WO2019130018A1

WO2019130018A1 - Methods of increasing yield and/or abiotic stress tolerance

Info

Publication number: WO2019130018A1
Application number: PCT/GB2018/053702
Authority: WO
Inventors: Hongning Tong; Chengcai Chu; Wenchao YIN; Yunhua XIAO; Dapu LIU; Guoxia ZHANG
Original assignee: Institute of Genetics and Developmental Biology of CAS; Institute of Crop Sciences of CAAS
Current assignee: Institute of Genetics and Developmental Biology of CAS; Institute of Crop Sciences of CAAS
Priority date: 2017-12-25
Filing date: 2018-12-19
Publication date: 2019-07-04
Anticipated expiration: 2020-06-25
Also published as: CN107868123B; CN107868123A

Abstract

The invention relates to methods of increasing yield and/or abiotic stress tolerance in a plant, transgenic plants with increased yield and/or abiotic stress tolerance and methods for making such plants. In particular, the invention comprises increasing the expression or activity of AGO2, particularly to a moderate level.

Description

Methods of increasing yield and/or abiotic stress tolerance

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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.

Under normal growth conditions, 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.

There is therefore a need to increase both crop yield and stress resistance. The present invention addresses this need.

SUMMARY OF THE INVENTION

Here, we identified an Argonaute member protein, AG02, as a candidate for achieving this goal. AG02 is a potential interacting protein of DLT, a putative GRAS family transcriptional factor protein. Strikingly, overexpression of AG02 produced plants simultaneously having enlarged grain size and enhanced salt tolerance. Thus, our study suggested a novel genetic cascade simultaneously controlling grain weight and stress resistance, and demonstrated its tremendous potential for enhancing grain production under either normal or adverse conditions.

We have previously shown that DLT plays a positive role in regulating BR responses. To explore the regulatory mechanism of DLT function, we performed a yeast two hybrid screening using DLT as a bait protein. Using this approach, we obtained a segment corresponding to the C-terminal amino acid 858-1034 of AG02 (AG02-C177). Further analysis showed that the AG02 C-terminal containing the PIWI domain, but not the N- terminal containing PAZ domain, can interact with the DLT C-terminal containing the conserved GRAS domain, but not the variable N-terminal. As a control of the interaction, we found that DLT cannot interact with the C-terminal of either AG03 or AG07, the two closest homologs of AG02, and also, the AG02 C-terminal cannot interact with either SLR1 or MOC1 , the two homologs of DLT belonging to GRAS family proteins, suggesting the DLT-AG02 interaction is specific and could have biological significance. Subcellular localization analysis showed that YFP-AG02 can be evidently observed in the nucleus of rice protoplast or tobacco leaf epidermal cells. The interaction was further verified using BiFC in rice protoplasts.

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. We were curious whether AG02 can modulate DLT activity to regulate gene expression. We selected a number of genes with altered expression in dlt mutant based on RNA-seq data and fused their promoters with a LUC reporter to test the DLT effects on their activities. We obtained three candidate promoters which could be enhanced by DLT in tobacco leaves. Interestingly, we found that AG02 alone can also activate these promoters. When DLT and AG02 were co-expressed, the activities of the promoters were greatly enhanced. These results suggested that AG02 can enhance DLT activity to regulate gene expression.

We have experimentally demonstrated that overexpression of AG02 increases grain size, grain weight, grain length and increases 100-grain weight by between 20 and 30% compared to wild type plants. At the same time, the resistance of plants to high salt stress and bacterial leaf blight and black streak dwarf is significantly increased. After culturing in 200 mM sodium chloride salt solution, almost 100% of wild-type plants do not survive. However, the survival rate of transgenic plants overexpressing AG02 was between 50%-80%. As such, overexpression of AG02 simultaneously increases yield and abiotic resistance.

In one aspect of the invention, there is provided a method of increasing at least one of yield and/or abiotic stress tolerance in a plant, the method comprising increasing the expression or activity of AG02 (argonaute member protein) in said plant. In one embodiment, only yield is increased.

In a preferred embodiment, the said increase is compared to a control or wild-type plant.

In one embodiment, the yield is grain yield. In another embodiment, the abiotic stress tolerance is salt tolerance.

In one embodiment, 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. Preferably, the nucleic acid sequence comprises SEQ ID NO: 2 or 3 or a functional variant or homologue thereof.

In a preferred embodiment, the regulatory sequence is a promoter, preferably a CaMV 35S promoter.

In another embodiment, the AG02 polypeptide comprises at least one modification that affects protein function. Preferably the AG02 polypeptide comprises at least one peptide tag.

In one embodiment, the plant is a crop plant. Preferably, the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.

In another aspect of the invention, there is provided a method for making a transgenic plant having increased yield and/or abiotic stress tolerance, the method 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.

In an alternative aspect, there is provided a method for making a transgenic plant having increased yield and/or abiotic stress tolerance, the method 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). Alternatively, 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).

In one embodiment, the nucleic acid sequence encoding an AG02 polypeptide comprises SEQ ID NO: 2 or 3 or a functional variant or homologue thereof.

In another embodiment, the regulatory sequence is a promoter, preferably a CaMV 35S promoter.

In yet another embodiment, the AG02 polypeptide comprises at least one modification. Preferably, the at least one modification leads to moderate overexpression of AG02 (for example as defined herein). In one example, the at least one modification affects protein function. Preferably, the AG02 polypeptide comprises at least one peptide tag.

In one embodiment the plant is a crop plant. Preferably, the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.

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

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

In an alternative aspect, there is provided 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). Alternatively, 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).

In a preferred embodiment, the plant is characterised by an increase in yield and/or abiotic stress tolerance compared to a control plant.

In one embodiment, the plant is characterised by an increase in grain yield. In another embodiment, the plant is characterised by an increase in salt tolerance.

In another preferred embodiment, the regulatory sequence is a promoter, preferably a CaMV 35S promoter.

In another aspect of the invention, there is provided 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 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.

There is also provided a vector comprising the nucleic acid construct as described above.

There is further provided a host cell comprising the nucleic acid construct or the vector described above. Preferably the host cell is a bacterial or plant cell.

There is also provided a transgenic plant expressing the nucleic acid construct described above. Preferably 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

There is further provided the use of the nucleic acid construct or the vector described above, to increase yield and/or abiotic stress tolerance in a plant.

In a further aspect of the invention there is provided a protein, which is the following (a) or (b):

(a) a protein consisting of an amino acid sequences shown in SEQ ID NO: 1 of the sequence list;

(b) a protein derived from the amino acid sequence of SEQ ID NO: 1 having a substitution and/or deletion and/or addition of one or a few amino acid residues and having the same function as SEQ ID NO: 1.

Also provided is a gene encoding the above protein. In one embodiment, the gene is a DNA molecule according to any one of the following (1)-(3):

(1) a DNA molecule having the sequence of SEQ ID NO: 2 or 3;

(2) a DNA molecule that can hybridise under stringent conditions to the above DNA molecule and encoding the protein of SEQ ID NO: 1 ;

(3) a DNA molecule having 90% or more homology to the DNA sequence defined in (1) or (2) or (3) and encoding the protein of SEQ ID NO: 1.

Also provided is a recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria comprising the above gene.

Also provided is the use of the protein or gene described above to regulate yield and/or tolerance in a plant, preferably abiotic stress tolerance. Preferably the plant is a dicotyledonous or monocotyledonous plant.

In another aspect of the invention there is provided 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. In a further aspect of the invention there is provided 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).

In a further aspect, there is provided the use of the protein, gene or method described above in plant breeding. Preferably, the purpose of 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.

BRIEF DESCRIPTION OF THE FIGURES

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

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.

Figure 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.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

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

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

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. These terms also encompass a gene. The term "gene" or“gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.

The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

The term "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. Encompassed by the aforementioned terms are 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. Also included within the term is 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.

For the purposes of the invention, a“genetically altered plant” or“mutant plant” is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant. In one embodiment, 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. In one embodiment, the mutagenesis method is targeted genome modification or genome editing. In one embodiment, 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. Therefore, in this example, these traits are conferred by the presence of an altered plant genome, for example, a mutated endogenous AG02 gene or promoter. In one embodiment, 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. In other words, the genetically altered plant can be described as transgene-free.

Nonetheless, in an alternative embodiment, the genetically altered plant is a transgenic plant. For the purposes of the invention, "transgenic", “transgene” or "recombinant" 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

(a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or

(b) 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. In the case of 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. Preferably, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In a preferred embodiment, the plant is a cereal. In another embodiment the plant is Arabidopsis. In one example, the monocotyledonous plant may be a plant of the Poales. The Poales plant may be a gramineous plant.

In a most preferred embodiment, the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet. In one embodiment the plant is from the genus Oryza, preferably rice, and in one example Zhonghua 11 rice. In another embodiment the plant is soybean.

The term "plant" as used herein 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). The term "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. In another aspect of the invention, there is provided a product derived from a plant as described herein or from a part thereof.

In a most preferred embodiment, 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.

In an alternative embodiment, 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.

In one aspect of the invention, there is provided 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. In one embodiment there is provided a method of increasing both yield and abiotic stress tolerance, meaning that yield can be increased under conditions of abiotic stress tolerance. As described below, abiotic stress tolerance may be selected from drought, salinity, wind, high or low temperature or high light. In a preferred embodiment, 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). As used herein high salt stress can be considered to be at least 100-250mM NaCI.

The term "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, and 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. All parameters are relative to a wild-type or control plant. The skilled person would be able to measure any of the above yield parameters using known techniques in the art. As used herein“grain” may also be referred to herein as“seed” and such terms can be used interchangeably.

In a preferred embodiment, 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. For example, 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. In a preferred embodiment, yield is increased by between 5 and 15%, more preferably between 8 and 10% compared to a control plant. In a preferred embodiment, 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.

As used herein“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. In other words, 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.

In a preferred embodiment, said stress is salt stress. As such, preferably 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. We have found, as shown in Figure 4, that overexpressing AG02 increases salt tolerance ability. In one example,“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. Typically, 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. Alternatively, 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). As used herein survival can be considered to be the ability to produce new green leaves after treatment. In one embodiment, 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,

13-fold- 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold and 20-fold compared to 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.

We have also found, as shown in Figure 5, that overexpression of AG02 increases ABA (abscisic acid) sensitivity. This has been shown by an increase in the level of reduction in shoot and root length in response to increasing ABA concentrations compared to the level of reduction in control plants. ABA sensitivity is associated with various types of abiotic stress resistance such as salt, drought or cold. Accordingly, in another aspect of the invention there is provided a method of increasing ABA sensitivity in a plant, the method comprising increasing the expression or activity of AG02 in a plant. In one embodiment, 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-

14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold and 20-fold compared to the ABA sensitivity in a control plant or wild-type plant.

In another aspect of the invention, there is also provided a method of increasing at least one of plant height and leaf length, preferably in the roots, the method comprising increasing the expression or activity of AG02 in a plant.

In one embodiment, 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. In a preferred embodiment, 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.

In an alternative embodiment, 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. Preferably, such mutation is introduced using targeted genome editing. Preferably, 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. To achieve effective genome editing via introduction of site-specific DNA DSBs, four major classes of 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). Meganuclease, 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.

Upon delivery into host cells via the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in host gene promoters and activate transcription. Their targeting specificity is determined by a central domain of tandem, 33-35 amino acid repeats. This is followed by a single truncated repeat of 20 amino acids. The majority of naturally occurring TAL effectors examined have between 12 and 27 full repeats.

These repeats only differ from each other by two adjacent amino acids, their repeat- variable di-residue (RVD). The RVD that determines which single nucleotide the TAL effector will recognize: one RVD corresponds to one nucleotide, with the four most common RVDs each preferentially associating with one of the four bases. Naturally occurring recognition sites are uniformly preceded by a T that is required for TAL effector activity. TAL effectors can be fused to the catalytic domain of the Fokl nuclease to create a TAL effector nuclease (TALEN) which makes targeted DNA double-strand breaks (DSBs) in vivo for genome editing. 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. (Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting; Nucleic Acid Research 39, 2011) describes a set of customized plasmids that can be used with the Golden Gate cloning method to assemble multiple DNA fragments. As described therein, the Golden Gate method uses Type IIS restriction endonucleases, which cleave outside their recognition sites to create unique 4 bp overhangs. Cloning is expedited by digesting and ligating in the same reaction mixture because correct assembly eliminates the enzyme recognition site. Assembly of a custom TALEN or TAL effector construct and involves two steps: (i) assembly of repeat modules into intermediary arrays of 1-10 repeats and (ii) joining of the intermediary arrays into a backbone to make the final construct.

Another genome editing method that can be used according to the various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art, for example in US 8,697,359 and references cited herein. In short, CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR- associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA). Three types (I- III) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of 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. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, 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. Finally, 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. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used.

The single guide RNA (sgRNA) 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. In plants, 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.

Thus, 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.

Thus, it is understood, as those skilled in the art will appreciate, that the aspects of the invention, including the methods and uses described herein, encompass not only a 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. For example, 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. Similarly, 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. Each of the proposed modifications is well within the routine skill in the art, as is determination or retention of biological activity of the encoded products.

In one embodiment, a functional variant has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%,

46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,

61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%,

76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,

91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the non-variant nucleic acid or amino acid sequence. The skilled person will also understand that the invention is not limited to a 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.

The term homolog, as used herein, 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%,

41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%,

56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,

71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%,

86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to the amino acid represented by SEQ ID NO: 1 or to the nucleic acid sequences as shown by SEQ ID NOs: 2 or 3. In one embodiment, 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%,

84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In one example, 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.

Functional variants of AG02 homologs as defined in SEQ ID NOs 5, 7, 9, 11 , 13, 15, 17, 19, 21 and 23 or SEQ ID NOs 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 are also within the scope of the invention.

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). AG02 may also be referred to as DIP2, and such terms can be used interchangeably.

In a preferred embodiment, the PAZ domain comprises the following sequence: AGPVLDLVQKSVRYLDYRTTLNKHQLDTLKNELKGQRVTVNHRRTKQKYIVKGLTDKP ASQITFVDSESGQTKKLLDYYSQQYGKVI EYQM LPCLDLSKSKDKQNYVPI ELCDLLE GQRYPKASLNRNSDKTLKEMA (SEQ ID NO: 30) In another preferred embodiment, the PIWI domain comprises the following sequence:

EIAQQFGISLDVQMMEVTGRTLPPPSLKLGTSSGQPPKFNIDQPNCQWNLTRKRLAE

GGVLQCWGVVDFSADSGQYALNGNMFIDKIVRKCCDLGVQMNRNPCIVQLLDMEVL

SDPHQLFEELNKAKQAAASKKQKLQLLFCPMSDQHPGYKTLKLICETQLGIQTQCFLS

FLANKQQGQDQYMSNLALKINGKIGGSNIQLFGESLPRISGAPYMFIGADVNHPSPGN

VESPSIAAWASVDQGASKYVPRIRAQPHRCEVIQHLGDMCKELIGVFEKRNRVKPQR

IIYFRDGVSDGQFDMVLNEELADMEKAIKTKDYSPTITVIVAKKRHHTRLFPKDLNQQQ

TKNGNVLPGTWDTGWDPAAYDFYLCSHNGLIGTSRPTHYYSLLDEHGFASDDLQK

LVYNLCFVFARCTKPVSLATPVYYADLAAYRGRLYY (SEQ ID NO: 31)

In one embodiment, 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%,

53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%,

68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%,

83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,

98%, or at least 99% overall sequence identity to the amino acid represented by SEQ ID NO: 1 and has at least a PAZ and/or PIWI domain, or a domain 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%, 53%, 54%, 55%,

56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,

71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%,

86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to SEQ ID NO: 30 or 31.

Two 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. 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. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Non-limiting examples of 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, for example when overexpressed in a plant.

Thus, the 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. In this manner, 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. In 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. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

Typically, 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. In one example, 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. In a further embodiment, 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.

Accordingly, in summary a variant can be considered to comprise one of the following:

(a) a 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;

(b) a 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;

(c) a nucleic acid sequence encoding an AG02 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 and has at least a PAZ and/or PIWI domain, or a domain 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 SEQ ID NO: 30 or 31 ; or

(d) a 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).

As used herein““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. In one embodiment, 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. For example, 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).

In one embodiment, however, the level of expression or the level of increase in activity is moderate. In one example, 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. Alternatively, moderate expression may comprise increasing levels of expression or activity by up to 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x,

18x, 19x, 20x, 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x,

35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, 50x, 51x,

52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x or 60x when compared to the level of expression in a control or wild-type plant. In one example the level of AG02 overexpression is sufficient to increase yield in a plant compared to a control plant. Accordingly, in one example 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).

In one example, 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. In one example, a protein tag may be fused to the C-terminal of AG02 to impair protein function by affecting protein structure or protein-protein interactions. In one embodiment, 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. Examples of 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. In one embodiment, the tag is a FLAG-tag, and comprises the amino acid sequence: DYKDDDDK (SEQ ID NO: 32).

In one aspect of the invention, a moderate increase in activity of AG02 may be achieved by the post-translational addition of at least one synthetic tag as described herein. Alternatively, 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. Preferably, the insertion is achieved using ZFNs, TALENs or CRISPR/Cas9.

Table 1 : Examples of protein tags:

In one example, 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. In alternative example, 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.

In a further example, 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. Preferably the mutation is any mutation that leads to an increase in AG02 expression as defined above. Examples of mutations include substitutions, deletions and additions of at least one nucleic acid. By“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). In one embodiment, the sequence of the AG02 promoter comprises or consists of a nucleic acid sequence as defined in SEQ ID NO: 26. In the above embodiments an‘endogenous’ nucleic acid may refer to the native or natural sequence in the plant genome.

In another aspect of the invention, 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. In one example, 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.

By“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.

As used herein, a“mutation” may be an addition, deletion or substitution of one or more nucleotides (or bases).

In one embodiment, 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.

In another example, moderate overexpression of AG02 can be achieved by choice of the regulatory sequence as described in further detail below.

As described herein, the nucleic acid construct preferably comprises a regulatory sequence. Preferably the regulatory sequence is operably linked to the nucleic acid sequence of interest. In one example, the nucleic acid sequence of interest is an AG02 nucleic acid sequence as defined herein.

As used herein, the term "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. The term "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.

The term "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. Encompassed by the aforementioned terms are 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. Also included within the term is 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 term "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. For expression in plants, 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. In one embodiment, 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.

For the identification of functionally equivalent promoters, 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.

The term "operably linked" as used herein 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.

For example, the nucleic acid sequence may be expressed using a promoter that drives overexpression. Overexpression according to the invention means that the transgene is expressed at a level that is higher than expression of endogenous counterparts driven by their endogenous promoters. However, the level of overexpression can be varied by using different types of promoter. For example, 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. Examples of constitutive promoters 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. Alternatively, 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. Furthermore, 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.

