synthetic tag by genome editing.
• the insertion is achieved using ZFNs, TALENs or CRISPR/Cas9.
• a moderate increase in activity of AG02 is achieved by the expression of a construct comprising a nucleic acid as defined in SEQ ID NO: 24 or a functional variant thereof.
• a moderate increase in activity of AG02 is achieved by the insertion of at least one copy of the nucleic acid as defined in SEQ ID NO: 24 or a functional variant thereof.
• moderate overexpression of AG02 can be achieved by introducing at least one mutation into the endogenous AG02 promoter and/or the open reading frame, preferably the main open reading frame, or the regulatory sequence of AG02, as defined herein, to increase expression of AG02.
• the mutation is any mutation that leads to an increase in AG02 expression as defined above.
• mutations include substitutions, deletions and additions of at least one nucleic acid.
• AG02 promoter is meant a region extending for at least 5 kbp, preferably at least 2.5 kbp, more preferably at least 2kbp upstream of the ATG codon of the AG02, preferably the AG02 ORF (open reading frame).
• the sequence of the AG02 promoter comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 26.
• an‘endogenous’ nucleic acid may refer to the native or natural sequence in the plant genome.
• a moderate increase in activity of AG02 may be achieved by introducing at least one mutation into the open reading frame, preferably the main open reading frame, to alter the activity of the resulting AG02 protein.
• the introduction of at least one mutation results in an AG02 protein with increased activity compared to an endogenous AG02, resulting in a moderate increase in activity compared to the endogenous AG02.
• At least one mutation is meant that where the AG02 gene (and promoter) is present as more than one copy or homoeologue (with the same or slightly different sequence) there is at least one mutation in at least one gene. Preferably all genes are mutated.
• a“mutation” may be an addition, deletion or substitution of one or more nucleotides (or bases).
• the mutation is introduced using targeted genome editing. That is, in one embodiment, the invention relates to a method and plant that has been generated by genetic engineering methods as described above, and does not encompass naturally occurring varieties or generating plants by traditional breeding methods. In an alternative embodiment, the mutation is introduced using any mutagenesis technique, such as T-DNA insertion or TILLING.
• Plants obtained or obtainable by such method which carry a functional mutation in the endogenous AG02 promoter locus are also within the scope of the invention.
• moderate overexpression of AG02 can be achieved by choice of the regulatory sequence as described in further detail below.
• the nucleic acid construct preferably comprises a regulatory sequence.
• the regulatory sequence is operably linked to the nucleic acid sequence of interest.
• the nucleic acid sequence of interest is an AG02 nucleic acid sequence as defined herein.
• regulatory sequence is used interchangeably herein with “promoter” and all terms are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated.
• regulatory sequence also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
• promoter typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in the binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid.
• transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue- specific manner.
• transcriptional regulatory sequence of a classical prokaryotic gene in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences.
• regulatory element also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
• a "plant promoter” comprises regulatory elements which mediate the expression of a coding sequence segment in plant cells.
• the promoters upstream of the nucleotide sequences useful in the nucleic acid constructs described herein can also be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3'-regulatory region such as terminators or other 3' regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoter is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms.
• the nucleic acid molecule is, as described above, preferably linked operably to or comprises a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern.
• the regulatory sequence is a tissue specific promoter. Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development.
• the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant.
• Suitable well-known reporter genes are known to the skilled person and include for example beta-glucuronidase or beta- galactosidase.
• operably linked refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
• the nucleic acid sequence may be expressed using a promoter that drives overexpression.
• Overexpression means that the transgene is expressed at a level that is higher than expression of endogenous counterparts driven by their endogenous promoters.
• the level of overexpression can be varied by using different types of promoter.
• overexpression may be carried out using a strong promoter, such as a constitutive promoter.
• a "constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ.
• constitutive promoters examples include the cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter, rubisco small subunit, maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2 or any promoter that gives enhanced expression.
• CaMV35S or 19S cauliflower mosaic virus promoter
• rice actin promoter examples include the cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter, rubisco small subunit, maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2 or any promoter that gives enhanced expression.
• enhanced or increased expression can be achieved by using transcription or translation enhancers or activators and may incorporate enhancers into the gene to further increase expression.
