High yield crop plants and methods for producing same

Abstract

The present invention relates to crop plants, particularly to plants of the Solanaceae family including tomato (Solarium lycopersicum) plants genetically engineered to have reduced expression and / or activity of SnRK2.3 protein and producing higher yield compared to a corresponding crop plant not engineered to have reduced expression and / or activity of the SnRK2.3 protein.

Description

[0001] HIGH YIELD CROP PLANTS AND METHODS FOR PRODUCING SAME

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to crop plants, particularly to plants of the Solanaceae family including tomato (Solarium lycopersicum) plants genetically engineered to have reduced expression and / or activity of SnRK2.3 protein and producing higher yield compared to corresponding crop plants not engineered to have reduced expression and / or activity of the SnRK2.3 protein.

[0004] BACKGROUND OF THE INVENTION

[0005] Throughout their life cycle, plants are constantly exposed to environmental challenges such as abiotic stress of drought, cold, and high salinity. In response to these challenges, plants employ a variety of defense mechanisms to ensure their survival. The activation of these mechanisms is often regulated by phytohormones that induce gene expression, leading to biochemical and physiological changes that alleviate the stress conditions. The phytohormone abscisic acid (ABA) regulates various plant processes, including seed development and dormancy, the vegetative to reproductive phase transition, and responses to environmental stresses. Under water stress, i.e., drought conditions, increasing levels of ABA reduce the transpiration rate and water loss by inducing stomata closure in the leaves. However, this salvage act reduces the leafs gas exchange, thus restricting photosynthesis.

[0006] ABA is synthesized from the xanthophyll violaxanthin in a pathway that begins in the plastids and completed in the cell cytoplasm. The ABA signaling machinery comprises a family of ABA protein receptors PYRABACTIN RESISTANCE 1 / PYR1- EIKE / REGUEATORY COMPONENT OF ABA RECEPTOR (PYR / PYL / RCAR) belonging to the START protein superfamily, protein phosphatases (PP2Cs), and sucrose non-fermenting 1 (SNF-l)-related kinases (SnRK2s), which activate downstream genes (Cutler S R et al., 2010. Annu. Rev. Plant Biol., 61, 651-79; Umezawa T et al., 2010. Plant and Cell Physiology, 51, 1821-1839). Under non-stressed conditions, PP2C inactivates SnRK2 by dephosphorylation, thus silencing ABA signaling. Once environmental conditions or developmental cues induce ABA biosynthesis, the ABA- bound PYR / PYL / RCAR receptors interact with PP2C and inhibit phosphatase activity. SnRK2 is then released from the PP2C suppression and activates downstream effectors through phosphorylation (Fig. 1). These effectors include transcription factors, such as ABRE BINDING proteins / ABRE-BINDING FACTORS (AREB / ABFs), which are suggested to be part of the ABA core- signaling pathway, and membrane proteins such as ion channels essential for ABA-mediated stomatai closure in response to water-deficit stress.

[0007] SnRK2s family members have been discovered to regulate abiotic stress response via the abscisic acid (AB A) -independent and dependent signaling pathways (Chong E et al., 2022. Stress Biology 2, 44). It has been shown that the tomato ortholog to the kinase Open Stomata 1 (S1OST1, Solyc01gl08280), positively regulates drought tolerance and promotes flowering under drought stress conditions. Mutants produced using CRISPR / Cas9 editing targeted to the first and second exons of S1OST1 showed reduced drought tolerance, late flowering under both normal and drought conditions, and smaller plant size compared to the corresponding wild type plants (Chong L et al., 2022. The Plant Cell, 34, 2001-2018). Ei et al. showed that SlSnRK2.3 interacts with S1SUI1 to modulate high temperature tolerance via ABA- controlling stomatai movement in tomato (Li Y et al., 2022. Plant Science, 321, 111305). In watermelon, it was suggested that ClSnRK2.3 might be a negative regulator in fruit ripening (Wang J et al., 2023. Journal of Integrative Biology, 65(10), 2336-2348).

[0008] There is a great need and it would be highly beneficial to have crop plants having increased fruit yield. Furthermore, due to public concerns about the use of transgenic plants, there is a great and unmet need for high-yield crop plants that are not classified as transgenic plants.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention answers the above-described needs, providing crop plants, particularly plants of the Solanaceae family including tomato (Solarium lycopersicum) plants genetically engineered to have reduced expression and / or activity of SnRK2.3 protein and elevated yield compared to corresponding plants having non-engineered expression of the SnRK2.3 protein. The present invention is based in part on the unexpected finding that tomato plants expressing inactive SnRK2.3 protein truncated, and optimally also modified at its carboxyl (C)-terminus produced higher fruit yield compared to control tomato plants expressing the intact protein, particularly when the plants are grown under customary irrigation practice of commercially grown tomato plants. The truncated protein is a result of a mutation inserted within the seventh exon of the SnRK2.3 encoding gene using gene editing techniques, with two independent mutations showing equivalent results. The high- yield production of the inactive-protein harboring tomato plants is highly unexpected, as hitherto published finding described a negative effect of null- or reduced expression mutants of SnRK2.3 on plant size, flowering time, and abiotic stress tolerance.

[0011] According to certain aspects, the present invention provides a crop plant comprising at least one genetically engineered cell having a reduced expression and / or activity of an SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1 compared to the protein expression and / or activity in a corresponding non-engineered cell, wherein the yield of the crop plant is higher compared to the yield of a control plant grown under the same conditions.

[0012] According to certain embodiments, the SnRK2.3 protein is encoded by SnRK2.3 gene, the SnRK2.3 gene comprising a nucleic acid sequence having at least 70% identity to the nucleic acid sequence set forth in SEQ ID NO:2.