According to a more preferred embodiment, the promoter is a CaMV 35S promoter. In one embodiment, the CaMV 35S promoter comprises the sequence defined in SEQ ID NO: 27 or a variant thereof. In alternative embodiments, 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). Preferably, the promoter chosen drives moderate overexpression as described herein.

Accordingly, in another aspect of the invention, there is provided a method of screening a plant population for moderate overexpression of an AG02, as described herein, and selecting such plants for subsequent propagation. Moderate overexpression 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. When the AG02 gene is used to construct a recombinant expression vector, any constitutive, tissue-specific, or inducible promoters may be added to their transcription initiation nucleotides, either alone or in combination with other plant promoters. In addition, when using the AG02 gene to construct a recombinant expression vector, an enhancer may also be used, including a translational enhancer or a transcriptional enhancer. These 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. To facilitate the identification and screening of transgenic plant cells or plants, 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.

In another aspect of the invention there is provided nucleic acid construct comprising a nucleic acid sequence as defined herein, wherein preferably, said nucleic acid sequence is operably linked to a regulatory sequence. In a preferred embodiment, the nucleic acid sequence encodes an AG02 protein as defined in SEQ ID NO: 1 or a functional variant or homologue thereof. In one aspect of the invention, 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. In another aspect, 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.

Thus, in a further aspect, 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.

In another aspect, 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.

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

In one embodiment, 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. In one embodiment, 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. For example, the OsAG02 construct can be expressed in another plant that is not rice, such as soybean. Alternatively, an endogenous nucleic acid construct is expressed in the transgenic plant. For example, the OsAG02 construct can be expressed in rice. Accordingly, in one embodiment, 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. In either of these embodiments, the plant is a transgenic plant.

In a further aspect of the invention there is provided a method of producing a plant with increased yield and/or abiotic stress resistance compared to a control plant, the method comprising introducing and expressing a nucleic acid construct comprising a nucleic acid encoding an AG02 polypeptide as described above.

Accordingly, in one embodiment, the method comprises

a. selecting a part of the plant;

b. transfecting at least one cell of the part of the plant of paragraph (a) with at least one nucleic acid construct as described herein or

c. regenerating at least one plant derived from the transfected cell or cells; d. selecting one or more plants obtained according to paragraph (c) that show increased expression of AG02 (preferably moderate levels of overexpression, as defined herein).

Any of the nucleic acid sequences of nucleic acid constructs described herein may be introduced into said plant through a process called transformation. The term "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. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). 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.

The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plants is now a routine technique in many species. Advantageously, 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. According to the invention, the nucleic acid is preferably stably integrated in the transgenic plants genome and the progeny of said plant therefore also comprises the transgene.

To select transformed plants, 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. For example, 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. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.

Following DNA or nucleic acid transfer and regeneration, 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. Alternatively or additionally, 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. For example, a first generation (or Ti) transformed plant may be selfed and homozygous second-generation (or T₂) transformants selected, and the T₂ plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion). In a further embodiment of any of the methods described herein, 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. In other words, the method may involve the step of screening the plants for the desired phenotype.

In a further aspect of the invention there is provided a plant obtained or obtainable by the above described methods.

While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

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

The foregoing application, and all documents and sequence accession numbers cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents 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 is now described in the following non-limiting examples.

EXAMPLE I: Obtaining AG02 Protein and its Encoding Gene

Zhonghua 11 : reference: “Xiao Y, Liu D, Zhang G, Tong H and Chu C(2017) Brassinosteroids Regulate OFP1 , a DLT Interacting Protein, to Modulate Plant Architecture and Grain Morphology in Rice. Front. Plant Sci. 8:1698. doi:10.3389/fpls.2017.01698”; the public can obtain them from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. pCAMBIA2300-35S-eGFP vector: reference:“Xiao Y, Liu D, Zhang G, Tong H and Chu C(2017) Brassinosteroids Regulate OFP1 , a DLT Interacting Protein, to Modulate Plant Architecture and Grain Morphologyin Rice. Front. Plant Sci.8:1698. doi:10.3389/fpls.2017.01698”; the public can obtain them from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

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.

EXAMPLE II: Obtaining AG02 Gene Overexpressed Transgenic Plants

1. Extraction of total RNA from seedlings of wild-type rice Zhonghua 11 and reverse transcription to cDNA.

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.

AG02FL-F:5’- CCC GGG ATG GAG CAC GAG CGC GGT G-3’; (SEQ ID NO: 38)

AG02FL-R:5’- TCT AGA GAT GAA GAA CAT GTT GTC CAC CAG A G-3’ (SEC ID NO: 39)

In the primer AG02FL-F and the primer AG02FL-R, the underlined portions are Xmal and Xbal enzyme digestion sites, respectively.

3. Recovery of enzyme-digested products by double digestion of the PCR amplification product obtained in step 2 with the restriction enzymes Xmal and Xbal.

4. Recovery of vector backbone of about 10 kb by double digestion of the pCAMBIA2300-35S-eGFP vector with the restriction enzymes Xmal and Xbal.

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.

6. Transforming agrobacterium AGL1 with the recombinant vector pCAMBIA2300-35S- eGFP-AG02 obtained in step 5 to obtain recombinant bacteria, transforming the recombinant bacteria into the callus in Zhonghua 11 according to the method in reference“Hiei Y.Ohta S.Komari T.Kumashiro T. Efficient transformationof rice(Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 1994; 6:271-82”, as specifically shown in steps 7-14.

7. 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.

8. Resuspending the precipitation obtained in step 7 by using AAM liquid medium containing 0.1 mM acetosyringone, and culturing at 28°C without light and at 150 rpm in a shaker to OD₆oo _nm=0.4 and obtaining an infection liquid.

9. Picking well-grown, granulated Zhonghua 11 callus and immersing it into the infection liquid obtained in step 8, and culturing at 28°C and at 150-200 rpm for 20 min; removing the callus and blotting up excess bacteria liquid with sterile filter paper; spreading the callus tissue on a sterile plate containing multi-layer filter paper, drying on a super-clean bench (the callus was dispersed without agglomeration), and then transferring the callus to the NB solid medium and culturing for 2-3 day at 26°C in the dark.

10. After completing step 9, 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.

11. After completing 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.

12. After completing step 11 , 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/m².s) and culturing to obtain regenerated plants.

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/m².s) until they were rooted and transferred to a greenhouse for cultivation, obtaining the TO transgenic plants.

14. Extracting the leaf total DNA of TO transgenic plants obtained in step 13 as a template and using a pair of primers consisting of NPT-F primers and NPT-R primers to carry out PCR amplification; screening the TO positive transgenic plants (positive plant PCR amplification product size is 582bp).

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.

15. Extracting total RNA from leaves of T2 plants of several overexpressed transgenic lines obtained from rice Zhonghua 11 (ZH11) and step 14 and reverse-transcribing into cDNA; using cDNA as a template and using qRT-PCR method to detect the expression of AG02 gene (Actin gene was used as the reference gene); detecting expression of Actin gene by using primer pairs consisting of primers ACT-F and ACT-R primers and detecting expression of AG02 gene by using primer pairs consisting of primer AG02-F and primer AG02-R.

AG02-F: 5’-AGC CAA GGT CAA ATT GTT GG-3’; (SEC ID NO: 42)

AG02-R: 5’-CTC CTT GTC TGA AGC CTT GG-3’; (SEC ID NO: 43)

ACT-F: 5’-TGC TAT GTA CGT CGC CAT CCA G-3’; (SEC ID NO: 44)

ACT-R: 5’-AAT GAG TAA CCA CGC TCC GTC A-3’ (SEC ID NO: 45) 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. Using the 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.

EXAMPLE III. Phenotypic Analysis of AG02 Gene Overexpressed Transgenic Plants

I. Statistics of Grain Size and Grain Weight

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.

The grain size and grain weight harvested at the ripening stage of the test plants were counted and the results are shown in Figures 2A and 2F.

The results showed that compared to the wild type, the grain length and grain weight of the transgenic lines increased significantly, especially in the two A20X-4 and A20X-8 plants, the 100-grain weight increasing by 30.8% and 22.7%, respectively. There was no significant difference between the phenotypes of empty vector plants and wild-type plants.

II. Salt Stress Resistance Test

Seeds of T2 plants of wild-type Zhonghua 11 (ZH11) and transgenic lines (A20X-4, A20X-7, A20X-8, A20X-14, A20X-18) were soaked in the dark at 37°C and germinated. Each half of each transgenic line and of wild type were spread on a 96-well plastic plate with the bottom removed. Each plant was set up with 4 replicates and cultured in a light incubator for 10 days using 1/2 MS liquid medium (temperature 30°C, light 12h/darkness 12h, fresh 1/2MS liquid medium every 2 days), replaced with 1/2MS liquid medium containing 200 mM sodium chloride at 10th day, continuing cultivation for 10 days, and observing phenotype.

The results are shown in Figure 6. These results show that under conditions of salt stress, wild type plants were nearly completely dead while most of the transgenic lines were still alive and the survival rate reached up to 50%-80%. There was no significant difference between the empty vector plants and the wild type plants, indicating that the AG02 gene can increase salt stress resistance in plants. EXAMPLE IV

To analyse AG02 function in plants, we produced a number of allelic AG02 mutants using CRISPR/CAS9. We noted that while AG02 showed some phenotypes at the early seedling development stage, we failed to observe an obvious difference at the latter stage. We thus further performed AG02 overexpression analysis using the 35S CaMV promoter in wild-type. Strikingly, we obtained five independent transgenic lines ( A20X for short) that showed an obviously enlarged grain size, and a 100-grain weight increase of around 20% compared with the wild-type. Gene and protein expression analysis verified the elevated expression of AG02 in the plants. Except the enlarged grain size, these plants could also have enhanced growth since both the plant height and leaf length were increased compared with the wild-type.

We considered that these transgenic plants might have increased grain yield due to grain enlargement. However, statistical data showed that in most cases the yield was not significantly altered. In addition, plant architecture appeared loose which was usually an unfavourable trait in field management. These negative effects could be attributed to strong expression of AG02. To tackle this problem, we tried in our overexpression analysis to fuse a Flag tag at the C-terminal of AG02 which was thought will impair protein function. As expected, we obtained a number of AG02-Flag overexpression plants (A2FOX for short) showing mildly increased grain size, and increased grain yield per plants at the T₀ generation. Importantly, these transgenes had no obvious effect on plant architecture. In two lines further analyzed, the grain yield per plant increased 10% and 8% respectively, in contrast to those without tag fusion which had an unaltered yield. These results suggested the potential value of AG02 to improve crop yield.

A previous study revealed that, among all the AGO members, AG02 is the only one induced by various stresses such as salt, drought and cold. Considering that DLT is unique among GRAS family proteins, we suspected that DLT may interact with AG02 to regulate stress responses. To test this possibility, we chose salt stress for our analysis of the plant response. Strikingly, we found that dlt is highly sensitive to salt stress. Repeatedly, in our tests using 200mM NaCI, the survival rates of wild-type plants remained between 50% and 100% whereas the dlt mutant plants were near all dead. The AG02 mutants also had increased sensitivity to salt stress, although to a much less extent.

Since DLT and AG02 positively regulate salt stress, we considered whether overexpression of these genes can enhance plant stress resistance. As reported previously, overexpression of DLT increased grain length and leaf growth. While we found that strong DLT-overexpression plants showed decreased tolerance to high salt concentration, possibly due to growth defects, the mild lines exhibited obviously increased stress resistance. Strikingly, AG02 overexpression plants showed greatly enhanced salt tolerance ability (Figure 5). Of two lines tested, survival rate of the plants remain above 50% when the wild-type plants are near all dead.

These beneficial traits attracted us to explore the underlying mechanisms. First, we analyzed the plant response to ABA, the well-known stress hormone. While dlt exhibited markedly decreased sensitivity to ABA treatment, AG02-overexpression plants showed greatly increased ABA sensitivity in terms of both shoot and root growth. This result suggested that both DLT and AG02 may mediate ABA response to modulate stress tolerant ability. Next, we performed transcriptome analysis using A20X-4 and wild-type seedlings. By RNA-seq, we identified 378 differentially expressed genes in A20X-4, with 116 up-regulated and 262 down-regulated compared with wild-type. Among them, 78 genes have been functionally reported in literatures (http://www.ricedata.cn/). and at least 31 of them were associated with stress responses, including a number of well-known players involved in stress and disease responses. These results strongly suggested the key role of AG02 in regulating stress response.

EXAMPLE V

AG02 overexpression can also be achieved in soybean. We have introduced 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.

SEQUENCE LISTING

SEQ ID NO: 1 0sAG02 amino acid sequence

MEHERGGGGRGRGRGRGGGRGGGGGDGRGGGYGGAGGGGVGGRGGRGPPGGG

GGRGYEPGGGRGYGGGGGGGGRGYGGGGGGGGYESGGGRGYGGGGRGYESGG

GRGPGGGGRGHESGGGGGRGGNVWAQPGRGRGGAPAPAPAPAPAARRIQDEGAA

RSSGTVERIASTEVVRVQPPAPPVAVSRSGTRVPMRRPDGGGSVSKAKVKLLVNHFIV

KYRQASTVFHYDIDIKLDISSPKASDKELSKGDFLTVKDELFKDESFRRLSSAVAYDGKR

NLFTCAELPDGLFRVKVRSRTYIVSVEFKKKLPLSQLSELPVPREVLQGLDVIVREASSW

RKIIIGQGFYSQGRSVPIGPDVVALKGTQQTLKCTQKGLILCVDYSVMPFRKAGPVLDLV

QKSVRYLDYRTTLNKHQLDTLKNELKGQRVTVNHRRTKQKYIVKGLTDKPASQITFVDS

ESGQTKKLLDYYSQQYGKVIEYQMLPCLDLSKSKDKQNYVPIELCDLLEGQRYPKASLN

RNSDKTLKEMALIPASSRKEEILELVNADDGPCRGEIAQQFGISLDVQMMEVTGRTLPP

PSLKLGTSSGQPPKFNIDQPNCQWNLTRKRLAEGGVLQCWGVVDFSADSGQYALNGN

MFIDKIVRKCCDLGVQMNRNPCIVQLLDMEVLSDPHQLFEELNKAKQAAASKKQKLQLL

FCPMSDQHPGYKTLKLICETQLGIQTQCFLSFLANKQQGQDQYMSNLALKINGKIGGSNI

QLFGESLPRISGAPYMFIGADVNHPSPGNVESPSIAAVVASVDQGASKYVPRIRAQPHR

CEVIQHLGDMCKELIGVFEKRNRVKPQRIIYFRDGVSDGQFDMVLNEELADMEKAIKTK

DYSPTITVIVAKKRHHTRLFPKDLNQQQTKNGNVLPGTVVDTGVVDPAAYDFYLCSHNG

LIGTSRPTHYYSLLDEHGFASDDLQKLVYNLCFVFARCTKPVSLATPVYYADLAAYRGRL

YYEGMMMSQPPPSSAASASSASSSGAGASDFRSFPALHEDLVDNMFFI

SEQ ID NO: 2 OsAG02genomic nucleic acid sequence

CTCTTCTGCTTTTT AGCGAAAT CCGAGAAAAAAATTGCCTGCAAAAAAAAAAATCGAA

ACGTCGTTCTGATCGATCGGCCGTTCGAGTTCACGGCGGAGATGGAGCACGAGCG

CGGTGGCGGTGGCCGCGGCCGCGGGAGGGGTCGCGGTGGCGGGCGTGGCGGC

GGTGGCGGCGATGGTCGCGGAGGCGGTTATGGTGGTGCTGGTGGTGGTGGTGTC

GGCGGGCGCGGTGGGCGTGGGCCTCCTGGTGGTGGTGGTGGACGCGGGTACGA

GCCCGGCGGCGGGCGTGGGTACGGTGGCGGCGGCGGCGGTGGTGGACGTGGGT

ATGGCGGCGGAGGCGGCGGTGGTGGGTACGAGTCCGGCGGTGGGCGTGGGTAT

GGCGGCGGTGGACGTGGGTATGAATCCGGCGGTGGGCGTGGACCTGGCGGCGG

CGGCCGTGGGCACGAGTCCGGCGGTGGCGGTGGCCGCGGCGGGAACGTGTGGG

CGCAGCCGGGGAGAGGGCGCGGAGGAGCCCCCGCCCCGGCGCCGGCGCCAGCA CCAGCAGCGAGGAGGATCCAGGACGAGGGGGCCGCGAGGTCGTCGGGTACCGTT GGT AAGCGTCTCACCAAGT AT AATTGGTGCTTCT GAAT GT ACCAT CACTTTTTTTTTT GTT AT GTCACGAAACACTGCAGGGAAGCAT CT AGGATTT AGAAAGTCCCT AGGACG TT CAGTTT CTT ACTT GAAAGTGCAT CAT G AGCAACATTTGCT CT GT CATTTCCAT CT G GGAAGTCGTTTTGGAAAAGCAAAATTTCTAGGGGGGACTACAACTTGTTGATCCATT TT GTT CT AACT GCTCGTT AAGTTGCT G ATTTT GCTT ACTCCT AT GT G AACTT G ACCT G TTTTT GTTT CTAACGGTT CTT CATTTTCCTT CAG AGCGCATTGCTT CT ACT G AGGTT G T AAGAGT ACAACCACCTGCACCCCCAGTTGCT GT GTCTCGT AGT GGCACGCGT GT G CCAATGCGAAGACCT GATGGTGGAGGCTCAGT ATCGAAAGCCAAGGT CAAATT GTT GGT GAACCATTTT AT AGTT AAGT ACCGACAGGCAT CAACT GTTTTT CACT AT GACAT A GACATCAAGCTTGATATAAGTTCCCCCAAGGCTTCAGACAAGGAGCTATCCAAGGG AGATTTTCTT ACT GT CAAGGACGAGCTCTTCAAGGAT GAGAGCTTTCGGCGGCTTT C AT CAGCT GTT GCTT AT GAT GGAAAAAGAAATTT ATTT ACTT GTGCT GAGCT ACCAGAT GGTTT GTTTCGT GT CAAAGT CCGTT CACGG ACTT ACATT GT ATCTGT GG AGTT CAAG AAGAAGCTT CCTTT GAGCCAACT CT CGGAACTGCCT GTGCCCAGAGAGGTCTTGCA GGGGCTTGATGTCATTGTGCGTGAGGCCTCTAGCTGGCGCAAGATTATCATTGGTC AGGGATTTT ACTCGCAGGGCCGCAGT GT GCCCATT GGGCCGGAT GTT GT AGCTCTC AAAGGAACCCAGCAGACCCT GAAAT GCACT CAGAAAGGACT GATCCTTT GT GTGGA CT ATT CGGTT ATGCCGTTT CGCAAAGCT GG ACCT GT GTT GG AT CTT GTT CAG AAGTC T GT G AG ATACCTT G ACT ACAGG ACAACACT AAACAAACACCAATT GG ACACTTT G AA GAATGAACTCAAAGGCCAGCGTGTCACTGTAAATCATAGGAGGACAAAGCAGAAGT ACATT GTT AAAGGTTT GACT GAT AAACCTGCAAGT CAG AT AACTTTT GT AG ATT CT GA AT CAGGACAGACCAAGAAGCTT CTT GATT ACT ATT CGCAGCAGT AT GGCAAGGTT AT T GAGT AT CAAATGCTTCCATGCTTGGATTT GAGCAAGAGCAAGGACAAGCAAAACT A T GT GCCGATT GAATT GT GT GAT CTT CTT GAAGGGCAGAGAT ACCCAAAAGCAAGCTT AAAT AGGAATT CT GAT AAAACACT GAAAGAAAT GGCTTT GATCCCTGCCTCAAGT AG GAAGGAGGAGATTCT GGAGTTGGT GAATGCT GACGATGGGCCTTGCAGGT AACT GT TT CAACTTTT G AGTTCCAACT CTTT CCACT GTTTGCAAACGCCT AATT GAT CTT G AAA CTT AACAT GTTGCT AT GATT AATT AGT AGT AGTGGTGGT AGT ACAACT AT AG ATTCGT GGATTCAATGGAAAT ACTTCTCTGCGT GAACGACCT AGT ACCACAAAACTTGGGTT A AT AT AT GTGT GCGAAACAACAAGAGACTT AACT ACCT CAAAT ATT GT AT GGGT CAAAT GGTT GACT CATT GTT CT CCTT GT CACATTT GT CTTT CT GAT ATTT AACT GTT ATT ACT C ATTTTTTT CCTTGCT AAGGG AG AACAT AAA CTT AACT GAT CTTT GT CATTCGGTTTT G G AG AAAATT AAT G AACT ACT CTT GAT CATTTT AACTT CAG AACT AG AG AT AACT GAT G TT AT CTT ATCAT GT ACT AATT ACT ATTTTT ATT CCAGGGGT GAAATTGCT CAGCAGTT CGGG ATTT CTTT GG AT GT ACAAAT G ATGG AAGT CACTGGT AGG ACCCTTCCT CCTCC