• an inducible expression system may be used, where expression is driven by a promoter induced by environmental stress conditions (for example the pepper pathogen-induced membrane protein gene CaPIMPI or promoters that comprise the dehydration-responsive element (DRE), the promoter of the sunflower HD-Zip protein gene Hahb4, which is inducible by water stress, high salt concentrations and ABA or a chemically inducible promoter (such as steroid- or ethanol-inducible promoter system)).
• the promoter may also be tissue- specific.
• the types of promoters listed above are described in the art. Other suitable promoters and inducible systems are also known to the skilled person.
• the promoter is a CaMV 35S promoter.
• the CaMV 35S promoter comprises the sequence defined in SEQ ID NO: 27 or a variant thereof.
• the promoter is an ACTIN 1 promoter (preferably comprising the sequence defined in SEQ ID NO: 28 or a variant thereof) or a Ubiquitin promoter (preferably comprising the sequence defined in SEQ ID NO: 29 or a variant thereof).
• the promoter chosen drives moderate overexpression as described herein.
• a method of screening a plant population for moderate overexpression of an AG02, as described herein, and selecting such plants for subsequent propagation can be measured using methods well known to skilled person, including PCR and Western blotting analysis.
• a nucleic acid construct or recombinant expression vector, as described herein, can be constructed by using existing plant expression vectors.
• the plant expression vector includes binary agrobacterium vector and vectors that can be used for microprojectile bombardment of plants and the like.
• any constitutive, tissue-specific, or inducible promoters may be added to their transcription initiation nucleotides, either alone or in combination with other plant promoters.
• an enhancer may also be used, including a translational enhancer or a transcriptional enhancer.
• Enhancer regions may be ATG start codons or adjacent region start codons, but it must be the same as the reading frame of the encoding sequence to ensure the correct translation of the entire sequence.
• the sources of translation control signals and initial codons are extensive and can be either natural or synthetic.
• the translation initial region can be from a transcription initial region or a structural gene.
• the plant expression vectors used may be processed, for example, by adding genes expressing enzymes that produce colour changes or luminescent compounds in plants, antibiotic markers having resistance, or marked genes antichemical reagents etc.
• the recombinant expression vector may be a recombinant plasmid obtained by inserting a DNA molecule shown by nucleotides at position 1-3102 from the 5’end of SEQ ID NO: 2 or 3 into the multiple cloning site of the pCAMBIA2300-35S-eGFP vector.
• the recombinant expression vector may specifically be a recombinant plasmid obtained by replacing the small fragment between the Xmal and Xbal digestion sites of the pCAMBIA2300-35S-eGFP vector with DNA molecules shown by nucleotides at position 1-3102 from 5’end of SEQ ID NO: 2 or 3.
• nucleic acid construct comprising a nucleic acid sequence as defined herein, wherein preferably, said nucleic acid sequence is operably linked to a regulatory sequence.
• the nucleic acid sequence encodes an AG02 protein as defined in SEQ ID NO: 1 or a functional variant or homologue thereof.
• the nucleic acid sequence as defined herein further comprises at least one mutation, preferably resulting in an AG02 protein with decreased activity compared to the endogenous AG02, hence causing a moderate increase in AG02 activity overall.
• the invention relates to an isolated host cell transformed with nucleic acid construct or vector as described above.
• the host cell may be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell.
• the invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described below.
• the invention relates to a transgenic plant expressing the nucleic acid construct as described herein. Also described herein is a transgenic plant obtained or obtainable by the above-described methods.
• the invention relates to the use of a nucleic acid construct as described herein to increase yield and/or abiotic stress resistance in a plant.
• a genetically altered plant part thereof or plant cell characterised in that the plant has increased expression or activity of AG02 compared to a wild-type or control plant. More preferably, the plant is also characterised by an increase in yield and/or abiotic stress tolerance, preference salt tolerance, as described above.
• the plant expresses a polynucleotide "exogenous" to an individual plant that is a polynucleotide, which is introduced into the plant by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below.
• an exogenous nucleic acid is expressed in the plant which is a nucleic acid construct comprising a nucleic acid encoding a polypeptide sequence as defined in SEQ ID NO: 1 or a homolog or functional variant thereof and that is not endogenous to said plant but is from another plant species.
• the OsAG02 construct can be expressed in another plant that is not rice, such as soybean.
• an endogenous nucleic acid construct is expressed in the transgenic plant.
• the OsAG02 construct can be expressed in rice.