[0013] According to certain embodiments, the SnRK2.3 protein is encoded by a nucleic acid sequence having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO: 3 or a part thereof.

[0014] According to some embodiments, the SnRK2.3 protein is encoded by a nucleic acid sequence having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:4.

[0015] According to certain embodiments, the genetically engineered cell comprises within its genome homozygous mutant alleles of the SnRK2.3 encoding gene, wherein the mutant alleles encode an SnRK2.3 protein exhibiting reduced activity.

[0016] Any mutation as is known in the art for inserting a mutation into a plant gene can be used according to the teachings of the present invention, as long as the mutation results in reduced function of the encoded SnRK2.3 protein. The mutation may interfere in the transcription, translation, expression, and / or activity of the protein. The mutation can be a deletion, an insertion, a site specific mutation and the like.

[0017] According to certain embodiments, the mutant allele comprises a mutation within the seventh exon of the SnRK2.3 encoding gene. According to certain embodiments, the mutation is a site-specific mutation.

[0018] According to certain embodiments, the site-specific mutation is an insertion mutation, resulting in a pre-mature stop codon. According to certain additional or alternative embodiments, the insertion mutation results in a frame shift and in a premature stop codon.

[0019] According to certain embodiments, the mutant allele comprising the site specific insertion mutation encodes a truncated SnRK2.3 protein.

[0020] According to certain embodiments, the encoded truncated protein is lacking the last 100 to 192 amino acid residues at the carboxyl-terminus of SEQ ID NO:1.

[0021] According to certain embodiments the encoded truncated protein is lacking the last 100-110, 100-120, 100-130, 100-150, 100-160, 100-170, 100-180, 100-180, or 100-199 amino acid residues at the carboxyl terminus of SEQ ID NO: 1. Each possibility represents a separate embodiment of the present invention.

[0022] According to certain embodiments, the insertion mutation is insertion of Guanine (G) at position 1818 of the SnRK2.3 encoding gene having SEQ ID NO:2. According to certain embodiments, the mutant allele comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO:5, and having the nucleic acid sequence AGAATAGTGA at positions 1811-1820. According to certain exemplary embodiments, the mutant allele comprises the nucleic acid sequence set forth in SEQ ID NO:5.

[0023] According to certain embodiments, the mutant genomic allele results in the insertion of Guanine (G) at position 962 of the SnRK2.3 transcribed mRNA having the nucleic acid sequence set forth in SEQ ID NOG. According to certain embodiments, the mutant transcribed mRNA comprises a nucleic acid sequence having at least 75% identity to SEQ ID NO:6 and having the nucleic acid sequence AGTGACGGAA at positions 961- 970. According to certain exemplary embodiments the mutant transcribed mRNA comprises the nucleic acid sequence set forth in SEQ ID NO:6. According to certain embodiments, the coding sequence of the mutant truncated protein has insertion of Guanin (G) at position 585 of SEQ ID NO:4. According to certain embodiments, the coding sequence of the mutant truncated protein comprises the nucleic acid sequence set forth in SEQ ID NO:21.

[0024] According to certain embodiments, the encoded mutant truncated protein comprises an amino acid sequence having at least 90% identity to amino acids 1-194 of SEQ ID NO:1, wherein said mutant protein is missing amino acids 195-362 of SEQ ID NO:1. According to certain embodiments, the encoded mutant truncated protein comprises the amino acid sequence set forth in SEQ ID NO:9. Said protein is designated herein mutant SnRK2.3-C or ASnRK2.3-C.

[0025] According to certain additional or alternative embodiments, the insertion mutation is an insertion of Thymine (T) at position 1741 of the SnRK2.3 encoding gene having SEQ ID NO:2. According to certain embodiments, the mutant allele comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO:7, and having the nucleic acid sequence TTGTTGCATT at positions 1741-1750. According to certain exemplary embodiments, the mutant allele comprises the nucleic acid sequence set forth in SEQ ID NO:7.

[0026] According to certain embodiments, the mutant genomic allele results in the insertion of Guanine (G) at position 886 of the SnRK2.3 transcribed mRNA. According to certain embodiments, the mutant transcribed mRNA comprises a nucleic acid sequence having at least 75% identity to SEQ ID NO:8 and having the nucleic acid sequence CTCGGTTGTT at positions 881-890. According to certain exemplary embodiments, the mutant transcribed mRNA comprises the nucleic acid sequence set forth in SEQ ID NO:8.

[0027] According to certain embodiments, the coding sequence of the mutant truncated protein has insertion of Thymine (T) at position 509 of SEQ ID NO:4. According to certain embodiments, the coding sequence of the mutant truncated protein comprises the nucleic acid sequence set forth in SEQ ID NO:22. According to certain embodiments, the encoded mutant truncated protein comprises an amino acid sequence having at least 90% identity to amino acids 1-170 of SEQ ID NO:1, wherein said mutant protein is missing amino acids 176-362 of SEQ ID NO:1. According to certain embodiments, the encoded mutant truncated protein comprises the amino acid sequence set forth in SEQ ID NO: 10, said protein is designated herein mutant SnRK2.3-C or ASnRK2.3-D.

[0028] According to certain embodiments, the site-specific mutation is inserted by a geneediting method using artificially engineered nucleases.

[0029] According to certain embodiments, the artificially engineered nucleases are selected from the group consisting of meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs), and CRISPR / Cas, CRISPR / Cas homologous and CRISPR / Cas modified systems.

[0030] Insertion of site-specific mutations, particularly using gene-editing system, has the advantage of designing mutagenesis tools that do not have off-target effects.