CAGCCTAAAACTTGGCACCTCCAGTGGCCAACCCCCCAAATTCAATATTGATCAGC

CT AACTGCCAGTGGAACCTT ACGAGGAAAAGACT AGCAGAGGGCGGGGT GOT ACA

GTGCTGGGGCGTTGTGGACTTCAGTGCAGATTCTGGGCAGTACGCCCTGAATGGG

AACAT GTTT ATT GACAAGATT GTCAGGAAGTGCTGCGACCTT GGCGTACAGAT GAAC

CGT AACCCAT GCATT GT GCAACT GTT AGAT ATGGAGGTGCT AT CCGAT CCACATCAG

CTCTTCGAGGAGCTTAACAAAGCTAAGCAGGCGGCAGCCAGTAAGAAACAGAAGCT

GCAGCTCCT CTTCTGCCCAAT GT CT GATCAGCAT CCT GGGT ACAAGACGCT GAAGC

TT AT CT GCGAGACGCAGCT GGGGATCCAGACCCAGTGCTTCTT GAGCTTCCTCGCG

AACAAACAACAGGGACAGGACCAGTACATGTCCAACCTTGCTCTGAAGATCAACGG

CAAGATTGGAGGAAGCAACATCCAACTGTTTGGTGAATCGCTCCCGCGGATCTCCG

GCGCGCCATACATGTTCATCGGCGCCGACGTGAATCACCCATCGCCGGGGAACGT

CGAGAGCCCGTCGATTGCAGCAGTGGTGGCCTCGGTGGATCAAGGCGCCAGCAAG

T ACGTGCCAAGAATCCGCGCT CAGCCTCACCGCTGCGAGGT GATCCAGCACCT CG

GCGACATGTGCAAGGAGCTCATCGGCGTGTTCGAGAAGCGGAACCGCGTGAAGCC

CCAGAGGATCATCTACTTCCGCGACGGCGTCAGCGACGGTCAGTTCGACATGGTG

CTGAACGAGGAGCTGGCGGACATGGAGAAGGCGATCAAGACCAAGGACTACTCCC

CGACGATCACCGTGATCGTGGCCAAGAAGCGGCACCACACCAGGCTGTTCCCCAA

GGACCTGAACCAGCAGCAGACCAAGAACGGCAACGTGCTCCCCGGCACGGTGGTG

GACACCGGCGTGGTCGACCCGGCGGCGTACGACTTCTACCTGTGCAGCCACAACG

GGCTGATCGGGACGAGCCGGCCGACGCACTACTACAGCCTTCTGGACGAGCACGG

CTTCGCCTCCGACGACCTGCAGAAGCTGGTGTACAACCTCTGCTTCGTCTTCGCCC

GCTGCACCAAGCCGGTGTCGCTGGCCACGCCCGTCTACTACGCCGACCTCGCCGC

CTACCGCGGCAGGCTCTACTACGAGGGCATGATGATGTCGCAGCCGCCACCGTCT

TCCGCGGCGTCGGCGTCGTCGGCATCCTCCTCCGGCGCCGGCGCTTCCGACTTCA

GGAGCTT CCCGGCGCTGCACGAGGATCTGGTGGACAACAT GTT CTTCAT CT GACGA

CAGCCTTT CGT CGCATGGT CCGGT GGTCTCTCT CT GTT CTT AGTTT ACCT CTT ACT G

CGT GG AGT CT CAGGTCAT AG ACT CATT AGT CACAG ACT CACAGGTT GT CT AT CTTTT

G ACTTTT GTT G ACCGT GTCGTCGGT CTTGGT AGTTGGT AGTCTT AT ACAT ACT GTT G

AGGCTGCATT AT GGTTGCACAAGAAAGTTT AAGAGT ACT AT ACTGGT ACTCT GT ATT

TTGCAATTCTCGTGCGGATGCTATGGAGTAGGATGTTTCT

SEQ ID NO: 3: 0sAG02 cDNA nucleic acid sequence

ATGGAGCACGAGCGCGGTGGCGGTGGCCGCGGCCGCGGGAGGGGTCGCGGTGG

CGGGCGTGGCGGCGGTGGCGGCGATGGTCGCGGAGGCGGTTATGGTGGTGCTG GTGGTGGTGGTGTCGGCGGGCGCGGTGGGCGTGGGCCTCCTGGTGGTGGTGGTG GACGCGGGTACGAGCCCGGCGGCGGGCGTGGGTACGGTGGCGGCGGCGGCGGT GGTGGACGTGGGTATGGCGGCGGAGGCGGCGGTGGTGGGTACGAGTCCGGCGG TGGGCGTGGGTATGGCGGCGGTGGACGTGGGTATGAATCCGGCGGTGGGCGTGG ACCTGGCGGCGGCGGCCGTGGGCACGAGTCCGGCGGTGGCGGTGGCCGCGGCG GGAACGTGTGGGCGCAGCCGGGGAGAGGGCGCGGAGGAGCCCCCGCCCCGGCG CCGGCGCCAGCACCAGCAGCGAGGAGGATCCAGGACGAGGGGGCCGCGAGGTC GT CGGGT ACCGTT GAGCGCATTGCTT CT ACT GAGGTT GT AAGAGT ACAACCACCT G CACCCCCAGTTGCT GT GTCT CGT AGT GGCACGCGT GT GCCAATGCGAAGACCT GAT GGTGGAGGCT CAGT ATCGAAAGCCAAGGTCAAATT GTT GGT GAACCATTTT AT AGTT AAGT ACCGACAGGCATCAACT GTTTTTCACT AT GACAT AGACAT CAAGCTT GAT AT A AGTTCCCCCAAGGCTTCAGACAAGGAGCTATCCAAGGGAGATTTTCTTACTGTCAA GGACGAGCTCTTCAAGGATGAGAGCTTTCGGCGGCTTTCATCAGCTGTTGCTTATG ATGGAAAAAGAAATTT ATTT ACTT GTGCT GAGCT ACCAGATGGTTT GTTTCGT GTCAA AGTCCGTT CACGG ACTT ACATT GT AT CT GTGG AGTT CAAG AAG AAGCTTCCTTT GAG CCAACTCT CGGAACT GCCT GTGCCCAGAGAGGTCTTGCAGGGGCTT GAT GT CATT G TGCGTGAGGCCTCTAGCTGGCGCAAGATTATCATTGGTCAGGGATTTTACTCGCAG GGCCGCAGTGTGCCCATTGGGCCGGATGTTGTAGCTCTCAAAGGAACCCAGCAGA CCCT GAAAT GCACT CAGAAAGGACT GATCCTTT GT GTGGACT ATTCGGTT ATGCCGT TTCGCAAAGCT GGACCT GT GTTGGAT CTT GTTCAGAAGT CT GT GAGAT ACCTT GACT ACAGGACAACACT AAACAAACACCAATT GGACACTTT GAAGAAT GAACT CAAAGGCC AGCGT GT CACT GT AAAT CAT AGGAGGACAAAGCAGAAGT ACATT GTTAAAGGTTT GA CT GAT AAACCTGCAAGT CAGAT AACTTTT GT AGATT CT GAAT CAGGACAGACCAAGA AGCTT CTT GATT ACT ATT CGCAGCAGT ATGGCAAGGTT ATT GAGT ATCAAATGCTTC CAT GCTTGGATTT GAGCAAGAGCAAGGACAAGCAAAACT AT GTGCCGATT GAATT GT GTGATCTTCTTGAAGGGCAGAGATACCCAAAAGCAAGCTTAAATAGGAATTCTGATA AAACACT GAAAGAAAT GGCTTT GATCCCTGCCT CAAGT AGGAAGGAGGAGATTCT G GAGTTGGTGAATGCTGACGATGGGCCTTGCAGGGGTGAAATTGCTCAGCAGTTCG GGATTTCTTT GG AT GT ACAAAT GAT GGAAGTCACTGGT AGGACCCTTCCT CCTCCCA GCCT AAAACTTGGCACCTCCAGTGGCCAACCCCCCAAATTCAAT ATTGAT CAGCCT A ACT GCCAGT GGAACCTT ACGAGGAAAAGACT AGCAGAGGGCGGGGTGCT ACAGT G CTGGGGCGTTGTGGACTTCAGTGCAGATTCTGGGCAGTACGCCCTGAATGGGAAC AT GTTT ATT GACAAGATT GT CAGGAAGTGCT GCGACCTT GGCGT ACAGAT GAACCG T AACCCATGCATT GT GCAACT GTT AGATATGGAGGTGCT ATCCGAT CCACATCAGCT CTTCGAGGAGCTTAACAAAGCTAAGCAGGCGGCAGCCAGTAAGAAACAGAAGCTG CAGCT CCT CTT CT GCCCAAT GTCT GATCAGCAT CCT GGGT ACAAGACGCT GAAGCT

T AT CT GCGAGACGCAGCT GGGGATCCAGACCCAGTGCTT CTT GAGCTTCCT CGCGA

ACAAACAACAGGGACAGGACCAGTACATGTCCAACCTTGCTCTGAAGATCAACGGC

AAGATTGGAGGAAGCAACATCCAACTGTTTGGTGAATCGCTCCCGCGGATCTCCGG

CGCGCCATACATGTTCATCGGCGCCGACGTGAATCACCCATCGCCGGGGAACGTC

GAGAGCCCGTCGATTGCAGCAGTGGT GGCCT CGGT GGATCAAGGCGCCAGCAAGT

ACGTGCCAAGAATCCGCGCT CAGCCTCACCGCTGCGAGGT GATCCAGCACCT CGG

CGACATGTGCAAGGAGCTCATCGGCGTGTTCGAGAAGCGGAACCGCGTGAAGCCC

CAGAGGATCAT CT ACTTCCGCGACGGCGTCAGCGACGGT CAGTT CGACATGGTGC

T GAACGAGGAGCT GGCGGACATGGAGAAGGCGATCAAGACCAAGGACT ACTCCCC

GACGATCACCGTGATCGTGGCCAAGAAGCGGCACCACACCAGGCTGTTCCCCAAG

GACCTGAACCAGCAGCAGACCAAGAACGGCAACGTGCTCCCCGGCACGGTGGTGG

ACACCGGCGTGGTCGACCCGGCGGCGTACGACTTCTACCTGTGCAGCCACAACGG

GCTGATCGGGACGAGCCGGCCGACGCACTACTACAGCCTTCTGGACGAGCACGGC

TTCGCCTCCGACGACCTGCAGAAGCTGGTGTACAACCTCTGCTTCGTCTTCGCCCG

CTGCACCAAGCCGGTGTCGCTGGCCACGCCCGTCTACTACGCCGACCTCGCCGCC

TACCGCGGCAGGCTCTACTACGAGGGCATGATGATGTCGCAGCCGCCACCGTCTT

CCGCGGCGTCGGCGTCGTCGGCATCCTCCTCCGGCGCCGGCGCTTCCGACTTCA

GGAGCTT CCCGGCGCTGCACGAGGATCTGGTGGACAACAT GTTCTTCATCT GA

SEQ ID NO: 4: Setaria italica (foxtail millet) nucleic acid sequence (XM_004976750)

Atggattacgagcgcggaggcggtggtgggcgcggccgcgggaggggccgtggtggcggaggcagcgccggcggcg gcggaagaggaggtgggtacggcggccgcggaggaggtgggtacggcggccgcggaggaggcgggtacgacggc ggcggccgcggaggaggcgggtacgacggcggcggaggaggaggaggagggggatacgggcgttacgaaggagg aggctacggcgggggccgtggaggcggcggctaccacggcccgcctcgcggaggcggatatggcgcgggagggcgt ggccccggcggtggtggcagacaggcgtacggccccggcggtgggcgcggcgggagcgcgtgggcgccgccgccgg gttcggggagaggtcgcggcggcggcggcaacggagcggagtacgtccccgtcaccagggcgcccgcgccggcgcct acatcaatggggatcgcgcccaaggacaaggaggcgcttagcgcgtcggggtccgtcgaacgcattgactccagtgaatt ggcaagaggaaaaccttcatcatcactagttgccacgccttacgctggagcacgtgtgccaatgcaaagacctgatcgtgg aggctcatcatctcaagcaaatgtcaaacttttggtgaaccatttcattgtcaagtaccgaaaggcgacaacaatttttcattat gacatagacatcaagcttgatcaagcttcccctaaggcttcgggcaaggagctctccaaggcagaatttctttctgtcaagga tgagctcttcaaggacaccagctttcgacgtctttcgtcatgtgttgcttatgatggcggacgaaatctattcacttctgctgaactt ccagaaggtttatttcgtgtgagagttcggtccaagacctacattgtatctgtagatttgaa gaaacagctgccattaagtcaactctcagagttacctgtgcctagagaggtcttgcagggtcttgatgtcattgtgcgtgag gcctctagatggcgcaagattatggttggtaaaggattttactcaccaaatagcagtctggacattgggcagggtgctgtg gctctgaaaggagcactacagacccttaaacatactcagcaagggctgatcctatgtgttgactattcagttatgccgtttta caaagctgggccagtgatggatcttgttgagaagatagtggggcgccttgattacaggacaactctgaacaagtggcaa ctggaaaatttggagtatgagcttaaaggccgacgtgtgactgtgattcaccgcaggactaatcagaagtacattgtgca aggtttgacacccttgcctgccggccagttgacctttgtggatgctgaaaccgggcaaacgaataggcttgttgattattatg ctcagaaacatggcaaggtgattgagtatcagatgcttccatgcctggatttgagcaagagcaaggacaaagccaatca tgtgccaattgagctttgcactcttcttgaagggcagaggtatccaaaggcaaatttggataggaattctgatagaacacta aaatcggaggccctaattcctgcatttaagcggaggaaggagattctggacttggtgaatgctacagatgggccttgcagt ggtgaaatagcaccgcaatttgggatttccttggatgtgcaaatgactgaagtcatgggtaggatccttcccccacccaac ctaaaacttggcgcccccaatggccagaccagcaaattcagtatcaaccacgagagttgccagtggaaccttatgaata agaaactagtagagggctgggatcttcagtgctggggcatcgtggacttcagtgcacgtacttctcaccccagggagga gtccctcaatggatggatgtttgtggaaaagatagtcaggaagtgctgtgagctcggcatccggatgaacactgatccatg cttcgtgcacaagtcagaaatggcagtgctctctgatccacatcgactgcacgaggagctaaacaaagcaaaacaagc agcagtgagcaaggagcagaggctgcaactcctgttctgcccgatgtccgagcagcacccaggttacaagacactga agctgatttgtgacacgcaactggggatcctcacccagtgtttcctgagcaaaattgcgaacaagcagcagggccagga ccagtacatgaccaaccttgcccttaagatcaacagcaagcttgggggcagcaacgtccagctgtatgactcgctccca cgtgtcagcggtgcgccattcatgttcatcggagctgacgttaaccatccgtcacccgggaacgtggagagcccgtcaat tgcagctgtggtcgcgtctatcaactctggcgtcaacaagtacgtgtcaagaatccgcgcccagccacaccgctgcgag gtgatccagcagcttggtgagatctgcctggagctcattggagtctttgagaagcagaacagcgtcaagccaaagagga tcatctacttccgtgatggcgtgagcgacggccagttcgacatggtcctgaacgaggagctggcggacatggagaagg cgatcaaggtgaacggctactcgccgaccatcaccgtgatcgtggccaagaagcggcaccacacgcggctattcccta aggaccagggccagccgcagacgaagaacggcaacgtgccgcccggcactgtggtggacacgggcgtggtggac ccatcggcttacgacttctacctatgcagccacaacgggctgctggggacgagccggccgacgcactactacagcctg gtggacgagcacgggttcgggtcggacgacctgcagaagctgatatacaacctgtgctttgtgttcgcccggtgcaccaa gccggtgtcgctggcgacgcctgtctattacgccgaccttgcggcctaccgcggcaggctctactatgaggcgggcatga ggtcgggaacttttgaagcggggagcttcccgaggctgcacaaggacctggaggacaacatgttcttcatctga