• the plant expresses a nucleic acid comprising a nucleic acid encoding a polypeptide sequence as defined in SEQ ID NO: 1 or a homolog or functional variant thereof.
• the plant is a transgenic plant.
• a method of producing a plant with increased yield and/or abiotic stress resistance compared to a control plant comprising introducing and expressing a nucleic acid construct comprising a nucleic acid encoding an AG02 polypeptide as described above.
• the method comprises
• nucleic acid sequences of nucleic acid constructs described herein may be introduced into said plant through a process called transformation.
• introduction or “transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
• Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
• tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
• the polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome.
• the resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
• Transformation of plants is now a routine technique in many species.
• any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell.
• the methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like.
• Transgenic plants including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.
• the nucleic acid is preferably stably integrated in the transgenic plants genome and the progeny of said plant therefore also comprises the transgene.
• the plant material obtained in the transformation is, in certain embodiments, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants.
• the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying.
• a further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants.
• the transformed plants are screened for the presence of a selectable marker such as the ones described above.
• putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation.
• expression levels of the newly introduced nucleic acid may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
• the generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques.
• a first generation (or Ti) transformed plant may be selfed and homozygous second-generation (or T 2 ) transformants selected, and the T 2 plants may then further be propagated through classical breeding techniques.
• the generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
• the method may further comprise at least one or more of the steps of assessing the phenotype of the transgenic or genetically altered plant, measuring at least one of an increase in yield and/or abiotic stress resistance and comparing said phenotype to determine an increase in at least one of yield and/or abiotic stress resistance in a wild-type or control plant.
• the method may involve the step of screening the plants for the desired phenotype.
• Agrobacterium AGL1 Beijing Biomed Gene Technology Co., Ltd. The public can obtain them from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.
• Sequencing, segmentation, and functional verification of full-length genes of rice result in candidate clones that are sequenced to obtain the full-length sequences of the target clones as shown in SEC ID NO: 3 of the sequence list and proteins as shown in SEC ID NO: 1 of the encoding sequence list.
• the protein shown in SEQ ID NO: 1 of the sequence list was named as AG02 protein, which consists of 1034 amino acid residues.
• the encoding gene of AG02 protein was named as AG02 gene.
• the encoding region of AG02 gene was shown in SEC ID NO: 3 of the sequence list.
• step 2 Using cDNA obtained in step 1 as a template, PCR amplification using a primer pair consisting of the primer AG02FL-F and the primer AG02FL-R to obtain a PCR amplification product.
• the underlined portions are Xmal and Xbal enzyme digestion sites, respectively.
• step 5 Connecting the enzyme-digested products in step 3 to the vector backbone in step 4 to give the recombinant vector pCAMBIA2300-35S-eGFP-AG02; according to the sequencing results, describing the structure of the recombinant vector pCAMBIA2300- 35S-eGFP-AG02 as follows: replacing the small fragment between the Xmal and Xbal digestion sites of the pCAMBIA2300-35S-eGFP vector with DNA molecules shown by nucleotides at the position of 1-3102 from 5’end in the SEC ID NO: 3 of the sequence list.
• step 6 Inoculating the recombinant bacteria obtained in step 6 into a YEB liquid medium containing 50 mg/ml kanamycin and 50 mg/ml rifampicin, and culturing at 200 rpm for 3 days in the dark to obtain recombinant bacteria suspension; collecting precipitation by centrifugation for 3 min at 4,000 rpm.
• step 10 inoculating the callus on NB solid medium containing 150 mg/L G418 and 400 mg/L cefotaxine and culturing at 26°C for 3.5 weeks in the dark.
• step 10 the viable calluses were transferred to NB solid medium containing 200 mg/L G418 and 200 mg/L cefotaxine and culturing at 26°C for 3.5 weeks in the dark.
• the viable calluses were transferred to a differentiation medium (NB minimal medium, 2 mg/L 6-BA, 1 mg/L NAA) containing 200 mg/L G418 at 26°C, low light (light intensity about 150 umol/m 2 .s) and culturing to obtain regenerated plants.
• NB minimal medium 2 mg/L 6-BA, 1 mg/L NAA
• low light low intensity about 150 umol/m 2 .s
• step 13 After completing step 13, culturing the regenerated plants in the seedling medium containing 200mg/L G418 (1/2MS, 0.5mg/L NAA, 0.25mg/L MET) on 26°C, weak light (light intensity About 150 umol/m 2 .s) until they were rooted and transferred to a greenhouse for cultivation, obtaining the TO transgenic plants.