[0031] Thus, according to certain exemplary embodiments, the crop plants of the invention having an increased yield are obtained by inserting a mutation within allele of the SnRK2.3 encoding gene using CRISPR / Cas system.

[0032] Since most genome-editing techniques can leave behind minimal traces of DNA alterations evident in a small number of nucleotides as compared to transgenic plants, crop plants created through gene editing could avoid the stringent regulation procedures commonly associated with genetically modified (GM) crop development, and are typically defined as non-transgenic crop plants.

[0033] According to certain embodiments, the yield of said crop plant is at least 5% higher compared to the yield of the control plant.

[0034] According to certain embodiments, the control plant does not comprise the at least one genetically engineered cell having a reduced expression and / or activity of an SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1, and is of said crop plant species. It is to be explicitly understood that the control plant according to the teachings of the present invention may have gone through genetic engineering alteration(s), as long as the control plant comprises unmodified SnRK2.3 encoding gene.

[0035] According to certain embodiments, the control plant is a plant comprising a wildtype SnRK2.3 protein.

[0036] According to certain embodiments, the crop plant of the present invention and the control plant are grown under irrigation conditions as are known to be suitable for the crop plant.

[0037] According to certain embodiments, the crop plant is selected from the group consisting of a field crop plant, including cereal plant, and an ornamental plant.

[0038] According to certain embodiments, the crop plant is of the family Solanaceae. According to some embodiments, the Solanaceae plant is selected from the group consisting of tomato (Solanum lycopersicuni), eggplant (Solanum melongend), potato (Solanum tuberosum), tobacco (Nicotiana tabacum), pepper (Capsicum annum), and tomatillo (Physalis philadelphica and Physalis ixocarpa). Each possibility represents a separate embodiment of the present invention.

[0039] According to certain exemplary embodiments, the crop plant is tomato (Solanum lycopersicum). According to these embodiments, the yield of the tomato plant is tomato fruit. A higher yield shall mean higher number of fruits per plant, the average mass of the fruits is about the same average mass of the fruits of a control plant, higher average mass of the fruits compared to the average mass of the fruit in a control plant having about the same number of fruits, or a combination of more fruits per plant and higher mass per fruit.

[0040] According to certain embodiments, the tomato plant is a commercial cultivar plant. According to additional or alternative embodiments, the tomato cultivar is an elite cultivar. According to certain embodiments, the tomato plant has a determinate growth habit.

[0041] The present invention further provides seeds as well as cells and tissue cultures derived therefrom, wherein crop plants grown from said seeds or regenerated from said tissue culture each comprises at least one genetically engineered cell having a reduced expression and / or activity of an SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 compared to the protein expression and / or activity in a corresponding non-engineered cell, wherein the yield of the plant is higher compared to the yield of a control plant grown under the same conditions.

[0042] According to further certain aspects, the present invention provides a method for producing a crop plant having an enhanced yield production, the method comprises genetically engineering at least one cell of the crop plant to have reduced expression and / or activity of SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1 compared to a nonengineered cell, thereby producing a crop plant having an enhanced yield production compared to a control plant.

[0043] The SnRK2.3 protein and polynucleotides encoding same are as described herein above.

[0044] According to certain embodiments, the method comprises introducing at least one mutation in the plant endogenous allele of SnRK2.3 encoding gene wherein the mutation confers reduced activity of the encoded SnRK2.3 protein.

[0045] The at least one mutation can be any mutation as is known in the art and described hereinabove, including an insertion, a deletion, and a combination thereof.

[0046] According to certain embodiments, the mutation is inserted into the seventh exon of the SnRK2.3 encoding gene. According to certain exemplary embodiments, the mutation within seventh exon of the SnRK2.3 encoding gene results in a C-terminus truncated encoded protein as described hereinabove.

[0047] According to certain embodiment, the at least one mutation is introduced by genome editing using at least one artificially engineered nuclease. According to certain embodiments, the artificially engineered nuclease is selected from the group consisting of meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR / Cas systems, including CRISPR / Cas homologous and CRISPR / Cas modified systems.

[0048] According to certain exemplary embodiments, the at least one mutation is introduced by genome editing using the CRISPR / Cas9 system.

[0049] According to certain embodiments, the control plant is of the same species and does not comprise the at least one genetically engineered cell.

[0050] According to certain embodiment, the crop plant produced by the methods of the present invention produces at least 5% higher yield compared to the yield produced by the control plant.

[0051] The crop plant species are as described hereinabove.

[0052] It is to be understood that any combination of each of the aspects and the embodiments disclosed herein is explicitly encompassed within the disclosure of the present invention.

[0053] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0054] BRIEF DESCRIPTION OF THE FIGURES

[0055] FIG. 1 is a schematic description of the core ABA signal transduction pathway in plants. PYR / PYL / RCAR, ABA receptors; PP2C, type 2C protein phosphatase; SnRK2, SNF1- related protein kinase 2.

[0056] FIG. 2 is a schematic description of the final Binary plasmid vector used to edit the SnRK2.3 gene in tomato. The map indicates the site of two guide-RNAs, the Cas9- encoding gene and details on the components between the right and left borders comprising the T-DNA sequence.

[0057] FIG. 3 shows the exon-intron structure of the gene for SlSnRK2.3 (Solyc01gl08280) on Chromosome #1 (sgn.cornell.edu / feature / 17707754 / details). Arrow indicates the direction of transcription.

[0058] FIG. 4 shows alignment of the amino acid sequence of SnRK2.3 protein in the wild type tomato (line M82, SEQ ID NO:1) and isogenic mutations ASnRK2.3-C (SEQ ID NO:9) and ASnRK2.3-D (SEQ ID NO: 10).