SEQ ID NO: 5 Setaria italica amino sequence

MDYERGGGGGRGRGRGRGGGGSAGGGGRGGGYGGRGGGGYGGRGGGGYDGG

GRGGGGYDGGGGGGGGGYGRYEGGGYGGGRGGGGYHGPPRGGGYGAGGRGP

GGGGRQAYGPGGGRGGSAWAPPPGSGRGRGGGGNGAEYVPVTRAPAPAPTSMGI

APKDKEALSASGSVERIDSSELARGKPSSSLVATPYAGARVPMQRPDRGGSSSQAN

VKLLVNHFIVKYRKATTIFHYDIDIKLDQASPKASGKELSKAEFLSVKDELFKDTSFRRL

SSCVAYDGGRNLFTSAELPEGLFRVRVRSKTYIVSVDLKKQLPLSQLSELPVPREVLQ GLDVIVREASRWRKIMVGKGFYSPNSSLDIGQGAVALKGALQTLKHTQQGLILCVDYS

VMPFYKAGPVMDLVEKIVGRLDYRTTLNKWQLENLEYELKGRRVTVIHRRTNQKYIVQ

GLTPLPAGQLTFVDAETGQTNRLVDYYAQKHGKVIEYQMLPCLDLSKSKDKANHVPIE

LCTLLEGQRYPKANLDRNSDRTLKSEALIPAFKRRKEILDLVNATDGPCSGEIAPQFGI

SLDVQMTEVMGRILPPPNLKLGAPNGQTSKFSINHESCQWNLMNKKLVEGWDLQCW

GIVDFSARTSHPREESLNGWMFVEKIVRKCCELGIRMNTDPCFVHKSEMAVLSDPHR

LHEELNKAKQAAVSKEQRLQLLFCPMSEQHPGYKTLKLICDTQLGILTQCFLSKIANKQ

QGQDQYMTNLALKINSKLGGSNVQLYDSLPRVSGAPFMFIGADVNHPSPGNVESPSI

AAVVASINSGVNKYVSRIRAQPHRCEVIQQLGEICLELIGVFEKQNSVKPKRIIYFRDGV

SDGQFDMVLNEELADMEKAIKVNGYSPTITVIVAKKRHHTRLFPKDQGQPQTKNGNV

PPGTWDTGWDPSAYDFYLCSHNGLLGTSRPTHYYSLVDEHGFGSDDLQKLIYNLC

FVFARCTKPVSLATPVYYADLAAYRGRLYYEAGMRSGTFEAGSFPRLHKDLEDNMFFI

SEQ ID NO: 6 Sorghum bicolor protein argonaute 2 nucleic acid sequence ((LOC8063972) XM_002447052 )

atggagcacgagcgcggccgtggccgcgggcggggacgtggaggacggggaggtggtggcggcggcggcggcg gcggaggaggaggacgcggaggtgccggcgggtacgggcggcatggaagcggcggcgacgaccgcggcggag gcggatacggctcgcgcggaggcgagtacggcggtggaggcggatacggcccccgcggtggtgagtacggcggcg gaggctacggagggggcagtggaggcggtggctatcaccaagggcctcgcggaggcgagtacggcggtggaggcg gcggatacgggacccgcggtggcgagtacggcggcggaggaggaggaggggtgtacgggcacgacggaggagg ctacggagggggcagtggaggcggtggctatcaccaagggcctcgcggaggcggaggtggaggcggccgcggag ggcgcggccccggcgcgggcggcggccaggcgtacgcgtccggcggtggccgcggaggcaacgcgtgggcgccg gcgccgggtgctgggagaggtcgtggagttggcggcggcgcggccgagtacgcccccgtcagggggcccgctcccg cgcctgcggcgagggcggttgctgttgcgcccagggacaaggaggcgccgagttcgtcgggatccgttgaacgcatca catccagtgaattggccagagtagaaccaccagcatccacactagctgcgacatcttcagttggcacacgagtgccaat gcagagacctgattctggaggttcattatctcaagcaaaggtcaaacttttggtgaatcacttcattgtcaactaccgagaa gtgtcaactatttttcactatgacataagcatcaagcttgatgaagcttcccctaaggcttcgggcaaagaactctccaagg cagaatttctttctgtcaaggatgaactcttcagggaaagcagtttacggcgtctttcctcatgtgttgcttatgatggtggaag aaacctctacacttctgctgaactgccagcaggtttatttcgtgtgagagttcgatcaaagacctacattgtatcagtagattt gaagaagcagctgccattaagtcaactctcagagttacctgtgcctagagaggtcttgcagggtcttgatgttgttgtgcgtg aggcctccagatggcgcaagattatccttggtagaggattttactcaccaagcagcagtgtagacattgggcagggtgct gtagctatgaaaggaacccagcagacccttaaatacactcagcaagggctgatcctatgtgttgactattcagttatgcca ttttacaaagctgggccggtgatggatattgttcagaaattagtaccccaccttgattaccggacaacactgaacaggagg caactggaaaatctgattgaggagctcaaaggccgacgtgtgactgtggttcatcggaggactaatcagaagtacacag tgcaaggcttgacacccttacctgccatccagatgacctttgtggatgctgaatctggccaaacgaagaggcttgtggatta ttatgctcagaaacatgacaaggtgattgagtaccagatgcttccatgcttggatttgagcaagagcaaggacaaaccaa atcatgtaccaattgagctctgcactcttcttgaaggacagaggtttccaaaagcaaacttggataagaattctgacaggat actgaaaggaaaggctctaattcctccatctcatcggaggaatgagattcaagacttggtgaatgcttcggatggaccttg cagaggagaaattgcacagcaatttgggatttccttggatttacgaatgacagaagtcacgggtaggatccttcccccac caaatctcaaacttggggcatccaatggccacatgtccaaattcagtatggatcagaactgccagtggaatcttgtgaag aagagactagtagagggccgggatcttcagtgctggggcattgtcgacttcagtgctgagccgtctcacccccggcagg agcccctcaatggaaggatgtttgttgacaagatcgtgaggaagtgctgtgagcttggtatccaaatgaactctaatccttg cttcatacacatatcaaagatggcagtgctctccgatccacatcgactaaaggaggagctaaacaaagcaaaacaggc tgcagtgagcaagaagcagaggttgcagctccttttctgcccgatgtccgagcagcacccagggtacaagacactgaa gctgatttgtgacacacagctcgggatcctgacccagtgtttcctgagcgaccgcgcaaacaagccaaatgggcagga ccagtacatgaccaaccttgctctcaagattaatggcaagcttgggggcagcaacgttcagctgtttgactcgcttccacg ggtcggtggtggggcacctttcatgttcatcggtgctgacgttaaccacccgtcacccgggaacgtggagagcccatcaa tcgcaggcgtggttgcatctatcaacagcggtgccaacaagtatgtgtcaagaatccgtgcacagccacaccgctgcga ggtgatccagcagctgggtgagatctgcctggagctcattggagtctttgtgaagataaatcgcgtgaagccacagaaga tcatctacttccgtgacggcgtgagtgacgggcagtttgacatggtcctgaacgaggagctggctgacctggagaaggc aatcaaggtggacggctatgcacctaccatcactgtgatcgtggccaagaagcggcaccacacgcggctgttccccaa ggaccagggccagcagcagacgaagactgggaacgtgccgcctggcacggtggtggacactggtgtggttgacccgt ctgcatacgacttctacctgtgcagccacaccgggcttctagggacgagcaggccgacacactactacagcctggtgga cgagcacggcttcgggtctgacgacctgcagaagctgatctacaacctgtgcttcgtgttcgcgcggtgcaccaagccgg tgtcactggcgacgcccgtctactatgctgacctcgtggcgtaccgtggaagggtctactacgaggcagccatgatggtgt cccagcgagggatggggtcggcttcttcagcttcctcgacctcctctgctgggactgttgacttcactaacttcccgaggttg cacaaggatgtggaggacaacatgttcttcatctga

SEQ ID NO: 7 Sorghum bicolor protein argonaute 2 amino acid sequence ((LOC8063972) XM_002447052 )

MEHERGRGRGRGRGGRGGGGGGGGGGGGGRGGAGGYGRHGSGGDDRGGGGY

GSRGGEYGGGGGYGPRGGEYGGGGYGGGSGGGGYHQGPRGGEYGGGGGGYGT

RGGEYGGGGGGGVYGHDGGGYGGGSGGGGYHQGPRGGGGGGGRGGRGPGAG

GGQAYASGGGRGGNAWAPAPGAGRGRGVGGGAAEYAPVRGPAPAPAARAVAVAP

RDKEAPSSSGSVERITSSELARVEPPASTLAATSSVGTRVPMQRPDSGGSLSQAKVK

LLVNHFIVNYREVSTIFHYDISIKLDEASPKASGKELSKAEFLSVKDELFRESSLRRLSS

CVAYDGGRNLYTSAELPAGLFRVRVRSKTYIVSVDLKKQLPLSQLSELPVPREVLQGL

DVVVREASRWRKIILGRGFYSPSSSVDIGQGAVAMKGTQQTLKYTQQGLILCVDYSV

MPFYKAGPVMDIVQKLVPHLDYRTTLNRRQLENLIEELKGRRVTVVHRRTNQKYTVQ

GLTPLPAIQMTFVDAESGQTKRLVDYYAQKHDKVIEYQMLPCLDLSKSKDKPNHVPIE LCTLLEGQRFPKANLDKNSDRILKGKALIPPSHRRNEIQDLVNASDGPCRGEIAQQFGI

SLDLRMTEVTGRILPPPNLKLGASNGHMSKFSMDQNCQWNLVKKRLVEGRDLQCWG

IVDFSAEPSHPRQEPLNGRMFVDKIVRKCCELGIQMNSNPCFIHISKMAVLSDPHRLKE

ELNKAKQAAVSKKQRLQLLFCPMSEQHPGYKTLKLICDTQLGILTQCFLSDRANKPNG

QDQYMTNLALKINGKLGGSNVQLFDSLPRVGGGAPFMFIGADVNHPSPGNVESPSIA

GWASINSGANKYVSRIRAQPHRCEVIQQLGEICLELIGVFVKINRVKPQKIIYFRDGVS

DGQFDMVLNEELADLEKAIKVDGYAPTITVIVAKKRHHTRLFPKDQGQQQTKTGNVPP

GTWDTGWDPSAYDFYLCSHTGLLGTSRPTHYYSLVDEHGFGSDDLQKLIYNLCFVF

ARCTKPVSLATPVYYADLVAYRGRVYYEAAMMVSQRGMGSASSASSTSSAGTVDFT

NFPRLHKDVEDNMFFI

SEQ ID NO: 8 Zea mays argonaute 2 nucleic acid sequence ((LOC 103642054) Sequence ID: XM_008665369.3 )

atggagtacgagcgcggggaccgggccaagcggggccgtggaggtggcggcggcggcggcggcggcggaggac gcggaggtggcggcgagtacgggcggcagcacggaggcgcgtacggcggaggcggatggcacgggcacgacga aggaggaggctacggcgggggccgtggaggcggtggctactatcaccaagggcctcgcggccgcggagggtggcg cggccccggcgcgggcggcagccaggcgtacgggtccggcggcggccgcgcgtgggcgccggggccgggtgagg ggagaggccttggggttggccgcgagtacgcccccgtcaggcgggcagcgccggcgcctgcgcctgcgcctaggga ggttgcgactgcgcccatggtcaaggaagcgccgagttcgtcgggatccgttgaacgcatcacatctagtgaattggccg gagtagaaccactagcatccacactagctgcgacatcttctgttggcacacgagtgccaatgcagagacctgactgtggt ggcgcattatctcaagcaaaggtcaaactcttggtgaatcactttattgtcaactaccaaaaggtgtcaactatttttcactat gacataaacatcaagcttgatgaagcttcctctaaggcttcaggcaaagaactctcgaaggcagaatttctttctgtcaag gatgagctcttcagggaaagcagtttacggcgtctttcctcatgtgttgcttatgatggtggaagaaatctcttcacttctgctg aactgccagcaggtttatttcgtgtgagagttcgatcaaaggcctacattgtatcagtagatttgaagaagcagctgccatta agtcaactctcagatttacctgtacctagagaggtcttgcagggtcttgatgttgttgtgcgtgaggcctccagatggcgcaa ggttatccttggtagaggattttactcaccaagcagcagtatagacattgggcagggtgttgtagctatgaaaggaaccca gcagacacttaaatacactcaacaagggttgaacctgtgtgttgattattcagttatgccattttacaaagctggaccggtga tggaccttgttcacaaaatagtggggtaccttgattatcgaacaactctgaacaagaggcaaatggaaaatctggttgatg agcttaaaggccgacgtgtaactgtgattcatcggaggactaatcagaagtacacagtgcaaggcttgacacccttacct gccagccagatgacctttgtggatgctgaatccggacaaacaaagtgtcttgtggagtattatgctcagaaacatggcatt gtgattgagtatcagatgctgccatgcttggatttgagcaagagcaaggacaaaccgaatcatgtcccaattgagctctgc actcttcttgaaggacagaggtttccaaaagcaaacttggataagaattctggcaggatactaaaaggaaaggctctaat tcctgcatccaatcggaggaaagagattctagacttggtgaatgcttcggatggaccttgcagaggagaaattgcacagc gatttgggatttccttggatttacgaatgacagaagtcacgggtaggatccttcccccaccaaacctcaaactcggggcat ccaatggccagacctccaaattcagtatcgatcagaactgccagtggaaccttgtgaagaagagactcgtagagggcc gggatcttcagtgttggggcattgtcgacttcagtgctgagccgtctgacccccagcaggagcccctcaatggaaggatgt ttattgagaagattgtgaggaagtgctgtgagcttggtatccgtatgaactccaacccatgcttcgtacacaaatctaagatg gcagtgctctccgatccgcatcgactacaggaggagctaaacaaggcaaaacaggctgcagtgagcaagaagcag aggttgcagctccttttctgcccgatgtccgagcagcatccagggtacaagacactgaagctgatttgcgatacacagctt gggatcatgacccagtgtttcctgggcgaccgcgcaaacaagccgaatgggcaggaccagtacatgaccaaccttgc cctcaagataaacggcaagcttgggggcagcaacgtccagctgttcgactcgctcccacgggtcggtggggcacctttc atgttcatcggtgctgacgtcaaccacccgtcaccggggaacgtggagagcccatcgattgcagccgtggttgcgtctat caactccggtgtcagcaagtacgtgacaagaatccgtgcccagccgcaccgctgtgaggtgatccagcagctcggcga gatctgcctggagctcatcggagtcttcgagaagcgaaaccgcgtgaagccgcagaagatcatctacttccgcgacgg cgtgagcgacgggcagttcgacatggtcctgaacgaggagctggcggacctggagaaggcgatcaaggtgggcggc tacgcgccgaccgtcaccgtgatcgtggccaagaagcggcaccacacgcgcctgttccccaaggaccccagccagc cgcagacgaagaacgggaacgtgccgcccggcacggtggtggacacgggcgtggtggacccgtccgcgtacgactt ctacctgtgcagccacgccgggatcctgggcacgagcaggccgacgcactactacagcctggtggacgagcacggct tccggtccgacgacctgcagaagctggtctacaacctctgcttcgtgttcgcgcggtgcaccaagcccgtgtcgctggcg acgcccgtctactacgccgacctcgcggcgtaccgtggcaggctctactacgaggcggccatgatgccgtcccaccag cgagggacggggtcggcgtcctcgggctcctccgctgggacttttggcgtcactaacttcccgaggctgcacaaggatgt ggagaacaacatgttcttcatctga

SEQ ID NO: 9 Zea mays argonaute 2 amino acid sequence ((LOC 103642054) Sequence ID: XM_008665369.3 )

MEYERGDRAKRGRGGGGGGGGGGGRGGGGEYGRQHGGAYGGGGWHGHDEGG

GYGGGRGGGGYYHQGPRGRGGWRGPGAGGSQAYGSGGGRAWAPGPGEGRGLG

VGREYAPVRRAAPAPAPAPREVATAPMVKEAPSSSGSVERITSSELAGVEPLASTLAA

TSSVGTRVPMQRPDCGGALSQAKVKLLVNHFIVNYQKVSTIFHYDINIKLDEASSKASG

KELSKAEFLSVKDELFRESSLRRLSSCVAYDGGRNLFTSAELPAGLFRVRVRSKAYIV

SVDLKKQLPLSQLSDLPVPREVLQGLDVWREASRWRKVILGRGFYSPSSSIDIGQGV

VAM KGTQQTLKYTQQG LN LCVDYSVM PFYKAG PVMDLVH KIVGYLDYRTTLN KRQM

ENLVDELKGRRVTVIHRRTNQKYTVQGLTPLPASQMTFVDAESGQTKCLVEYYAQKH

GIVIEYQMLPCLDLSKSKDKPNHVPIELCTLLEGQRFPKANLDKNSGRILKGKALIPASN

RRKEILDLVNASDGPCRGEIAQRFGISLDLRMTEVTGRILPPPNLKLGASNGQTSKFSI

DQNCQWNLVKKRLVEGRDLQCWGIVDFSAEPSDPQQEPLNGRMFIEKIVRKCCELGI

RMNSNPCFVHKSKMAVLSDPHRLQEELNKAKQAAVSKKQRLQLLFCPMSEQHPGYK

TLKLICDTQLGIMTQCFLGDRANKPNGQDQYMTNLALKINGKLGGSNVQLFDSLPRVG

GAPFMFIGADVNHPSPGNVESPSIAAVVASINSGVSKYVTRIRAQPHRCEVIQQLGEIC

LELIGVFEKRNRVKPQKIIYFRDGVSDGQFDMVLNEELADLEKAIKVGGYAPTVTVIVA KKRHHTRLFPKDPSQPQTKNGNVPPGTWDTGWDPSAYDFYLCSHAGILGTSRPTH

YYSLVDEHGFRSDDLQKLVYNLCFVFARCTKPVSLATPVYYADLAAYRGRLYYEAAM

MPSHQRGTGSASSGSSAGTFGVTNFPRLHKDVENNMFFI

SEQ ID NO: 10 Aegilops tauschii subsp. tauschii argonaute 2-like nucleic acid sequence ((LOC109771941), Sequence ID: XM_020330637.1)