• NPT-F 5’-TCC GGT GCC CTG AAT GAA CT-3’; (SEQ ID NO: 40)
• NPT-R 5’-GGC GAT ACC GTA AAG CAC GA-3’ (SEC ID NO: 41)
• the TO plants selfing to give T 1 plants.
• the T 1 plants selfing to obtain T2 plants.
• T1 generation plants and T2 generation plants were also identified using primers NPT- F and NPT-R primers. If a TO generation plant was tested, the T1 generation plants and T2 generation plants tested by PCR were identified as positive, and the TO plant and its selfing offspring were a homozygous over-expressing transgenic line.
• Figure 2c shows the relative expression of the AG02 gene.
• the results show that, compared to the wild type, expression of AG02 gene is increased in the 5 transgenic lines (A20X-4, A20X-7, A20X-8, A20X-14, A20X-18). 16.
• pCAMBIA2300-35S-eGFP vector instead of the recombinant vector pCAMBIA2300-35S-eGFP-AG02 and carrying out the operation according to step 6 to step 14 to obtain the empty vector plants.
• Plants to be tested T2 plants of the wild-type Zhonghua 11 (ZH11), transgenic lines (A20X-4, A20X-7, A20X-8, A20X-14, A20X-18), and empty vector plants.
• AG02 overexpression can also be achieved in soybean.
• a rice AG02 coding sequence for example, the nucleic acid sequence defined in SEQ ID NOs: 2 or 3, or a nucleic acid sequence coding for the polypeptide sequence defined in SEQ ID NO: 1 into a binary vector after a 35S promoter with fusion of Flag tag at the N-terminal of AG02.
• This can be used to recreate a 35S-Flag-AG02 plasmid (transgene) which can be used to transform soybean (cv. William82) to cause overexpression of rice AG02 in soybean to improve yield and/or increased stress resistance, preferably abiotic stress tolerance, compared to wild type soybean.
• SEQ ID NO: 2 OsAG02genomic nucleic acid sequence
• GCAGCTCCT CTTCTGCCCAAT GT CT GATCAGCAT CCT GGGT ACAAGACGCT GAAGC
• CTTCGCCTCC GACGACCTGCAGAAGCTGGTGTACAACCTCTGCTTCGTCTTCGCCC
• SEQ ID NO: 3 0sAG02 cDNA nucleic acid sequence
• SEQ ID NO: 4 Setaria italica (foxtail millet) nucleic acid sequence (XM_004976750)
• SEQ ID NO: 6 Sorghum bicolor protein argonaute 2 nucleic acid sequence ((LOC8063972) XM_002447052 )
• SEQ ID NO: 7 Sorghum bicolor protein argonaute 2 amino acid sequence ((LOC8063972) XM_002447052 )
• SEQ ID NO: 8 Zea mays argonaute 2 nucleic acid sequence ((LOC 103642054) Sequence ID: XM_008665369.3 )
• SEQ ID NO: 9 Zea mays argonaute 2 amino acid sequence ((LOC 103642054) Sequence ID: XM_008665369.3 )
• Triticum aestivum cultivar Chinese Spring nucleic acid sequence (Sequence ID: AK335299.1)
• Triticum aestivum cultivar Chinese Spring amino acid sequence (Sequence ID: AK335299.1)
• SEQ ID NO: 14 Hordeum vulgare subsp. Vulgare nucleic acid sequence (Sequence ID: AK364273.1) atggattacgagcaaggcggcggcggtggccgcggccgcggaagatctcgcggcggcggagggcgtggcggggc gcccggtggctacgggcctcaaggaggcggcggaggcggaggctacggaggaggctacggaggaggcggtcaaggccggggcg ctcagggaagcggtggagggtacggaggaggcggaggcggaggctacgggccccaggggggcttggagggccgc ggaggtggctacgcccaggggggcttggagggccgc ggaggtggctacgcgctcgcggc
• SEQ ID NO: 15 Hordeum vulgare subsp. Vulgare amino acid sequence (Sequence ID: AK364273.1)
• SEQ ID NO: 16 Brachypodium distachyon argonaute 2-like nucleic acid sequence ((LOC 100834773) Sequence ID: XM_010228966.2)

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