[0059] FIG. 5 shows water loss over time in detached leaves from the SnRK2 mutant plants compared with the control plants (M82) and the ABA deficiency mutant hp3 plant. Detached leaflets were weighed and placed on plates at the 28°C. Data shown are means ± SE (n=3).

[0060] FIG. 6 shows the relative water content (RWC) of leaves (RWC, %) of the three SnRK2 mutant plants (SnRK2.2, SnRK2.3, SnRK2.4) and wild type plant under 15 days of control regular watering (right panel) and under drought stress conditions. The data represent mean values ± SE (n=8).

[0061] FIG. 7 demonstrates stomatai aperture in leaf epidermis from M82 (WT), sitiens, ASnRK2.3 and ASnRK2.4 plants in response to ABA treatment. The stomatai pore ratio was measured after incubation for 2 h in a buffer containing either no treatment or with 10 pM ABA. Data are means ± SE of triplicate experiments with at least 50 stomata per genotype and per treatment. Student's t-test, *P< 0.05.

[0062] FIG. 8 shows the stomatai conductance (gsw) of ASnRK2.3 and M82 plants in the field, measured using Li-600. Plants were grown under irrigated, wet conditions (left) or nonirrigated, dry conditions (right). Compared with WT, the phenotypic differences were statistically significant with Student's t-test, **P< 0.001.

[0063] FIG. 9 shows the transpiration rate of ASnRK2.3-D, ASnRK2.4, and M82 plants grown in the greenhouse under a controlled environment. Plants were grown on the lysimeter system in a greenhouse with a period of full irrigation. Transpiration rate measurement of ASnRK2.3-D, ASnRK2.4, and M82 plants were taken at 8:00 (left) and 13:00 (right). Statistical analysis performed with ANOVA: Ordered difference between ASnRK2.3 and ASnRK2.4 and ASnRK2.3 and M82, P<0.001.

[0064] FIG. 10 shows the fruit yield of wild type (M82) and mutant (ASnRK2.3-C) plants grown in Akko, 2019, under irrigation conditions. Data represent average +SE, p-value =052431.

[0065] FIG. 11 shows the fruit yield of wild type (M82) and mutants (ASnRK2.3-C and ASnRK2.3-D) plants grown in Akko, 2021, under irrigation conditions. Data represent average +SE, *** SnRK2.3D p <0.001.

[0066] FIG. 12 shows the fruit yield in randomly planted mutants (ASnRK2.3-C and ASnRK2.3- D) and the WT (M82) plants under irrigated and non-irrigated growth conditions. ( +SE, a, p-value 0.099957 (not significant); b, p>0.05; c, p-value = 0.107559 (non-significant); d, p-value < 0.01.

[0067] DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention provides crop plants which show high yield production, particularly Solanaceae plants including tomato (Solarium lycopersicum) plants having high fruit yield. The high yield is attributed to a reduced function of the ABA-receptor sucrose non-fermenting 1 (SNF-l)-related kinase SnRK2.3. Exemplified in tomato, plants comprising mutated SnRK2.3 gene encoding a truncated form of the SnRK2.3 protein missing C-terminal amino acids showed higher yield compared to tomato plants comprising intact protein.

[0069] Since SnRK2.3 homologs in a variety of plant species (SnRK2.3 orthologs) are conserved, reducing the expression and / or activity of SlSnRK2.3 or orthologs thereof according to the teachings of the present invention may lead to improved yield in a variety of crop plant species, particularly crop plants of the Solanaceae family.

[0070] Definitions

[0071] The terms “comprise”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0072] The term “consisting of’ means “including and limited to”.

[0073] The term “consisting essentially of’ means that the composition, method, or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0074] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a part” with reference to a polynucleotide may include a plurality of polynucleotide parts, including mixtures thereof.

[0075] The term “about” as used herein refers to a numeric value ± 10%.

[0076] As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of "and / or" and not the exclusive sense of "either / or."

[0077] The term “plant” is used herein in its broadest sense. It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a root, stem, shoot, leaf, flower, petal, fruit, etc.

[0078] As used herein, the term “crop plant” refers to a plant with at least one part having commercial value. The term encompasses plants producing edible fruit (including vegetables), plants producing grains (as a food, feed and for oil production), plant producing flowers and ornamental plants, legumes, root crops, tuber crops, leafy crops, and the like. According to certain exemplary embodiments the crop plant is a tomato plant. According to certain exemplary embodiments, the tomato plant is Solarium lycopersicum crop plant. The term “crop plant” further denote a plant having a biological status other than a “wild” status, which “wild” status indicates the original non-cultivated or natural state of a plant or accession. The term “crop plant” (for cultivated plants) includes, but is not limited to, semi-natural, semi-wild, traditional cultivar, landrace, breeding material, research material, breeder's line, synthetic population, hybrid, founder stock / base population, inbred line (parent of hybrid cultivar), segregating population, mutant / genetic stock, and advanced / improved cultivar. The term as used herein includes registered as well as non-registered lines. According to certain embodiments, the crop plants of the present invention are elite cultivar plants, particularly tomato elite cultivars.

[0079] According to certain exemplary embodiments of the present invention, the tomato plant has a determinate growth pattern. As used herein, the terms “determinate growth pattern” or “determinate growth habit” of a tomato plant refers to tomato plant characterized by a genetically programmed growth habit in which the main stem and all lateral branches terminate in a flower cluster after a specific number of nodes. The cessation of vegetative growth results in a compact, bush-like plant structure, with the majority of the fruit ripening over a relatively short period. Commercial determinate tomato plant varieties are typically processing tomato varieties.