atggattacgagcaaggcggcggcggtggccgcggccgcgggagatctcgcggcggcggcgggcgtggcggggcg cccggtggctacgggcctcagggaggcggcggaggctacggaggaggcggtcaaggccggggcgctcagggaag cggtggaggatacggaggaggcggaggctacgggccccaagggggcatggagggccgcggaggcggctacgcg cctcgcggcggaggttgcgctcctcgcgggggcggcgagggccgcggacgaggctatggtcctcagggaggcggcg atggccgcggaggcggctacggcggaggcgggccgggagggcgtggtcctgccggtggtgggcgggggtacggcc ccggcggtgggcgtggtgcgaacgcgtgggcgcagcctgggagagggcccgccggaggacccggggaatacgtcc cggtgagggcgccggcgccagcgtctgcggcgaggagggttgagtccgaggaggcggggggctcatctggatccgtc gaacgcatcagctccaaagaagtgtcaaaattagaaccattagcgcccccagttgctgtgtctcccaatggcatacgtgt gccaatgcaaagacctgatggtggtggatcattatgtcaagccagggtccagcttttggtaaaccacttcatagtcaagta cccaaagctgtcaaccttttttcactatgacatagatatcaagttcgatccagcgtcttcaaaggtttctggcaaggagctctc caatgcagattttctttctgcaaaggccgagcttttcaaggatgacagctttcggcagctttcatcggctgttgcttatgatgga aagagaaacctatttactgttgcgcagctgccagaaggtttattccgtgtcagagttcgttcaaagacctacattgtatcagt agagttcaagaagcagcttcctctaagccaactctcggagctgcctgtggctagggagatcttacagggtcttgatgtcatt gtgcgtgaggcttctagctggagcaagattatacttggtcatggattttactcaccacaaagcaaagtggatatggggtca ggtgttgtatctatgaaaggaacccagcagacccttaaacacactcagcagggactggtcctatgtgttgactattcagtta tgccatttcgcagagatggacctgttctggatatcgtgcggcagtttataaagccccttccccttgactacaggacagcact gaacaagactcatcgggaaaagttggtgtatgagctcaaaggccagcgtgttactgtgtcccaccggaagactaagca gaaatacactattcaaggcttcactgatttgccagccagccagatcacctttttagattcagaatcagggcagtcaaggaa acttgttgactattttgctcagcagtatggcaaggtgattgagtatcagatgcttccatgcctggatttgagcaagagcaaag acaaacccaattatgtgcccattgagctttgtaagcttgttgaaggacagaggtacccaatggcaaatttgaataaggaca ctgatagagcactgaaaggcaaggctttaattaaggcagctgagcggaagtgggagattgagactgcggtcaaagctg aagatgggccttgcaggggagaaatagctcaacagtttgggatatctttggatgtgaagatgatggaagtcaccggcag ggtcctttctcctcccatgctgacactcggctcctccagaggagcccccggcaatttaagtatcactccatctaattgccagt ggaacctcatggggaagaaactggtagaaggtaaagcacttcagtgctggggcattgtggacttcagtgcacggccatc tcacaacaagcagcaagcgcttgatgggaacatgtttattaactatatcgtcaggaagtgctgtgaccttggcattcagatg aacaagacagcatgctttgtgcatctatcggcaatgtcagtgctatcagatccacaccagttgtacgaggagctgaacaa agctaagcaggctgcggtgaagaagaaccagaagctgcagctcctcttctgcccgatgtctgaacagcatcctgggtac aagacactgaagctgatctgtgagacacagctggggatccaaacccagtgtttcttgagccacctcgcaaacaaaactc agggccaggaccagtacatgtccaaccttgctctcaagatcaacagcaagctcgggggcatcaacgtccagctccagg acaagctcccactggacaacggtgctccttacatgttcatcggcgcagatgtcaaccacccgtcacccgggaatgtgga aagcccgtcgatagcagctgtggttgcgtccatgaataggggcgccaccaagtacgtgcctagaatccgcgcccagcc gcaccgctgtgaggtgatcaagaacctcggcgagattgttcaagagctcatcggtgtctttgagaagaaggccggcgta aagcctcagagaatcatttacttccgtgatggcgtgagtgatggacagttcgagatggtccttaacgaggagcttgcagac atggagaaggtgatcaaggtgaagggctactcgccgacgatcaccgtgattgtggccaagaagcggcaccacacgc ggctgttccccaaggagcacaacgagccgctgcagacgaagaacggcaatgtgctccctggcacggtggtggacac cagaattgttgaccctgtgacgtacgacttctacctgtgcagccacaacgggctgatagggaccagccggccgacgcac tactacaacctgatggacgaacacggctacgggtcggacgacctgcagaggctggtgtacaacctctgcttcgtgttcgc gcgctgcaccaagcccgtatctctggcgacgcccgtctactacgctgacctggcggcataccgtggcaggctctactatg agggcatgctggcgtcccagccccaggtgcgcagctcgtcgtcctcggcctcctctgcagctgcgggtgcttccgacttca gcaacttcccgatgctgcacgtggatcttcaggacaacatgttcttcatctga

SEQ ID NO: 11 Aegilops tauschii subsp. tauschii argonaute 2-like amino acid sequence ((LOC109771941), Sequence ID: XM_020330637.1)

MDYEQGGGGGRGRGRSRGGGGRGGAPGGYGPQGGGGGYGGGGQGRGAQGSG

GGYGGGGGYGPQGGMEGRGGGYAPRGGGCAPRGGGEGRGRGYGPQGGGDGR

GGGYGGGGPGGRGPAGGGRGYGPGGGRGANAWAQPGRGPAGGPGEYVPVRAP

APASAARRVESEEAGGSSGSVERISSKEVSKLEPLAPPVAVSPNGIRVPMQRPDGGG

SLCQARVQLLVN H FIVKYPKLSTFFH YDI Dl KFDPASSKVSGKELSNADFLSAKAELFKD

DSFRQLSSAVAYDGKRNLFTVAQLPEGLFRVRVRSKTYIVSVEFKKQLPLSQLSELPV

AREILQGLDVIVREASSWSKIILGHGFYSPQSKVDMGSGWSMKGTQQTLKHTQQGL

VLCVDYSVMPFRRDGPVLDIVRQFIKPLPLDYRTALNKTHREKLVYELKGQRVTVSHR

KTKQKYTIQGFTDLPASQITFLDSESGQSRKLVDYFAQQYGKVIEYQMLPCLDLSKSK

DKPNYVPIELCKLVEGQRYPMANLNKDTDRALKGKALIKAAERKWEIETAVKAEDGPC

RGEIAQQFGISLDVKMMEVTGRVLSPPMLTLGSSRGAPGNLSITPSNCQWNLMGKKL

VEGKALQCWGIVDFSARPSHNKQQALDGNMFINYIVRKCCDLGIQMNKTACFVHLSA

MSVLSDPHQLYEELNKAKQAAVKKNQKLQLLFCPMSEQHPGYKTLKLICETQLGIQTQ

CFLSHLANKTQGQDQYMSNLALKINSKLGGINVQLQDKLPLDNGAPYMFIGADVNHPS

PGNVESPSIAAVVASMNRGATKYVPRIRAQPHRCEVIKNLGEIVQELIGVFEKKAGVKP

QRIIYFRDGVSDGQFEMVLNEELADMEKVIKVKGYSPTITVIVAKKRHHTRLFPKEHNE

PLQTKNGNVLPGTVVDTRIVDPVTYDFYLCSHNGLIGTSRPTHYYNLMDEHGYGSDD

LQRLVYNLCFVFARCTKPVSLATPVYYADLAAYRGRLYYEGMLASQPQVRSSSSSAS

SAAAGASDFSNFPMLHVDLQDNMFFI SEQ ID NO: 12 Triticum aestivum cultivar: Chinese Spring nucleic acid sequence (Sequence ID: AK335299.1)

atggattacgagcaaggcggcggcggtggccgcggccgcgggagatctcgcggcggcggcgggcgtggcggggcg cccggtggctacgggcctcagggaggcggcggagactacggaggaggcggtcaaggccggggcgctcagggaag cggtggaggatacggaggaggcggaggctacgggccccaagggggcatggagggccgcggaggcggctacgcg cctcgcggcggaggttacgctcctcgcgggggcggcgagggccgcggacgaggctatggtcctcagggaggcggcg atggccgcggaggcggctacggcggaggcgggccgggagggcgtggtcctgccggtggtgggcgggggtacggcc cgggcggtgggcgtggtgcgaacgcgtgggcgcagcctgggagagggcccgccggaggacccggggaatacgtcc cggtgagggcgccggcgccagcgtctgcggcgaggagggttgagtccgaggaggcggggggctcatctggatccgtc gaacgcatcagctccaaagaagtgtcaaaattagaaccattagcgcccccagttgctgtgtctcccaatggcatacgtgt gccaatgcaaagacctgatggtggtggatcattatgtcaagccagggtccagcttttggtaaaccacttcatagtcaagta cccaaagctgtcaaccttttttcactatgacatagatatcaagttcgatccagcgtcttcaaaggtttctggcaaggagctctc caatgcagattttctttctgcagaggccgagcttttcaaggatgacagctttcggcagctttcatcggctgttgcttatgatgga aagagaaacctatttactgttgcgcagctgccagaaggtttattccgtgtcagagtttgttcaaagacctacattgtatcagta gagttcaagaagcagcttcctctaagccaactctcggagctgcctgtggctagggagatcttacagggtcttgatgtcattg tgcgtgaggcttctagctggagcaagattatacttggtcatggattttactcaccacaaagcaaagtggatatggggtcag gtgttgtatctatgaaaggaacccagcagacccttaaacacactcagcagggactggtcctatgtgttgactattcagttat gccatttcgcagagatggacctgttctggatatcgtgcggcagtttataaagccccttccccttgactacaggacagcactg aacaagactcatcgggaaaagttggtgtatgagctcaaaggccagcgtgttactgtgtcccaccggaagactaagcag aaatacactattcaaggcttcactgatttgccagccagccagatcacctttttagattcagaatcagggcagtcaaggaaa cttgttgactattttgctcagcagtatggcaaggtgattgagtatcagatgcttccatgcctggatttgagcaagagcaaaga caaacccaattatgtgcccattgagctttgtaagcttgttgaaggacagaggtacccaatggcaaatttgaataaggacac tgatagagcactgaaaggcaaggctttaattaaggcagctgagcggaagtgggagattgagactgcggtcaaagctga agatgggccttgcaggggagaaatagctcaacagtttgggatatctttggatgtgaagatgatggaagtcaccggcagg gtcctttctcctcccatgctgacactcggctcctccagaggagcccccggcaatttaagtatcactccatctaattgccagtg gaacctcatggggaagaaactggtagaaggtaaagcacttcagtgctggggcattgtggacttcagtgcacggccatct cacaacaagcagcaagcgcttgatgggaacatgtttattaactatatcgtcaggaagtgctgtgaccttggcattcagatg aacaagacagcatgctttgtgcatctatcggcaatgtcagtgctatcagatccacaccagttgtacgaggagctgaacaa agctaagcaggctgcggtgaagaagaaccagaagctgcagctcctcttctgcccgatgtctgaacagcatcctgggtac aagacactgaagctgatctgtgagacacagctggggatccaaacccagtgtttcttgagccacctcgcaaacaaaactc agggccaggaccagtacatgtccaaccttgctctcaagatcaacagcaagctcgggggcatcaacgtccagctccagg acaagctcccactggacaacggtgctccttacatgttcatcggcgcagatgtcaaccacccgtcacccgggaatgtgga aagcccgtcgatagcagctgtggttgcgtccatgaataggggcgccaccaagtacgtgcctagaatccgcgcccagcc gcaccgctgtgaggtgatcaagaacctcggcgagattgttcaagagctcatcggtgtctttgagaagaaggccggcgta aagcctcagagaatcatttacttccgtgatggcgtgagtgatggacagttcgagatggtccttaacgaggagcttgcagac atggagaaggtgatcaaggtgaagggctactcgccgacgatcaccgtgattgtggccaagaagcggcaccacacgc ggctgttccccaaggagcacaacgagccgctgcagacgaagaacggcaatgtgctccctggcacggtggtggacac cagaattgttgaccctgtgacgtacgacttctacctgtgcagccacaacgggctgatagggaccagccggccgacgcac tactacaacctgatggacgaacacggctacgggtcggacgacctgcagaggctggtgtacaacctctgcttcgtgttcgc gcgctgcaccaagcccgtatctctggcgacgcccgtctactacgctgacctggcggcataccgtggcaggctctactatg agggcatgctggcgtcccagccccaggtgcgcagctcgtcgtcctcggcctcctctgcagctgcgggtgcttccgacttca gcaacttcccgatgctgcacgtggatcttcaggacaacatgttcttcatctga

SEQ ID NO: 13 Triticum aestivum cultivar: Chinese Spring amino acid sequence (Sequence ID: AK335299.1)

MDYEQGGGGGRGRGRSRGGGGRGGAPGGYGPQGGGGDYGGGGQGRGAQGSG

GGYGGGGGYGPQGGMEGRGGGYAPRGGGYAPRGGGEGRGRGYGPQGGGDGR

GGGYGGGGPGGRGPAGGGRGYGPGGGRGANAWAQPGRGPAGGPGEYVPVRAP

APASAARRVESEEAGGSSGSVERISSKEVSKLEPLAPPVAVSPNGIRVPMQRPDGGG

SLCQARVQLLVN H FIVKYPKLSTFFH YDI Dl KFDPASSKVSGKELSNADFLSAEAELFKD

DSFRQLSSAVAYDGKRNLFTVAQLPEGLFRVRVCSKTYIVSVEFKKQLPLSQLSELPV

AREILQGLDVIVREASSWSKIILGHGFYSPQSKVDMGSGWSMKGTQQTLKHTQQGL

VLCVDYSVMPFRRDGPVLDIVRQFIKPLPLDYRTALNKTHREKLVYELKGQRVTVSHR

KTKQKYTIQGFTDLPASQITFLDSESGQSRKLVDYFAQQYGKVIEYQMLPCLDLSKSK

DKPNYVPIELCKLVEGQRYPMANLNKDTDRALKGKALIKAAERKWEIETAVKAEDGPC

RGEIAQQFGISLDVKMMEVTGRVLSPPMLTLGSSRGAPGNLSITPSNCQWNLMGKKL

VEGKALQCWGIVDFSARPSHNKQQALDGNMFINYIVRKCCDLGIQMNKTACFVHLSA

MSVLSDPHQLYEELNKAKQAAVKKNQKLQLLFCPMSEQHPGYKTLKLICETQLGIQTQ

CFLSHLANKTQGQDQYMSNLALKINSKLGGINVQLQDKLPLDNGAPYMFIGADVNHPS

PGNVESPSIAAVVASMNRGATKYVPRIRAQPHRCEVIKNLGEIVQELIGVFEKKAGVKP

QRIIYFRDGVSDGQFEMVLNEELADMEKVIKVKGYSPTITVIVAKKRHHTRLFPKEHNE

PLQTKNGNVLPGTVVDTRIVDPVTYDFYLCSHNGLIGTSRPTHYYNLMDEHGYGSDD

LQRLVYNLCFVFARCTKPVSLATPVYYADLAAYRGRLYYEGMLASQPQVRSSSSSAS

SAAAGASDFSNFPMLHVDLQDNMFFI

SEQ ID NO: 14: Hordeum vulgare subsp. Vulgare nucleic acid sequence (Sequence ID: AK364273.1) atggattacgagcaaggcggcggcggtggccgcggccgcggaagatctcgcggcggcggagggcgtggcggggc gcccggtggctacgggcctcaaggaggcggcggaggctacggaggctacggaggaggcggtcaaggccggggcg ctcagggaagcggtggagggtacggaggaggcggaggcggaggctacgggccccaggggggcttggagggccgc ggaggtggctacgcgcctcgcggcggaggttacgctcctcgcggcggacgaggctatggtcctcagggaggcggcga gggccgcggaggcggctacggcggaggcgggccgggagggcgtggtcctggcggtggtgggcgggggtacggcc ccggcggtgggcgtggtgggaacgcctgggcgcagcctggcagagggcccgccggaggacccggggattacgccc cggtgagggcgccggcgccggcaccagcgtctgcggcaaggagggttgggtccgaggaggcggggggctcatctgg atccgtcgaacggatcagctccaaagaagtggcaaaattagaaccattagcccccccagttgctgcgtctcccaatggc atacgggtgccaatgcggagacctgatgatggtggatcattatgtcaagccagggtccagcttttggtaaaccacttcata gtcaagtacccaaagttgtcaaccttttttcactatgacatagatatcaagttcgatccagcgtcttcaaaggtttctggcaag gagctgtccaatgcagattttctttctgcaaaggctgagcttttcaaggatgacagctttcggcagctttcatcagctgttgctt atgatggaaagagaaacctatttactgctgcacaactgccagaaggtttattccgtgtcagagttcattcaaagacctacat tgtgtcagtagagtttaagaagcagcttcctttaagccaaatctcagagttgcctgtggctagggagatcttacagggtcttg atgtcattgtgcgcgaggcttctacctggcgcaagattatacttggtcatggattttactcaccagacagcaaagtggatatg gggtcaggtgttgtgtctatgaaaggaacccagcagacccttaaacatactcagcagggactggtcctatgtgtcgactat tcagttatgccatttcgcaaagatggacctgttctggatattgtgcggcagtttataaagccccttccccttgactacaggaca gcactgaacaagactcatcgggaaaagttggtgtatgagctcaaaggccaacgtattactgtgtcccaccggaagacta agcagaagtacactattcaaggcttcactgatttgccagccagccagatcacctttttagattcagaatcaggacagacaa agagactcgttgactatttttctcagcagtatggcaaggtgatcgagtatcagatgctaccatgcctggatttgagcaagag cagagataaaccaaactatgtgcccattgagctttgtaagcttgttgaaggacagaggtatccaatggcaaatttgaaca aggacactgagagagcactgaaaggcaaggctttaatcaaggcagctgagcgaaagtgggagattgagaccgcggt caaagccgaagatgggccttgcaggggagaaatagctcaacagtttgggatctctttggatgtaaagatgatggaagtc accggcagggtccttactcctccctcgctgacactcggctcctccagaggaggtcctggcaatatcagtatcactccatcta actgccagtggaacctcatggggaagaaactggtagaaggcaaagcactccagtgttggggcattgtcgacttcagtgc acggccatctcacaacaagcagcaaccgcttgatgggaacatgtttattaactatatcgtcaggaagtgctgtgaccttgg cattcagatgaacaagacagcatgttttgtgcatctatcagaaatgtcagtgctatcggatccacaccaactgcacgagg agctgaacaaagcaaagcaggctgcggtgaagaagaaccagaagctgcaactcctcttctgcccgatgtctgagcag catcatgggtacaagacactgaagctgatctgcgagacgcagctggggatccaaacccagtgtttcttgagccacctcg caaacaaaacccagggccaggaccagtacatgtccaaccttgctctcaagatcaacggcaagctcgggggcatcaac acccagctccaggacaagctcccactggacaacggtgttccttacatgttcataggcgcagacgtgaaccacccatcac ctgggaatggggagagcccgtcgatagcagccgtggttgcgtccatgaataggggcgccaccaagtatgtgcctagaa tccgtgcccagccacaccgctgtgaggtgatcaagaacctcggtgagattgtccaagagcttattggtgtctttgagaaga aggccggcgtaaagcctcagaggatcatttacttccgtgatggcgtgagtgatgggcagttcgagatggtccttaacgag gagcttgcggacatggagaacgtgatcaaggtgaagggctactctccaacgattaccgttattgtggccaagaagcggc accacacgcggctgttccccaaggagcacaatgagccgctgcagacgaagaacggcaatgtgctccctggcacggtg gtggacaccagaattgttgaccccgtgacgtacgacttctacctgtgcagccacaacgggctgattgggaccagccggc cgacgcactactacaacctgatggacgagcatggctacgggtcagacgacctgcagaggctggtgtacaacctctgctt cgtgttcgcgcgctgcaccaagcccgtgtcgctggcgacgcccgtctactacgccgacctggcggcgtaccgcggcag gctctactatgagggcatgctggcgtcccagccccaggtgcgcagctcgtcgtcgtcggcctcctccacagcggcgggg gcttgtgacttcagcaacttcccgacgctgcacgtggatcttcaggacaacatgttcttcatctga

SEQ ID NO: 15: Hordeum vulgare subsp. Vulgare amino acid sequence (Sequence ID: AK364273.1)

MDYEQGGGGGRGRGRSRGGGGRGGAPGGYGPQGGGGGYGGYGGGGQGRGAQ

GSGGGYGGGGGGGYGPQGGLEGRGGGYAPRGGGYAPRGGRGYGPQGGGEGRG

GGYGGGGPGGRGPGGGGRGYGPGGGRGGNAWAQPGRGPAGGPGDYAPVRAPA

PAPASAARRVGSEEAGGSSGSVERISSKEVAKLEPLAPPVAASPNGIRVPMRRPDDG

GSLCQARVQLLVNHFIVKYPKLSTFFHYDIDIKFDPASSKVSGKELSNADFLSAKAELF

KDDSFRQLSSAVAYDGKRNLFTAAQLPEGLFRVRVHSKTYIVSVEFKKQLPLSQISELP

VAREILQGLDVIVREASTWRKIILGHGFYSPDSKVDMGSGWSMKGTQQTLKHTQQG

LVLCVDYSVMPFRKDGPVLDIVRQFIKPLPLDYRTALNKTHREKLVYELKGQRITVSHR

KTKQKYTIQGFTDLPASQITFLDSESGQTKRLVDYFSQQYGKVIEYQMLPCLDLSKSR

DKPNYVPIELCKLVEGQRYPMANLNKDTERALKGKALIKAAERKWEIETAVKAEDGPC

RGEIAQQFGISLDVKMMEVTGRVLTPPSLTLGSSRGGPGNISITPSNCQWNLMGKKLV

EGKALQCWGIVDFSARPSHNKQQPLDGNMFINYIVRKCCDLGIQMNKTACFVHLSEM

SVLSDPHQLHEELNKAKQAAVKKNQKLQLLFCPMSEQHHGYKTLKLICETQLGIQTQC

FLSHLANKTQGQDQYMSNLALKINGKLGGINTQLQDKLPLDNGVPYMFIGADVNHPSP

GNGESPSIAAVVASMNRGATKYVPRIRAQPHRCEVIKNLGEIVQELIGVFEKKAGVKP

QRIIYFRDGVSDGQFEMVLNEELADMENVIKVKGYSPTITVIVAKKRHHTRLFPKEHNE

PLQTKNGNVLPGTVVDTRIVDPVTYDFYLCSHNGLIGTSRPTHYYNLMDEHGYGSDD

LQRLVYNLCFVFARCTKPVSLATPVYYADLAAYRGRLYYEGMLASQPQVRSSSSSAS

STAAGACDFSNFPTLHVDLQDNMFFI

SEQ ID NO: 16 Brachypodium distachyon argonaute 2-like nucleic acid sequence ((LOC 100834773) Sequence ID: XM_010228966.2)

atggagcacgagcaaggcggcggccgcggccgcgggaaagctcgcggcggaggcgggcgtggcggtgggcgag ggggtggctacgggcctcaaggaggcggcggaggctacggacgaggtgagggccagggccgtggaggcggaggc ggctacgcgcctcgcgggagaggctacggccctcagggaggcggcgagggccgcggcggaggaggctacgggta ccagggccgcggcggaggaggctacggctctcagggaggcggcgagggccgcggaagaggctacgggcctcagg ggcgcggcgagggacggggaggaggctatggcgcccatgggcgcggcgagggaaggggtggaggctatggcgct caggggcgcggcgagggccttggagccggaggaggccacgggcctcagggaagcggagggcacgatggtgggc gtggtactggcggtggtggacgtgggtacgggccaggcggcggcagcgcgtgggcgcagccggggagagggcgtg gaggtggaggtggaggtggagcggtcccggcgtgggggccggcgccagcgccagctccagccccggaagcgagg aggatcaaagcagaggaggcggggagctcgtctggatccgtcgaacgcatatccgacaaagtggcaaaaatagaac catcagcaccccaagttgttgtgtctcctaatggcacacgggtgccaatgcaaagacctgatcgtggaggatcatcatttc gaaaccaggtccaacttttggtgaactacttcatagtcaactaccaaaatgtgtcaaccatatttcactacgacatagacat caagtttgatcaatcatcttcagagtcttcaggcaaggagctctccaatgcagattttcattctgcaaaggccgagcttttcaa ggatgacagctttcggaatctttcatcagctgttgcttatgatggaaagagaaatctatttacttgcactgaactgccagaag gcttattccgtgtgagagttcgttcacggacctacattgtatcagtagagttcaagaagcggcttcctttaaaccaacaccca caattgcctagggaggtcttgcagggtcttgatgtcgtcgtgcgcgaggattcaaggctgcacaatataatgcttggcnag ggattttactcacgggacaggattactaatctcgggcaggatgttgtagctatgaaaggaaccaaacagacccttaaaca cacccagcaagggctggtcctatgtgttgaccattcagttatgccattccgcaaagctggacctgtcctggatgttgtcagg cagttcataagaaggaaccttgactacaggacaaatctgaccgacagtgaatgggataatttgtcatctgaactgaaag gccaacgagttactgtgaaccaccggatgactaaccagaagtacactattcgaggcttaacaaaggaccctgccagaa tgatcacctttgaagattctgaatcagggcagcaaaagaggcttgttgattattttgctcagcagtatggcaaggtgactga gtatgagatgcttccatgcttggatttgtccaagacgaaaaagaactatgtgccgattgagctttgtgatttccttgaaggac agaggtatccaaaggcaaatttgccaaggaatattgatacaagcctgaaaaggatggctttaatcggtgtagatgaacg gaaggcagagattatgatgtcggtaggagctacacatgggccttgcaggggagagatagctgagaagttcggggtgtct ttggatgtaaagatgacagaagtcaccggtaggatccttcctcctccagtcctgaaacttggcggccccaaaggccaga cttgcaaattcaatatcaatcagcctacctgccaatggaacctcatgaggaataaactggtagaaggcaaggccctcaat tactggggcattatcgacttcagtacagattccaggcagccgcttcaccgagaaatgtttcttaattatatcgttggcaagtg ccgtgagnttggcattcagatggctgagaagccatgctatcagtattcctcagaaatggcagtgctatgggatgcagacg aattatacagggtgctgaacgaagcaaagcagtctgcagaagaaaagaagcaaaagctgcagctgctcttctgcccg atgtttgagcagcatccggggtacaagacactgaagctgatctgcgagacgaaactggggatccagacccagtgtttctt gagcaacgtcgcgaacaatccccggggccaggaccagtacatgtccaaccttgctctcaaaatcaacagcaagatcg gtgggagcaacgtccagctctttgatctgctcccaagggacacgggcgctcctttcatgctcatcggcgcggacgtgaac cacccgtcgcccggtaatctggagagcccgtctatagcagccgtggtggcgtccatggacaaaggagccaccaagtat gcgtccaggattcgcgcgcagccacaccgctgcgaggtgatcaagcacctcggcgagatctgccaagagctcatcagt gtctttgagaacaagaacaaggtcaagccacataagatcatttacttccgcgatggcgtgagcgatgggcagttcgacat ggtcctgaatgaggagctggcggacatggagaagaggatcaaggtgaacggctactcgccgacgatcaccgtcatcg tggccaagaagcggcaccacacgcggctgttccccaaggataagcaggcgggtaagggcaacgtgctccctggcac ggtggtggacaccaaagtggtcgacccctcggcatacgacttctacctgtgcagccacaacgggctgatcgggacgag ccggccgacgcactactacagcctcatggacgagcataactacagctcggacgacctccaaaagctgatctacaacct ctgcttcgtcttcgcccgctgcaccaagcctgtgtcgctggccacgcccgtctactatgccgacctggcggcgtaccgcgg caggctctactacgagggcatgacgatggcgttcccgacccaggagcgtgggtcttcatcctcgtcctcttcctccgcagc tgctcctgctcctgctccagagttcccgaggctgcacgaggatattcagaacaacatgttcttcatctga SEQ ID NO: 17 Brachypodium distachyon argonaute 2-like amino acid sequence ((LOC 100834773) Sequence ID: XM_010228966.2)

MEHEQGGGRGRGKARGGGGRGGGRGGGYGPQGGGGGYGRGEGQGRGGGGGY

APRGRGYGPQGGGEGRGGGGYGYQGRGGGGYGSQGGGEGRGRGYGPQGRGEG

RGGGYGAHGRGEGRGGGYGAQGRGEGLGAGGGHGPQGSGGHDGGRGTGGGGR

GYGPGGGSAWAQPGRGRGGGGGGGAVPAWGPAPAPAPAPEARRIKAEEAGSSSG

SVERISDKVAKIEPSAPQWVSPNGTRVPMQRPDRGGSSFRNQVQLLVNYFIVNYQN

VSTIFHYDIDIKFDQSSSESSGKELSNADFHSAKAELFKDDSFRNLSSAVAYDGKRNLF

TCTELPEGLFRVRVRSRTYIVSVEFKKRLPLNQHPQLPREVLQGLDVWREDSRLHNI

MLGXGFYSRDRITNLGQDWAMKGTKQTLKHTQQGLVLCVDHSVMPFRKAGPVLDV

VRQFIRRNLDYRTNLTDSEWDNLSSELKGQRVTVNHRMTNQKYTIRGLTKDPARMITF

EDSESGQQKRLVDYFAQQYGKVTEYEMLPCLDLSKTKKNYVPIELCDFLEGQRYPKA

NLPRNIDTSLKRMALIGVDERKAEIMMSVGATHGPCRGEIAEKFGVSLDVKMTEVTGR

ILPPPVLKLGGPKGQTCKFNINQPTCQWNLMRNKLVEGKALNYWGIIDFSTDSRQPLH

REMFLNYIVGKCREXGIQMAEKPCYQYSSEMAVLWDADELYRVLNEAKQSAEEKKQ

KLQLLFCPMFEQHPGYKTLKLICETKLGIQTQCFLSNVANNPRGQDQYMSNLALKINS

KIGGSNVQLFDLLPRDTGAPFMLIGADVNHPSPGNLESPSIAAVVASMDKGATKYASR

IRAQPHRCEVIKHLGEICQELISVFENKNKVKPHKIIYFRDGVSDGQFDMVLNEELADM

EKRIKVNGYSPTITVIVAKKRHHTRLFPKDKQAGKGNVLPGTVVDTKVVDPSAYDFYL

CSHNGLIGTSRPTHYYSLMDEHNYSSDDLQKLIYNLCFVFARCTKPVSLATPVYYADL

AAYRGRLYYEGMTMAFPTQERGSSSSSSSSAAAPAPAPEFPRLHEDIQNNMFFI

SEQ ID NO: 18 Soybean Glycine max argonaute 2-like nucleic acid sequence ((LOC 100805250), Sequence ID: XM_003556633.3) atggaaagaggtgcttacagaggccgtggccgtaacggtaatggtggccgtaacagttacggtggtggtcgcggtagtt acggtggtggccgtgacggtaacggtgaccataacagttacggtggtggccgcggtggtcgtagcagttacggtgatag aagttctccttctcaatcacagtggcaaccaaggtcaaacccttctttttcttctccaaaacctaacaattctcattctcaagttc aaacacaaactcaagctcatccagacattggatcttcgaagataccagagaagaaaatggacaccatcacacctgtgc gtaggcctgacaatgggggaacagttgcagtgcgaaagtgctatctccgtgtgaaccatttccctgtttccttcaatccaca gagtataattatgcattataatgtggaagtgaaggccaaggctcctccactgaagaacaatcgtcctcccaagaagatttc gaagtacgacttgtcattgattcgggataagctgttttccgacaattcactgccagcttcagcgtatgatggtgaaaagaac atcttcagcgcggtgcctttgccggaggaaacatttaccgtggacgtgtccaaaggagaggacgaaaggcctgtttcttat ttggtctctctgacattggtgagtaggctcgagcttcggaagttgagggattacctcagtggaagcgtgctttcgatccctag ggatgttttgcacggcttggatttggtggtgaaggaaaatccttcgaagcagtgtgtttccttggggcggtgcttcttccccatg aaccctcctttgaggaagaaagatcttaaccatggcataattgcgattggagggtttcagcagagtcttaagtctacttctca gggattgtccttgtgcctggactattcggttttgtcctttcggaagaagctgttggtgttggattttctgcacgagcatattaggg acttcaatttaagggagtttgggcggttcaggagacaagttgagcatgtacttattgggttgaaggttaatgttaaacaccgg aagacaaagcagaagtacactattactaggttgacacccaaggttacgagacatatcacattccctattttggatcccga gggccggaatcccccaaaggaagctactctggttggttactttctagagaagtatggtgtgaacattgaatacaaggacat tcctgccttggattttggaggcaacaagacgaattttgtgcctatggagttgtgtgagttggttgaggggcagagatatccca aagagaatttggacaaatatgctgccaaggacttaaaagacatgtcagtggctcctccaagggtgaggcaaagtacaa tacaagcaatggtaaactcagaggacggaccgtgcggaggtggtgttattaaaaattttggaatgagtgtcaacacttcc atgacaaatgtgacaggacgtgtaattcagcctccacaattgaagctaggtaatccaaatggccagactgttagtatgac acttgaagtagagaaatgtcagtggaatctagtgggacgatcaatggtggaaggcaagccagttgagtgttggggcattc ttgattttaccagccaggagtctggttggcgcaaattaaacagcaaacaattcattgagaaccttatgggtaagtatagaa aattgggtattggcatgaaggagccagtttggcgtgaacaatctagtatgtggagtcttggggattacaattcgctgtgtaaa ttacttgaaaatattgaggataaggttcaaaaaagatatcgacgaaaactacaatttcttctgtgtgtgatgtccgacaagc atcaaggttacaagtgcctcaaatggattgctgagaccaaggttggcatagtgacacaatgctgcttgtctggtattgctaat gaagggaaggaccaatatcttacaaatcttgccctcaagatcaatgccaaaattggaggaagtaatgtggagctcatca ataggctaccacactttgagggtgaaggtcatgttatgttcataggggctgatgtcaatcatccagcttcccgggacatcaa cagtccatcaattgctgctgtagttgccactgttaattggcctgctgcaaatcgctatgcagcacgtgtttgtgctcaaggtcat cgggttgagaaaattttgaattttgggagaatttgctatgaacttgtttcgtattacgataggctgaacaaagtcaggcctgaa aaaattgttgtctttcgtgatggcgtgagcgaaagtcaattccatatggttctcacagaggagttacaagatttgaaatcggt gtttagtgatgcaaattacttcccaaccatcactattattgtcgcacaaaagcgacatcaaactcgattttttcctgtgggtcca aaggatgggattcaaaatggcaatgtgtttccaggtacagttgtggacacaaaagtagtacatccttttgaatttgacttttac ctttgtagtcactatggaagcttgggtactagtaagcccactcactatcatgtcttatgggatgagcataaatttaactctgatg atttgcagaaactgatatatgacatgtgctttacctttgcaaggtgcactaaacctgtatctttagtccctccagtgtactatgct gatctcactgcatatagaggacggttatactatgaagcaatgaatcaaatgcaatctcctggttcagctgtgtcgtcttcatc atcacagattacttctttgtcaatttcttcaacaggctcaagtttaaatgatccggggtattacaagttgcatgctgatgtggaa aatataatgttcttcgtttga

SEQ ID NO: 19 Soybean Glycine max argonaute 2-like amino acid sequence (( LOC 100805250) , Sequence ID: XM_003556633.3)