[0080] As used herein the term “yield” with reference to a crop plant of the present invention, refers to the amount (e.g., as determined by weight or size) or quantity (numbers) of tissues or organs to be harvested which are produced per plant and / or per growing season. According to certain embodiments, the harvested organ is a fruit. An increased yield could affect the economic benefit one can obtain from the plant in a certain growing area and / or growing time. According to certain embodiments, the yield is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40% or more.

[0081] The term “SnRK2.3 protein” refers to a sucrose non-fermenting 1 (SNF-l)-related kinase. The terms “SnRK2.3 protein” and ^'SnRK2' .3 gene” encompass the tomato (Solarium lycopersicum SlSnRK2.3) protein / gene Solyc01gl08280.2 and orthologs thereof. SlSnRK2.3 protein Solyc01gl08280.2 comprises the amino acid sequence set forth in SEQ OD NO:1.

[0082] The terms “SnRK2.3 mutant” “SnRK2.3 mutant”, “ASnRK2.3”, and their plural forms are used herein interchangeably and refer to SnRK2.3 protein having reduced activity, encoded by SnRK2.3 gene or a part thereof comprising at least one mutation.

[0083] As used herein, the terms “activity” and “function” with reference to the SnRK2.3 protein or mutants thereof refer to the functional activity of the wild type SnRK2.3 protein as is known in the art. According to certain embodiments, the wild type SnRK2.3 protein is activated by ABA and positively regulates ABA signaling related to abiotic stress responses and developmental regulation.

[0084] The term “homozygous” as is used herein, means a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes.

[0085] As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid 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. Sequences which differ by such conservative substitutions are considered to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG. (Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 89(22), 10915-9, 1992).

[0086] Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN, BlastX or Blastp software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

[0087] According to some embodiments of the invention, the identity is a global identity, i.e., an identity over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof.

[0088] According to some embodiments of the invention, the term “homology” or “homologous” refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences; or the identity of an amino acid sequence to one or more nucleic acid sequence.

[0089] The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of RNA or a polypeptide. A polypeptide can be encoded by a full-length coding sequence or by any part thereof. The term “parts thereof’ when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, “a nucleic acid sequence comprising at least a part of a gene” may comprise fragments of the gene or the entire gene.

[0090] The term “gene” also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences.

[0091] The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”", and "isolated polynucleotide" are used interchangeably herein. These terms encompass isolated nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA or hybrid thereof, that is single- or double-stranded, linear or branched, and that optionally contains synthetic, non-natural or altered nucleotide bases. The terms also encompass RNA / DNA hybrids.

[0092] According to certain aspects, the present invention provides a crop plant comprising at least one genetically engineered cell having a reduced expression and / or activity of an SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1 compared to the protein expression and / or activity in a corresponding non-engineered cell, wherein the yield of the crop plant is higher compared to the yield of a control plant grown under the same conditions.

[0093] As used herein, the expression and / or activity of SnRK2.3 is “reduced”, “inhibited”, “down regulated” or "knocked down" if the level of the protein or its measured activity is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, %, at least 95%, at least 96% at least 97%, at least 98%, at least 99%, or more compared to its level in a control plant or compared to a predetermined threshold level. According to certain embodiments, the aforementioned terms do not include 100% inhibition or “loss of function” or “null function” protein.

[0094] As used herein, the term “control plant” refers to a plant comprising within its genome a gene encoding SnRK2.3 protein having at least 90% identity to SEQ ID NO:1, wherein the expression of the SnRK2.3 has not been artificially modified. It is to be explicitly understood that the control plant can comprise other modifications, for example modified expression and / or activity of proteins other that SnRK2.3. According to certain embodiments, the control plant is of the same species. As exemplified hereinbelow, two independent mutant alleles of SnRK2.3 gene were generated and analyzed, and all plants harboring either of the mutant alleles displayed the increased yield phenotype. According to certain embodiments, the control plant is a corresponding plant comprising wild-type SnRK2.3 protein.

[0095] According to certain embodiments, the non-modified SnRK2.3 protein comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to the amino acid sequence set forth in SEQ ID NO:1. Each possibility represents a separate embodiment of the present invention.

[0096] According to certain embodiments, the non-modified SnRK2.3 protein is encoded by an SnRK2.3 gene having at least 70%, at least 75%, least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to the nucleic acid sequence set forth in SEQ ID NO:2. Each possibility represents a separate embodiment of the present invention.

[0097] According to certain embodiments, the non-modified SnRK2.3 protein is encoded by a nucleic acid sequence having at least 75%, least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to the nucleic acid sequence set forth in SEQ ID NO:3. Each possibility represents a separate embodiment of the present invention.

[0098] According to some embodiments, the SnRK2.3 protein is encoded by a nucleic acid sequence having at least 75% identity, least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to the nucleic acid sequence set forth in SEQ ID NO:4. Each possibility represents a separate embodiment of the present invention.

[0099] According to certain embodiments, the genetically engineered cell comprises within its genome homozygous mutant alleles of the SnRK2.3 encoding gene, wherein the mutant alleles result in reduced function of the encoded SnRK2.3 mutant protein.

[0100] Any mutation(s) can be inserted into a gene / polynucleotide encoding SnRK2.3, including deletions, insertions, site specific mutations including nucleotide substitution and the like, as long as the mutation(s) result in down-regulation of the gene expression or in the production of less- functional protein.

[0101] As exemplified hereinbelow, the present invention demonstrates that tomato plants expressing SnRK2.3 protein truncated at its C-terminus, and grown in the field under common agricultural conditions produced higher fruit yield compared to corresponding tomato plants expressing the wild-type SnRK2.3 protein grown under the same conditions. The C-terminus truncation is a result of an insertion mutation leading the formation of a pre-mature stop codon, and optionally to a frame shift leading to a modified amino acid sequence compared to the wild type before the truncation point. Without wishing to be bound by any specific theory or mechanism of action, the truncation at the protein C-terminus may be a gain-of-function mutation leading to a protein that performs a new biological function or acts independently of its normal regulation within the plant cell.

[0102] According to certain embodiments, the C-terminus truncation is a result of a mutation within the 7thexon of the SnRK2.3 gene having the nucleic acid sequence set forth in SEQ ID NO:2 or of an ortholog thereof.

[0103] According to certain embodiments, the encoded truncated protein is missing the C- terminal 100 to 192 amino acid residues of SEQ ID NO:1.

[0104] According to certain embodiments the encoded truncated protein is missing the C- terminal 100-110, 100-120, 100-130, 100-150, 100-160, 100-170, 100-180, 100-180, or 100-199 amino acid residues of SEQ ID NO:1. Each possibility represents a separate embodiment of the present invention.

[0105] According to certain embodiments, the insertion mutation is insertion of Guanine (G) at position 1818 of the SnRK2.3 encoding gene having SEQ ID NO:2, before Guanine (G) at position 1818 of said SEQ ID NO:2 (genomic mutation 1818 insertion G).

[0106] According to certain embodiments, the mutant allele comprises a nucleic acid sequence having at least 70%, at least 75%, least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to SEQ ID NO:5, and having the nucleic acid sequence AGAATAGTGA at positions 1811-1820. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the mutant allele comprises the nucleic acid sequence set forth in SEQ ID NO:5.

[0107] According to certain embodiments, the mutant genomic allele results in the insertion of Guanine (G) at position 962 of the SnRK2.3 transcribed mRNA. According to certain embodiments, the mutant transcribed mRNA comprises a nucleic acid sequence having at least 75%, least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to SEQ ID NO:6 and having the nucleic acid sequence AGTGACGGAA at positions 961-970. According to certain exemplary embodiments the mutant transcribed mRNA comprises the nucleic acid sequence set forth in SEQ ID NO:6. Each possibility represents a separate embodiment of the present invention.

[0108] According to certain embodiments, the coding sequence of the mutant truncated protein has insertion of Guanin (G) at position 585 of SEQ ID NO:4. According to certain embodiments, the coding sequence of the mutant truncated protein comprises the nucleic acid sequence set forth in SEQ ID NO:21. According to certain embodiments, the mutant protein comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to amino acids 1-194 of SEQ ID NO:1, wherein said mutant protein is missing amino acids 195-362 of SEQ ID NO:1. Each possibility represents a separate embodiment of the present invention.

[0109] According to certain embodiments, the mutant protein comprises the amino acid sequence set forth in SEQ ID NO:9. Said protein is designated herein mutant SnRK2.3- C or ASnRK2.3-C.

[0110] According to certain additional or alternative embodiments, the insertion mutation is insertion of Thymine (T) at position 1741 of the SnRK2.3 encoding gene having SEQ ID NO:2, before Thymine (T) at position 1741 of said SEQ ID NO:2 (genomic mutation 1741 insertion T). According to certain embodiments, the mutant allele comprises a nucleic acid sequence having at least 70%, at least 75%, least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to SEQ ID NO:7, and having the nucleic acid sequence TTGTTGCATT at positions 1741-1750. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the mutant allele comprises the nucleic acid sequence set forth in SEQ ID NO:7.

[0111] According to certain embodiments, the mutant genomic allele results in the insertion of Guanine (G) at position 886 of the SnRK2.3 transcribed mRNA. According to certain embodiments, the mutant transcribed mRNA comprises a nucleic acid sequence having at least 75%, least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to SEQ ID NO:8 and having the nucleic acid sequence CTCGGTTGTT at positions 881-890. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the mutant transcribed mRNA comprises the nucleic acid sequence set forth in SEQ ID NO:8.

[0112] According to certain embodiments, the coding sequence of the mutant truncated protein has insertion of Thymine (T) at position 509 of SEQ ID NO:4. According to certain embodiments, the coding sequence of the mutant truncated protein comprises the nucleic acid sequence set forth in SEQ ID NO:22.

[0113] The T insertion not only results in the formation of a pre-mature stop codon within the coding sequence, but also in a frame shift leading to a modification of the amino acids at positions 171-176 of SEQ ID NO:1 from Leucine-Histidine-Serine- Glutamine-Proline (LHSQP) to Valine -Alanine-Phenylalanine-Threonine-Threonine (VAFTT).

[0114] According to certain currently exemplary embodiments, the mutant protein comprises an amino acid sequence having at least 90% identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or is identical to amino acids 1-170 of SEQ ID NO:1, wherein said mutant protein is missing amino acids 176-362 of SEQ ID NO: 1 and comprises the amino acids VAFTT at positions 171-175. Each possibility represents a separate embodiment of the present invention.

[0115] According to certain embodiments, the mutant protein comprises the amino acid sequence set forth in SEQ ID NO: 10. Said protein is designated herein mutant SnRK2.3- D or ASnRK2.3-D.

[0116] According to further certain aspects, the present invention provides a method for producing a crop plant having an enhanced yield production, the method comprises genetically engineering at least one cell of the crop plant to have reduced expression and / or activity of SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1 compared to a nonengineered cell, thereby producing a crop plant having an enhanced yield production compared to a control plant.

[0117] The SnRK2.3 protein and polynucleotides encoding same are as described hereinabove.