MERGAYRGRGRNGNGGRNSYGGGRGSYGGGRDGNGDHNSYGGGRGGRSSYGD

RSSPSQSQWQPRSNPSFSSPKPNNSHSQVQTQTQAHPDIGSSKIPEKKMDTITPVRR

PDNGGTVAVRKCYLRVNHFPVSFNPQSIIMHYNVEVKAKAPPLKNNRPPKKISKYDLS

LIRDKLFSDNSLPASAYDGEKNIFSAVPLPEETFTVDVSKGEDERPVSYLVSLTLVSRL

ELRKLRDYLSGSVLSIPRDVLHGLDLWKENPSKQCVSLGRCFFPMNPPLRKKDLNH

GIIAIGGFQQSLKSTSQGLSLCLDYSVLSFRKKLLVLDFLHEHIRDFNLREFGRFRRQV EHVLIGLKVNVKHRKTKQKYTITRLTPKVTRHITFPILDPEGRNPPKEATLVGYFLEKYG

VNIEYKDIPALDFGGNKTNFVPMELCELVEGQRYPKENLDKYAAKDLKDMSVAPPRV

RQSTIQAMVNSEDGPCGGGVIKN FGMSVNTSMTNVTGRVIQPPQLKLGNPNGQTVS

MTLEVEKCQWNLVGRSMVEGKPVECWGILDFTSQESGWRKLNSKQFIENLMGKYRK

LGIGMKEPVWREQSSMWSLGDYNSLCKLLENIEDKVQKRYRRKLQFLLCVMSDKHQ

GYKCLKWIAETKVGIVTQCCLSGIANEGKDQYLTNLALKINAKIGGSNVELINRLPHFEG

EGHVMFIGADVNHPASRDINSPSIAAVVATVNWPAANRYAARVCAQGHRVEKILNFG

RICYELVSYYDRLNKVRPEKIWFRDGVSESQFHMVLTEELQDLKSVFSDANYFPTITII

VAQKRHQTRFFPVGPKDGIQNGNVFPGTVVDTKVVHPFEFDFYLCSHYGSLGTSKPT

HYHVLWDEHKFNSDDLQKLIYDMCFTFARCTKPVSLVPPVYYADLTAYRGRLYYEAM

NQMQSPGSAVSSSSSQITSLSISSTGSSLNDPGYYKLHADVENIMFFV

SEQ ID NO: 20 Cotton Gossypium hirsutum argonaute 2-like nucleic acid sequence ((LOC107961816) Sequence ID: XM_016897971.1) atggagagaggtggtggtggctatagagaaagaggaattggcggaagaggtcgttactatggtggtggaagaggaag agcaagaggaagaggtagagatggcagcgctgataatcagcctccgtatcaggcgcccataggccaagggcaaagt ggggccaattggaacccaactaactctcaagtgggtccttatcaggacggcgatgcgaggggtggtcagactggtggtg ctcgtggtagaaggggagggtgggttcctcggcttccaccggagagaccgaattatagttatgaggcggaatcagccag cggcaggggcggcggaaatggtcgtgggcgagggaggaggcctttcaggcccgttcctgccccaacactggagcctg aagataacatacctgcattgcctcctagtcctcctcctccacagtctgtcccagattctgttgtgatgaatgtttcagagcagg tggcttctacatcattatcttccgagaatcaaaatagacataacccaattatgcgaccggataaaggtggaagaactgctc ttgcaactgtcaggcttaatgttaatcattttcgggtgaagttcaatccggaaagtatcataaggcattatgatgttgatgtcag gcaactggagtctcctaagaatggtcgccctgccaaactatctaagccattattgtctagtatccggaaaaaactattcact gataatcctcaattgccattgttgatgaccgcatatgatggtgaaaagaatatatttagtgctgttcgattacctgaaggaga attcaaggtggaattgtccgagggggaagacgtgatggcccgtacatacatcttttctataaagcttgtgaatgagctcaa gcttggcaagttgcgggattacttgtctaggaaagcctattcaatacctcgtgacatattacaggggatggatgtagttatga aggagaatccggtaatgcatatgattcctgctggccggagcttccatccttatgaatcttgtccagaagatgatcttggatat ggcatcacagcatctagaggaatccaatatagtctcaagcccacgtaccagggtctagcactttgcttggattattcagtttt agcatttcgcaagaaaatgccagttttagattttctagctgagcacattcctggttttaatgtgaatagttttcggaggtttagac gacttgttgatcacgccttgaaaaacctcaaggttaatgtaacccatcgacgtaccaaacagaagtatgttatagttgggtt gacgcgagatgacactcgcgatgtttcatttccagatgcgaatgatccgcaagtgcaagttagactcgttgattatttcagg gagaaatatgacaataatattagatttttggatattccttgcctagatttgagcaggaacaagcggatgaactatgtcccaat ggagtattgtgtcttagctgagggtcagatttatccaaaggatgatttggatcgagaagcagcttttatgttgaagaacatatc cctggcaaagccacatgaaagacaaagcaagatctgtggtatggttcaatccaaggatggaccatgtggtggaaacatt attcaaaattttggtatcgaagttgatactaggatgacaccagtggaaggacgtgtgattggacctccagttttgaagttggc tgctccaactactggcaaattgttgaagataacagttgacaaaaacacatgccagtggaacctggttagaaaagctgttgt ggtaggcaaagcaattcagtgctgggctgtgatagactttacccaggctgatcgatcgaaactgaactgtgactcattcatt ccaaagctgaggaataggtgcaggaatcttgggatgacaatggacaaccccatcttatatgaaccggctcgaatgcaa atattttctgatgggaatgcacttcttcaactgcttgaaaatgttacttgtcgagtacatgaacttggtagaggaaatttacaatt tctactttgtgtaatgtcgtggaaggatgatggctacaaatgtcttaagtggatctctgagacaaaaattggtgtggtgactca gtgttgcttgtccaaccatgcaaacaaggggcaggaccaataccttgctaatcttgctctgaaaatcaatgctaagcttgg gggcagcaatgtagagctgaatgaccggcttccccatttccatggtgaaggtcatgttatgtttattggagcagatgttaatc atcctggttctgataataagactagcccatccattgctgctgtagtagcaacaatgaactggcctcaagctaatcgttatgca gcaagggttcgaccccagttccatcgcaaagaacagatcctagagttcggtgaaatgtgtttggagcttgttgaatcttatg ctcgtctaaataatgttaaaccagaaaaaattgtgctcttccgtgatggggttagtgagggtcagtttgatatggttcttaatga agagttaatggatctcaagagtgcattccatagaatcaattattttcctacgatcacccttattgttgctcagaagcgacacc aaactcgtttctttcccgagggaaagtgggatgggggtcccactggtaatatttcacctggcacagttgttgacactaaaatt gtgcatcctttcgagtttgacttctatctttgtagtcactatggaagccttggtactagcaagcctacacactaccatgtcttgtg ggatgagcatgggttctcttctgatcagttgcaggagcttatctacaacatgtgctttacatttgcacggtgcacaaaaccgg tatctctggttccacctgtgtactatgccgaccttgttgcatatagggggcggctgtactaccaggcaatgattaaaagaca atctcccatttcaacattatcctcatcttcatcaatgacatcgtcatcatcaatgtcatctgcagcttcattccatgactggtttaa gctgcatgcaaacctggaaaacatgatgttttttctttga

SEQ ID NO: 21 Cotton Gossypium hirsutum argonaute 2-like amino acid sequence ((LOC107961816) Sequence ID: XM_016897971.1)

MERGGGGYRERGIGGRGRYYGGGRGRARGRGRDGSADNQPPYQAPIGQGQSGAN

WNPTNSQVGPYQDGDARGGQTGGARGRRGGWVPRLPPERPNYSYEAESASGRG

GGNGRGRGRRPFRPVPAPTLEPEDNIPALPPSPPPPQSVPDSVVMNVSEQVASTSLS

SENQNRHNPIMRPDKGGRTALATVRLNVNHFRVKFNPESIIRHYDVDVRQLESPKNG

RPAKLSKPLLSSIRKKLFTDNPQLPLLMTAYDGEKNIFSAVRLPEGEFKVELSEGEDVM

ARTYIFSIKLVNELKLGKLRDYLSRKAYSIPRDILQGMDWMKENPVMHMIPAGRSFHP

YESCPEDDLGYGITASRGIQYSLKPTYQGLALCLDYSVLAFRKKMPVLDFLAEHIPGFN

VNSFRRFRRLVDHALKNLKVNVTHRRTKQKYVIVGLTRDDTRDVSFPDANDPQVQVR

LVDYFREKYDNNIRFLDIPCLDLSRNKRMNYVPMEYCVLAEGQIYPKDDLDREAAFML

KNISLAKPHERQSKICGMVQSKDGPCGGNIIQNFGIEVDTRMTPVEGRVIGPPVLKLAA

PTTGKLLKITVDKNTCQWNLVRKAVWGKAIQCWAVIDFTQADRSKLNCDSFIPKLRN

RCRNLGMTMDNPILYEPARMQIFSDGNALLQLLENVTCRVHELGRGNLQFLLCVMSW

KDDGYKCLKWISETKIGVVTQCCLSNHANKGQDQYLANLALKINAKLGGSNVELNDRL PHFHGEGHVMFIGADVNHPGSDNKTSPSIAAWATMNWPQANRYAARVRPQFHRKE

QILEFGEMCLELVESYARLNNVKPEKIVLFRDGVSEGQFDMVLNEELMDLKSAFHRIN

YFPTITLIVAQKRHQTRFFPEGKWDGGPTGNISPGTVVDTKIVHPFEFDFYLCSHYGSL

GTSKPTHYHVLWDEHGFSSDQLQELIYNMCFTFARCTKPVSLVPPVYYADLVAYRGR

LYYQAMIKRQSPISTLSSSSSMTSSSSMSSAASFHDWFKLHANLENMMFFL

SEQ ID NO: 22 Medicago truncatula argonaute 2 nucleic acid sequence

(LOC11446921) Sequence ID: XM_003607808.3) atggatcaacgcggtttcagaaactcaaccggtggtaatagccataacggtaacagtaatggtggtggtggcagagga agaggaagagagtttcaacaacaatcaagtcctggtggtcgtggtggtcgagggtttactcaacaaccgcaaaaccgtt ggcaacaaaggccaaacccaccttcttccttcacaccttcaactcctgctccgatcaccaagcccaaccccaacaaccg tcaacccccagtccaaactcatccacaagtccaaactcatccacttccagatattggagctctgaaaataaaagagcag cccgtggaaaataccacaattggacccatgttccggcctgacaagggagggacagtgtcaattcgagactgcagacttc gtgtcaatcattttccggttgctttcaatccacaaagcattatcatgcactacgatgtggacgtgaaggcttctgtgccaccga gaaagggtttgcctccaaagaagatttccaagtctgacttgtctatgatcagggacaaattgtgtgctgatcatcctcagata ctgcctttgttgaagacttcatatgatggagagaagaacatcttcagttctgtgccattgcctgaagaaacttttaccgtggag gtctcaaaaggagaagatgaaagagctgtttcttatacggttactataacactggtgaacaaactcgagcttcgtaagttg agagattaccttagtggcaatgtgtattccattccgagggatattttgcagggaatggacttagtggtgaaggagaatccgg caaggcgcacagtttctttaggacggtgtttctttcccaccaatcctcccttaatacagagagatcttgaacccggaataatt gcaattggaggatttcaacacagtctcaagaccacggctcagggtttagctttgtgtcttgattattcagttttgtcttttcgaaa gaaaatgtcagtcttggatttcctccatgatcatattagaggtttcaatttggctgagttcaggaaatataagaaatttgtcgag gaggtactcttgggattgaaagtaaatgttactcacagaagaaccaaacagaaatatactattgctaagctaacagataa agatactcgccacatcactttccctattttggaccaagagggccaaaccccccctagaagcacctctcttcttgcctacttta aagataaacataactatgatattcaacacaaagatattcctgcattggattttgggggaaacaagactaatttcgtgcctat ggagctatgcgtcttggttgagggtcagcgatttcccaaagagtatttggacaagaatgctgccaagaacttgaaaaatat gtgcttggctagtccaagggacagggaatctacaatacaaatgatgatgaagtctagtgatggaccgtgcggcggtggt attcttcagaattttggaatgaatgtcaacacttccatgacaaatgtgaccggacgtgtaattggacctccaatgttgaagtta ggcgatccacgtggaaagagtactcctatgaaactggatccagagaaatgccattggaatcttgttggaaagtcaatggt agaaggaaaagctgttgaatgttggggcatccttgattttaccagcgatgcacctaattggtgcaaattaagaggcaatca gttcgttaacaatcttatggataagtacaggaagttggggattgtcatgaatgaacctgtttggcatgagtattctgcaatgtg gaaacttggggattacaatttactatgtgagttacttgaaaaaataaatgagaaggttcaaaaaaagtgtcgacggcggc tacaatttctcctttgtgtgatggccaacaaggatccaggttacaagagcctcaagtggattgctgagaccaaggttggcat agtgacacagtgctgcttatctggtaatgctaatgaagggaaagaccaatatctcacaaatcttgctttaaaaatcaatgca aaaattggaggcagtaacgttgagctcattaataggctcccacactttgaggatgaaagtcatgttatgtttataggggctg atgtcaatcatccaggttcccgggacacaaatagtccatcaattgttgcagtggttgccactactaactggccagctgcaa atcgctatgcagcacgtgtttgcgctcaagagcattgtacagagaaaattttgaattttggagagatttgccttgaccttgtta gacattatgagaagttgaacaaagtcaggccccaaaaaattgttatctttcgtgatggtgttagtgagagccaatttcacat ggttcttggcgaggagttaaaagatttgaagaccgtgtttcagcactcaaattactttccaactatcactcttattgtagctcaa aagcgccatcaaactcgattgtttcctgccggtgtaagggagggggctcccagtggaaatgtgttccctggaacagttgtg gacacaaaggtcgtacatccttttgaatttgacttttacctgtgtagtcactatggaagcctaggtacaagcaagcccactca ctatcatgtcttgtgggatgagcacaggtttacttctgataatttgcagaagctcatatatgatatgtgctttacctttgcaaggt gcactaaacctgtatctttagtccctccagtgtactatgctgaccttgctgcttacagaggaagactatactatgaagcaaag atgtcaactcaatctccatattcaactgtatcttcttcatcatcacctttagcttcttcatccatttcatccaccgcctcaatttcaa atgatccagggttttacaagctgcatcctgatacggaaaacggaatgttcttcgtctga

SEQ ID NO: 23 Medicago truncatula argonaute 2 amino acid sequence

( (LOC11446921) Sequence ID: XM_003607808.3)

MDQRGFRNSTGGNSHNGNSNGGGGRGRGREFQQQSSPGGRGGRGFTQQPQNR

WQQRPNPPSSFTPSTPAPITKPNPNNRQPPVQTHPQVQTHPLPDIGALKIKEQPVENT

TIGPMFRPDKGGTVSIRDCRLRVNHFPVAFNPQSIIMHYDVDVKASVPPRKGLPPKKIS

KSDLSMIRDKLCADHPQILPLLKTSYDGEKNIFSSVPLPEETFTVEVSKGEDERAVSYT

VTITLVNKLELRKLRDYLSGNVYSIPRDILQGMDLVVKENPARRTVSLGRCFFPTNPPLI

QRDLEPGIIAIGGFQHSLKTTAQGLALCLDYSVLSFRKKMSVLDFLHDHIRGFNLAEFR

KYKKFVEEVLLGLKVNVTHRRTKQKYTIAKLTDKDTRHITFPILDQEGQTPPRSTSLLAY

FKDKHNYDIQHKDIPALDFGGNKTNFVPMELCVLVEGQRFPKEYLDKNAAKNLKNMC

LASPRDRESTIQMMMKSSDGPCGGGILQNFGMNVNTSMTNVTGRVIGPPMLKLGDP

RGKSTPMKLDPEKCHWNLVGKSMVEGKAVECWGILDFTSDAPNWCKLRGNQFVNN

LMDKYRKLGIVMNEPVWHEYSAMWKLGDYNLLCELLEKINEKVQKKCRRRLQFLLCV

MANKDPGYKSLKWIAETKVGIVTQCCLSGNANEGKDQYLTNLALKINAKIGGSNVELIN

RLPHFEDESHVMFIGADVNHPGSRDTNSPSIVAVVATTNWPAANRYAARVCAQEHCT

EKILNFGEICLDLVRHYEKLNKVRPQKIVIFRDGVSESQFHMVLGEELKDLKTVFQHSN

YFPTITLIVAQKRHQTRLFPAGVREGAPSGNVFPGTWDTKWHPFEFDFYLCSHYGS

LGTSKPTHYHVLWDEHRFTSDNLQKLIYDMCFTFARCTKPVSLVPPVYYADLAAYRG

RLYYEAKMSTQSPYSTVSSSSSPLASSSISSTASISNDPGFYKLHPDTENGMFFV

SEQ ID NO: 24 Nucleic acid sequence of AG02-FLAG (3xFlag) ATGGAGCACGAGCGCGGTGGCGGTGGCCGCGGCCGCGGGAGGGGTCGCGGTG GCGGGCGTGGCGGCGGTGGCGGCGATGGTCGCGGAGGCGGTTATGGTGGTGC TGGTGGTGGTGGTGTCGGCGGGCGCGGTGGGCGTGGGCCTCCTGGTGGTGGTG GTGGACGCGGGTACGAGCCCGGCGGCGGGCGTGGGTACGGTGGCGGCGGCGG CGGTGGTGGACGTGGGTATGGCGGCGGAGGCGGCGGTGGTGGGTACGAGTCC GGCGGTGGGCGTGGGTATGGCGGCGGTGGACGTGGGTATGAATCCGGCGGTGG GCGTGGACCTGGCGGCGGCGGCCGTGGGCACGAGTCCGGCGGTGGCGGTGGC CGCGGCGGGAACGTGTGGGCGCAGCCGGGGAGAGGGCGCGGAGGAGCCCCCG CCCCGGCGCCGGCGCCAGCACCAGCAGCGAGGAGGATCCAGGACGAGGGGGC CGCGAGGTCGT CGGGT ACCGTT GAGCGCATTGCTTCT ACT GAGGTT GT AAGAGT A CAACCACCTGCACCCCCAGTTGCT GT GTCTCGT AGT GGCACGCGT GTGCCAAT G CGAAGACCT GAT GGTGGAGGCT CAGT ATCGAAAGCCAAGGT CAAATT GTT GGT GA ACCATTTT AT AGTT AAGT ACCGACAGGCAT CAACT GTTTTT CACT AT GACAT AGACA T CAAGCTT GAT AT AAGTTCCCCCAAGGCTTCAGACAAGGAGCT AT CCAAGGGAGA TTTTCTTACTGTCAAGGACGAGCTCTTCAAGGATGAGAGCTTTCGGCGGCTTTCAT CAGCT GTTGCTT AT GATGGAAAAAGAAATTT ATTT ACTT GTGCT GAGCT ACCAGAT GGTTT GTTT CGT GTCAAAGTCCGTT CACGG ACTT ACATT GTATCT GTGG AGTT CAA GAAGAAGCTTCCTTT GAGCCAACT CTCGGAACTGCCT GT GCCCAGAGAGGT CTT G CAGGGGCTTGATGTCATTGTGCGTGAGGCCTCTAGCTGGCGCAAGATTATCATTG GTCAGGGATTTTACTCGCAGGGCCGCAGTGTGCCCATTGGGCCGGATGTTGTAG CT CT CAAAGGAACCCAGCAGACCCT GAAATGCACT CAGAAAGGACT GAT CCTTT G T GTGGACT ATT CGGTTATGCCGTTT CGCAAAGCT GGACCT GT GTTGGATCTT GTTC AG AAGT CT GT GAG AT ACCTT G ACT ACAGG ACAACACT AAACAAACACCAATT GG AC ACTTT GAAGAAT GAACTCAAAGGCCAGCGT GT CACT GT AAATCAT AGGAGGACAA AGCAGAAGT ACATT GTT AAAGGTTT GACT GAT AAACCTGCAAGTCAGAT AACTTTT GT AG ATT CT GAAT CAGG ACAG ACCAAG AAGCTT CTT GATT ACT ATTCGCAGCAGT A TGGCAAGGTT ATTGAGT ATCAAATGCTT CCATGCTT GGATTT GAGCAAGAGCAAG GACAAGCAAAACT AT GTGCCGATT GAATT GT GT GAT CTTCTT GAAGGGCAGAGAT A CCC A AA AG C A AG CTT AA AT AG G A ATT CTG AT AAA AC ACT G AA AG A AAT G G CTTT G A T CCCTGCCT CAAGT AGGAAGGAGGAGATT CTGGAGTTGGT GAAT GCT GACGAT G GGCCTTGCAGGGGTGAAATTGCTCAGCAGTTCGGGATTTCTTTGGATGTACAAAT GATGGAAGTCACTGGTAGGACCCTTCCTCCTCCCAGCCTAAAACTTGGCACCTCC AGT GGCCAACCCCCCAAATTCAAT ATT GATCAGCCT AACT GCCAGT GGAACCTT A CGAGGAAAAGACTAGCAGAGGGCGGGGTGCTACAGTGCTGGGGCGTTGTGGAC TT CAGTGCAGATTCT GGGCAGT ACGCCCT GAATGGGAACAT GTTT ATTGACAAGA TT GTCAGGAAGT GCTGCGACCTTGGCGT ACAGAT GAACCGT AACCCATGCATT GT

GCAACT GTT AGAT AT GGAGGTGCT ATCCGAT CCA CAT CAGCT CTTCGAGGAGCTT

AACAAAGCTAAGCAGGCGGCAGCCAGTAAGAAACAGAAGCTGCAGCTCCTCTTCT

GCCCAAT GT CT GATCAGCAT CCTGGGT ACAAGACGCT GAAGCTT ATCTGCGAGAC

GCAGCTGGGGATCCAGACCCAGTGCTTCTTGAGCTTCCTCGCGAACAAACAACAG

GGACAGGACCAGT ACAT GT CCAACCTTGCT CT GAAGAT CAACGGCAAGATTGGAG

GAAGCAACATCCAACTGTTTGGTGAATCGCTCCCGCGGATCTCCGGCGCGCCATA

CATGTTCATCGGCGCCGACGTGAATCACCCATCGCCGGGGAACGTCGAGAGCCC

GTCGATTGCAGCAGTGGTGGCCTCGGTGGATCAAGGCGCCAGCAAGTACGTGCC

AAGAATCCGCGCTCAGCCTCACCGCTGCGAGGTGATCCAGCACCTCGGCGACAT

GTGCAAGGAGCTCATCGGCGTGTTCGAGAAGCGGAACCGCGTGAAGCCCCAGAG

GAT CAT CT ACTTCCGCGACGGCGTCAGCGACGGT CAGTT CGACATGGT GCT GAA

CGAGGAGCTGGCGGACATGGAGAAGGCGATCAAGACCAAGGACTACTCCCCGAC

GATCACCGTGATCGTGGCCAAGAAGCGGCACCACACCAGGCTGTTCCCCAAGGA

CCTGAACCAGCAGCAGACCAAGAACGGCAACGTGCTCCCCGGCACGGTGGTGGA

CACCGGCGTGGTCGACCCGGCGGCGTACGACTTCTACCTGTGCAGCCACAACGG

GCTGATCGGGACGAGCCGGCCGACGCACTACTACAGCCTTCTGGACGAGCACGG

CTT CGCCT CCGACGACCTGCAGAAGCT GGT GT ACAACCT CT GCTTCGT CTTCGCC

CGCTGCACCAAGCCGGTGTCGCTGGCCACGCCCGTCTACTACGCCGACCTCGCC

GCCTACCGCGGCAGGCTCTACTACGAGGGCATGATGATGTCGCAGCCGCCACCG

TCTTCCGCGGCGTCGGCGTCGTCGGCATCCTCCTCCGGCGCCGGCGCTTCCGAC

TTCAGGAGCTTCCCGGCGCTGCACGAGGATCTGGTGGACAACATGTTCTTCATCC

T CT AGAGGAT CT CGAGGCGCGCCGT CGACATGGACT ACAAGGACGACGAT GAT A

AGGGCAT GGACT ACAAGGACGACGAT GAT AAGGGCATGGACT ACAAGGACGACG

AT GAT AAGGGCGGT ACCCCGGGTT CGAAAT CGAT GGAT CCT ACGT AG

SEQ ID NO: 25 AG02 promoter sequence: 2-kbp plus 5’UTR sequence attagtacactttgtaggagattgtagatgaaaaatattaggtttttgtcggctaaattttgcatcgtaactctgtaagaaaatttt gatgaaaaatcaatatttattagtactaattacaaaaaagaacaagtattataaaaatgatagattaaatatgtttgaaatat tgtgaaaataatacgagaggtgatgattggatatgcttacacgttgatttgtacattttaataaattatagattatgttgtataact tgcgtgagctttgatacgtaattattgttagtggatgatgagttgacatgtttgtaggttaaattttagatttagatttagttgtttgg gtgtgttcgctattactagttccagaaccatccgcacgaaaaacggaacggtccattagcacgtgattaattaagtattagc tttttttccaaaaatggattaatttgatttttaaacaattttcgtatagaaactttttgtaaaaaacgcttagcagtttgaaaagcgt gtgcgtggaagagagaggggttaggaaaaggggtgtccgaacacacccgttgtcactatcccaactcacttttctcgtatt ttatgtgcacgttttctaaatggctaaacatcgtgttttttttaaaaaaaatctatataaaagttgttttaaaaattatattgattattc tttaaaaaatacttagtcaatcatacgctgataaaaccgcgtgtgaggtttgctttatgggaaaatatagatagcgcgtatatt aaagtaaattgcgaggacattgcttaattgccaagttattctttcctgtccttttgaatctggccctgcacctgcgatcagcgat cgagcaagcatcgagctgtagtaatgtcctctgccgatttgacgtgctgtcgtcatcgtcagttcgtcatcatcacctctcttta ctacggagatttcgaaggattttcgctgaatttgacatcgccgctggatcagatttgtggtgaaagctgataaaggaaagc catgtgtcgatcaacggacagtggtatactcctctcgttagagcaagtttaatagtacagccaactactaactctaaattatc tatagccaattcatataatagttgtttactatccaattaatacctggttccacctgtcatacacatatattatatcttggagtccgt actgcagctggctacagatctgtagcccgctgctcttctctctcttcctttatctctttaaaatatatttatagctggtttataatccg ttattgtacttgctcttactgttacatcacctcgtagttggactcgataaacagacgagattttgattaaagaggaactagtcatt tcaaaaaaaaaaaaaatccctgaccttcgtggtaaaacagaattaagaaatcttcaattggaaatgcaacataggctgt gtttagatccaaagtttagatccaaacttcagtccttttccatcacatcaacctgtcatacacacataacttttcagtcacatca tcttcaatttcaaccaaaatccaaactttgcgctgaactaaacacaaccatagataaatgtgtgtctctccagcctgattttgt gtaccaacaagagaaatgcagaaacaatggattaaaatagtgccgcatcttcaaattgcttattgcctgctcaaacattttt attgccttattaacatttatttcttggaattagggtttagggtttgctttatcgtgcaaacacctaagctgataaaggcaaaggta agaaggggaagggagtttctatggttcttgctggtgccgttggcgcactcccacgctgatgaggccaagtggtgtgtgctg gagaggtggcagtggggccaagctcgaaaagttaggtgttaggccaacggcgaggagaagaagaggaggcgacg attagagtttgtgtgtttgcatgaagtgtttgaaacgaaaattcacaaggtattctaggtttttttttcatatgtagagtaacgcta gggagtgacggcggaggtggtggggtcaagttcaagatattgggagctgtggcggcggaggaggagcgggggacta gggtttaggtgtttgcacgaattccaaagcgatccgacgtttcagaattcacaagatatttatgggctattttttttctaaatgta caatagtcagttacttcatttattcgtctcacattcctaatttccaattaagacagggacctaacagcaaaaaggccaaacc ctcccgcgacagaacgggcagtcgggcacgaccgagaccctcgcgcccacgcagcaacctccgtacacacgtcgc acttgacttgcgtggactccaacttcccattttacccctccgtgctcaaccaaattgccactaaaccgctcaccactcactaa tctatcaggggcaagatcgtattttgccactaatcccactaatccccctgcgcgcttctcgtataactactccccactgctctct tcgatcccctcttctgctttttagcgaaatccgagaaaaaaattgcctgcaaaaaaaaaaatcgaaacgtcgttctgatcg atcggccgttcgagttcacggcggag

SEQ ID NO: 26 2kb promoter sequence only gtggaagagagaggggttaggaaaaggggtgtccgaacacacccgttgtcactatcccaactcacttttctcgtattttatg tgcacgttttctaaatggctaaacatcgtgttttttttaaaaaaaatctatataaaagttgttttaaaaattatattgattattctttaa aaaatacttagtcaatcatacgctgataaaaccgcgtgtgaggtttgctttatgggaaaatatagatagcgcgtatattaaa gtaaattgcgaggacattgcttaattgccaagttattctttcctgtccttttgaatctggccctgcacctgcgatcagcgatcga gcaagcatcgagctgtagtaatgtcctctgccgatttgacgtgctgtcgtcatcgtcagttcgtcatcatcacctctctttactac ggagatttcgaaggattttcgctgaatttgacatcgccgctggatcagatttgtggtgaaagctgataaaggaaagccatgt gtcgatcaacggacagtggtatactcctctcgttagagcaagtttaatagtacagccaactactaactctaaattatctatag ccaattcatataatagttgtttactatccaattaatacctggttccacctgtcatacacatatattatatcttggagtccgtactgc agctggctacagatctgtagcccgctgctcttctctctcttcctttatctctttaaaatatatttatagctggtttataatccgttattgt acttgctcttactgttacatcacctcgtagttggactcgataaacagacgagattttgattaaagaggaactagtcatttcaaa aaaaaaaaaaatccctgaccttcgtggtaaaacagaattaagaaatcttcaattggaaatgcaacataggctgtgtttag atccaaagtttagatccaaacttcagtccttttccatcacatcaacctgtcatacacacataacttttcagtcacatcatcttca atttcaaccaaaatccaaactttgcgctgaactaaacacaaccatagataaatgtgtgtctctccagcctgattttgtgtacc aacaagagaaatgcagaaacaatggattaaaatagtgccgcatcttcaaattgcttattgcctgctcaaacatttttattgcc ttattaacatttatttcttggaattagggtttagggtttgctttatcgtgcaaacacctaagctgataaaggcaaaggtaagaag gggaagggagtttctatggttcttgctggtgccgttggcgcactcccacgctgatgaggccaagtggtgtgtgctggagag gtggcagtggggccaagctcgaaaagttaggtgttaggccaacggcgaggagaagaagaggaggcgacgattaga gtttgtgtgtttgcatgaagtgtttgaaacgaaaattcacaaggtattctaggtttttttttcatatgtagagtaacgctagggagt gacggcggaggtggtggggtcaagttcaagatattgggagctgtggcggcggaggaggagcgggggactagggttta ggtgtttgcacgaattccaaagcgatccgacgtttcagaattcacaagatatttatgggctattttttttctaaatgtacaatagt cagttacttcatttattcgtctcacattcctaatttccaattaagacagggacctaacagcaaaaaggccaaaccctcccgc gacagaacgggcagtcgggcacgaccgagaccctcgcgcccacgcagcaacctccgtacacacgtcgcacttgactt gcgtggactccaacttcccattttacccctccgtgctcaaccaaattgccactaaaccgctcaccactcactaatctatcag gggcaagatcgtattttgccactaatcccactaatccccctgcgcgcttctcgtataactactccccactgctctcttcgatcc c

SEQ ID NO: 27 CaMV 35S promoter aattccaatcccacaaaaatctgagcttaacagcacagttgctcctctcagagcagaatcgggtattcaacaccctcatat caactactacgttgtgtataacggtccacatgccggtatatacgatgactggggttgtacaaaggcggcaacaaacggc gttcccggagttgcacacaagaaatttgccactattacagaggcaagagcagcagctgacgcgtacacaacaagtcag caaacagacaggttgaacttcatccccaaaggagaagctcaactcaagcccaagagctttgctaaggccctaacaag cccaccaaagcaaaaagcccactggctcacgctaggaaccaaaaggcccagcagtgatccagccccaaaagagat ctcctttgccccggagattacaatggacgatttcctctatctttacgatctaggaaggaagttcgaaggtgaaggtgacgac actatgttcaccactgataatgagaaggttagcctcttcaatttcagaaagaatgctgacccacagatggttagagaggcc tacgcagcaggtctcatcaagacgatctacccgagtaacaatctccaggagatcaaataccttcccaagaaggttaaag atgcagtcaaaagattcaggactaattgcatcaagaacacagagaaagacatatttctcaagatcagaagtactattcca gtatggacgattcaaggcttgcttcataaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcctactgaa tctaaggccatgcatggagtctaagattcaaatcgaggatctaacagaactcgccgtgaagactggcgaacagttcata cagagtcttttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacactctggtctactccaaaaa tgtcaaagatacagtctcagaagaccaaagggctattgagacttttcaacaaaggataatttcgggaaacctcctcggatt ccattgcccagctatctgtcacttcatcgaaaggacagtagaaaaggaaggtggctcctacaaatgccatcattgcgata aaggaaaggctatcattcaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtg gaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgacatctccactgacgtaaaggatgacgca caatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacg

SEQ ID NO: 28 ACTIN1 promoter ctcgaggtcattcatatgcttgagaagagagtcgggatagtccaaaataaaacaaaggtaagattacctggtcaaaagt gaaaacatcagttaaaaggtggtataagtaaaatatcggtaataaaaggtggcccaaagtgaaatttactcttttctactatt ataaaaattgaggatgttttgtcggtactttgatacgtcatttttgtatgaattggtttttaagtttattcgcgatttggaaatgcatat ctgtatttgagtcggtttttaagttcgttgcttttgtaaatacagagggatttgtataagaaatatctttaaaaaacccatatgcta atttgacataatttttgagaaaaatatatattcaggcgaattccacaatgaacaataataagattaaaatagcttgcccccgt tgcagcgatgggtattttttctagtaaaataaaagataaacttagactcaaaacatttacaaaaacaacccctaaagtccta aagcccaaagtgctatgcacgatccatagcaagcccagcccaacccaacccaacccaacccaccccagtgcagcc aactggcaaatagtctccacccccggcactatcaccgtgagttgtccgcaccaccgcacgtctcgcagccaaaaaaaa aaaaagaaagaaaaaaaagaaaaagaaaaacagcaggtgggtccgggtcgtgggggccggaaaagcgaggag gatcgcgagcagcgacgaggcccggccctccctccgcttccaaagaaacgccccccatcgccactatatacatacccc cccctctcctcccatccccccaaccctaccaccaccaccaccaccacctcctcccccctcgctgccggacgacgagctc ctcccccctccccctccgccgccgccggtaaccaccccgcccctctcctctttctttctccgttttttttttcgtctcggtctcgatc tttggccttggtagtttgggtgggcgagagcggcttcgtcgcccagatcggtgcgcgggaggggcgggatctcgcggctg gcgtctccgggcgtgagtcggcccggatcctcgcggggaatggggctctcggatgtagatcttctttctttcttctttttgtggta gaatttgaatccctcagcattgttcatcggtagtttttcttttcatgatttgtgacaaatgcagcctcgtgcggagcttttttgtaggt agaagaggtac

SEQ ID NO: 29 Ubiquitin promoter aattagcttgcatgcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataa aaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataat ataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattga gtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttc atccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctc taaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaatt aaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgc cgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacgg catctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgc gtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcacggcagctacgggggatt cctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcg tgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgc tcgtcctccccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcat gtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgatt gctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgttt cgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttg gttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttgg atctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgt tgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcg ttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagtt acgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggc atatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatat acttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgt cgatgctcaccctgttgtttggtgttacttctgcaggtcgactctagaggatc

Claims

CLAIMS:

1. A method of increasing at least one of yield and/or abiotic stress tolerance in a plant, the method comprising increasing the expression or activity of AG02 (argonaute member protein 2) in said plant.

2. The method of claim 1 , wherein said increase is compared to a control or wild- type plant.

3. The method of claim 1 or 2, wherein the method increases yield in a plant.

4. The method of any preceding claim, wherein the yield is grain yield.

5. The method of any preceding claim wherein the abiotic stress tolerance is salt tolerance.

6. The method of any preceding claim, wherein 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.

7. The method of claim 6, wherein, the nucleic acid sequence comprises SEQ ID NO: 2 or 3 or a functional variant or homologue thereof.

8. The method of claim 6, wherein the nucleic acid construct comprises the nucleic acid sequence as defined in SEQ ID NO: 24.

9. The method of claim 6, wherein the regulatory sequence is a promoter, preferably a CaMV 35S promoter.

10. The method of any preceding claim wherein the AG02 polypeptide comprises at least one modification that negatively affects protein function

11. The method of claim 10, wherein the modification comprises at least one peptide tag.

12. The method of any preceding claim, wherein the plant is a crop plant.

13. The method of claim 12, wherein the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.

14. A method for making a transgenic plant having increased yield and/or abiotic stress tolerance, the method 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.

15. The method of claim 14, wherein the nucleic acid construct comprises the nucleic acid sequence as defined in SEQ ID NO: 24.

16. A method for making a transgenic plant having increased yield and/or abiotic stress tolerance, the method 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 target genome editing; or said mutation is 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.

17. The method of claim 16, wherein the mutation is introduced using ZFNs, TALENs or CRISPR/Cas9.

18. The method of any of claims 14 to 17, wherein, the nucleic acid sequence encoding an AG02 polypeptide comprises SEQ ID NO: 2 or 3 or a functional variant or homologue thereof.

19. The method of any of claims 14 to 18, wherein the regulatory sequence is a promoter, preferably a CaMV 35S promoter.

20. The method of any of claims 14 to 19 wherein the AG02 polypeptide comprises at least one modification that affects protein function

21. The method of any of claims 14 to 20, wherein the AG02 polypeptide comprises at least one peptide tag.

22. The method of any of claims 14 to 21 , wherein the plant is a crop plant.

23. The method of claim 22, wherein the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.

24. A plant obtained or obtainable by the method of any of claims 14 to 23.

25. A 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.

26. 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; or wherein the mutation is 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.

27. The transgenic plant of claim 26 wherein said mutation is introduced using ZFNs, TALENs or CRISPR/Cas9.

28. The transgenic plant of claim 25 or 26, wherein the plant is characterised by an increase in yield and/or abiotic stress tolerance compared to a control plant.

29. The transgenic plant of claim 25 or 26, wherein the plant is characterised by an increase in grain yield.

30. The transgenic plant of claim 25 or 26, wherein the plant is characterised by an increase in salt tolerance.

31. The transgenic plant of claim 25 or 26, wherein the regulatory sequence is a promoter, preferably a CaMV 35S promoter.

32. The transgenic plant of any of claims 25 to 31 , wherein the plant is a crop plant.

33. The transgenic plant of claim 32, wherein the plant is selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.

34. 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 homologue thereof wherein said nucleic acid sequence is operably linked to a regulatory sequence, wherein the regulatory sequence is a promoter, preferably a CaMV 35S promoter

35. The nucleic acid construct of claim 34, wherein the nucleic acid construct comprises the nucleic acid sequence as defined in SEQ ID NO: 24.

36. A vector comprising the nucleic acid construct of claims 34 or 35.

37. A host cell comprising the nucleic acid construct of claims 34 or 35 or vector of claim 36.

38. The host cell of claim 37, wherein the cell is a bacterial or plant cell.

39. A transgenic plant expressing the nucleic acid construct of claim 34 or 35 or the vector of claim 33.

40. The transgenic plant of claim 39, wherein the plant is a crop plant, preferably selected from rice, maize, wheat, soybean, barley, brassica, sugarcane, cotton, sorghum, alfalfa and millet.

41. The use of the nucleic acid construct of claim 34 or 35 or the vector of claim 36, to increase yield and/or abiotic stress tolerance in a plant.