[0118] According to certain embodiments, the method comprises introducing at least one mutation in the plant endogenous allele of SnRK2.3 encoding gene wherein the mutation confers reduced activity of the encoded SnRK2.3 protein.

[0119] Any method for mutagenesis as is known in the art can be used according to the teachings of the present invention including chemical mutagenesis, radio-mutagenesis and site directed mutagenesis, for example using genome editing techniques. According to certain embodiments, the plants of the present invention are produced by inserting a mutation within the SnRK2.3 gene using the CRISPR / Cas system, a CRISPR / Cas homologous and CRISPR / Cas modified systems.

[0120] The CRISPR / Cas system for genome editing contains two distinct components: a gRNA (guide RNA) and an endonuclease e.g., Cas9.

[0121] The gRNA is typically a 20-nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA / Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the gRNA / Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break. Comparable with other genome editing nucleases, Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the double- stranded brakes produced by CRISPR / Cas can undergo homologous recombination or nonhomologous end-joining (NHEJ).

[0122] The Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks in the genomic DNA.

[0123] A significant advantage of CRISPR / Cas is that the high efficiency of this system coupled with the ability to easily create synthetic gRNAs enables multiple genes to be targeted simultaneously. In addition, the majority of cells carrying the mutation present bi-allelic mutations in the targeted genes.

[0124] However, apparent flexibility in the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cut by Cas9.

[0125] Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’. With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or 'nick'. A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a doublestrand break, in what is often referred to as a 'double nick' CRISPR system. A doublenick can be repaired by either NHEJ or homology directed repair (HDR) depending on the desired effect on the gene target. Thus, if specificity and reduced off-target effects are crucial, using the Cas9 nickase to create a double-nick by designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA would decrease off-target effect as either gRNA alone will result in nicks that will not change the genomic DNA.

[0126] Modified versions of the Cas9 enzyme containing two inactive catalytic domains (dead Cas9, or dCas9) have no nuclease activity while still able to bind to DNA based on gRNA specificity. The dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains. For example, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.

[0127] There are number of publicly available tools to help choose and / or design target sequences as well as lists of bioinformatically determined unique gRNAs for different genes in different species such as the Feng Zhang lab's Target Finder, the Michael Boutros lab's Target Finder (E-CRISP), the RGEN Tools: Cas-OFFinder, the CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes and the CRISPR Optimal Target Finder.

[0128] In order to use the CRISPR system, both gRNA and Cas9 should be expressed in a target cell. The insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids.

[0129] According to certain embodiments, the crop plant is selected from the group consisting of a field crop plant including a cereal plant, and an ornamental plant.

[0130] According to certain embodiments, the field crop plant is of the family Solanaceae. According to some embodiments, the Solanaceae plant is selected from the group consisting of tomato (Solanum lycopersicuni), eggplant (Solanum melongend), potato (Solanum tuberosum) tobacco (Nicotiana tabacum), pepper (Capsicum annum), and tomatillo (Physalis philadelphica and Physalis ixocarpa).

[0131] According to certain exemplary embodiments, the crop plant is tomato (Solanum lycopersicum). According to these embodiments, the yield of the tomato plant is tomato fruit.

[0132] According to certain embodiments, the tomato plant is a commercial cultivar plant. According to additional or alternative embodiments, the tomato cultivar is an elite cultivar. According to certain embodiments, the tomato plant has a determinate growth pattern.

[0133] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention. EXAMPLES

[0134] Materials and Methods

[0135] Tomato lines

[0136] The tomato (Solarium lycopersicum) line M82 was used in this work for all the transformations and as the control. For water loss in detached leaves the high-pigment 3 (hp3) mutant line (el472) was used (Galpaz N et al., 2008. The Plant Journal, 53, 717- 730). hp3 is known for an ABA-deficient phenotype, i.e., it is a control for an impaired ABA signaling.

[0137] For the genetic engineering assays, plants were grown year-round in the Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem greenhouse in four-liter pots with commercial soil (Shaham, Givat Ada, Israel). Plants were tested at 12 weeks. Field trial with plants harboring the SnRK2.3 mutations of the present invention were grown as described in Example 4 hereinbelow.

[0138] Bacteria strains

[0139] Escherichia coli XL1 Blue strains were used for DNA cloning and plasmid preparations. E.coli was grown in Luria Bertani (LB) medium in suspension or agar plates at 37°C.

[0140] Agrobacterium tumefaciens GV3101 strain was used for DNA transformation of tomato following standard protocols (Sharma M K et al., 2009. J Biosci., 34, 423-433).

[0141] CRISPR-Cas9 editing of SnRK2 genes

[0142] Vector construction

[0143] The tomato genome contains eight SnRK2 genes (SOL Genomic Network, www.sgn.cornell.edu). For the SnRK2.3 gene, two gRNAs designed using the CRISPR- P v2.0 Web Tool (crispr.hzau.edu. cn / CRISPR2) were used (Table 1). The CRISPR / Cas9 knockout construct was built using the Golden Gate cloning platform (Werner S et al., 2012. Bioeng. Bugs., 3, 38-43). The plasmids used for constructing the vector are described in Table 2. The cloning protocol was previously described (Brooks C et al., 2014. Plant Physiology, 166, 1292-1297). The final pIAGM4723 binary vector contained a gene for plant kanamycin resistance, the SpCas9 coding domain sequence, and two guide RNA recognition sequences, designed using the CRISPR-P v2.0 Web Tool (crispr.hzau.edu. cn / CRISPR2 / ), and left and right borders of the T-DNA for Agrobacterium-mediated transformation (Figure 2).

[0144] Table 1: Primers for constructing SnRK2 binary plasmids Table 2. Plasmids used for building the pAGM4723 binary vector for gene editing.

[0145] Transformation

[0146] The transformation of tomato lines with the designed constructs was carried out using a protocol developed by Gupta and Van Eck, 2016 (Gupta S and Van Eck J, 2016. Plant Cell, Tissue and Organ Culture (PCTOC), 127, 417-423) with some modifications. The final binary vector was transfected into competent Agrobacterium tumefaciens strain GV3101 via electrophoresis and stored in a 50% glycerol stock at -80°C. Seeds of the desired tomato lines for transformation were sterilized in 6% sodium hypochlorite, 70% ethanol, and sterile ddH2O and germinated on Magenta dishes containing 50 mL Nitsch medium (N0223, Duchefa Biochemie). After 10 days, the cotyledons were removed from the seedlings and placed on Feeder plates containing Murashige and Skoog (MS) Medium, including Vitamins (M0222, Duchefa Biochemie) with aceto syringone for two days until transformation. Following transformation through exposure to suspension- culture-grown Agrobacterium cells for 15 minutes at room temperature, the cotyledons were placed adaxially on new Feeder plates. After 48 hours, the leaves were taken and placed abaxially on SL-1 selection plates with Jones medium (M0256, Duchefa Biochemie), 400 mg / L ticarillin, 1 mg / L zeatin, 100 mg / L kanamycin, and 0.7% plant agar. After two weeks on SL-1 plates, the cotyledons were transferred to SL-2 selection plates (Jones medium with 250 mg / L ticarillin, 100 mg / L kanamycin, 1 mg / L zeatin, 0.5 mg / L zeatin riboside, 0.1 mg / L IAA, and 0.8% agar) until callus formation. The calli were then placed on SL-3 selection plates (Jones medium with 250 mg / L ticarillin, 70 mg / L kanamycin, 0.15 mg / L zeatin, 0.1 mg / L IAA, 0.1 mg / L IB A, and 0.8% agar) until shoot apex formation. After shoot apexes were observed protruding from the calli, they were transferred to Magenta plates containing 50 mL rooting medium (Nitsch medium with 150 mg / L ticarillin, 50 mg / L kanamycin, and 2 mg / L IB A) until primary root formation and apical shoot maturation. After approximately two weeks in the rooting medium, the regenerated plants were transferred to a transparent container containing water, wet soil, and humid conditions for another two weeks before moving to a four-liter pot with commercial potting soil in the greenhouse. The transgenic plants were genotyped through PCR using primers specific for the integrated T-DNA via sequencing.

[0147] DNA extraction, sequencing, and genotyping of individuals

[0148] DNA was extracted from cotyledons and young leaves ground using a BeadBeater (2x for 15 seconds at maximum speed) in 300 pL 2x CTAB buffer (2% cetyl trimethylammonium bromide, 1% polyvinyl pyrrolidone, 100 mM Tris-HCl, 1.4 M NaCl, and 20 mM EDTA). The DNA was then separated from the ground leaf tissue using 300 pL chloroform and cleaned using 300 pL isopropanol and 500 pL 70% ethanol. The genomic DNA, dissolved in 120 pL sterile ddH2O, was stored at -20°C. The primers for the analysis are collected and presented in Table 3. All DNA sequencing was performed by the Genomic Center of the Silberman Institute in Givat Ram, The Hebrew University of Jerusalem, Israel, using an ABI Prism® 377 DNA sequencer (Perkin Elmer; www.perkinelmer.com). SnapGene® Viewer software (GSL Biotech LLC) was used to analyze the sequence histogram.

[0149] Table 3: Primers for genotyping the transgenic events

[0150] Stomatai conductance in field-grown mutant plants using Li-600

[0151] The stomatai conductance (gsw) was measured using a porometer / fluorometer instrument (Portable Photosynthesis System LL600, Licor, USA) in at least three leaves from three different plants of each genotype.

[0152] Water loss in detached leaves

[0153] The rate of water loss in detached leaflets was measured for well-watered plants of M82 (wild-type), SnRK2 mutants, and hp3 plants. Leaflets were detached, placed on open petri dishes at room temperature, and weighed using an analytical scale once every hour. The water loss (%) was calculated: net weight (time = x) - net weight (time = 0)

[0154] %Aw (time =X) = - X 100 net weight (time = 0)

[0155] Relative water content (RWC)

[0156] M82 (wild-type) and mutant plants were grown under two conditions: a dry state in which the plants stopped being watered for 15 days and a normal state in which the plants were grown under continuous irrigation conditions. One leaflet from the first fully expanded leaves of eight plants per line (mutant or M82) was cut from a plant on days 0, 5, 13, and 15. The fresh weight (FW) of the leaf was measured immediately after cutting. Then, the leaflet was immersed in ddbhO and incubated at room temperature. After five hours, the leaf was taken out and weighed to obtain turgid weight (TW). Afterward, the leaf was put in a drying oven at 60°C for 72 hours and weighed to determine the dry weight (DW). Relative water content was calculated: (RWC in %) = [(FW - DW) / (TW - DW)] * 100 (Barrs H D and Weatherley P E, 1962. Aust. J. Biol. Sci., 15, 413-428).

[0157] Stomatai

[0158] The stomatai aperture was determined by following an established protocol (Hassidim et al., 2017. Plant Physiology, 175, 1864-1877). Leaves from each plant (three plants from each genotype) were blended with 40 mL of opening solution in a Waring blender (Waring Commercial). Treatment with an “opening solution” that contains 48 mM KC1, 0.1 mM CaC12, and 5 mM MES, pH 6.1, was performed as a control treatment. The blended material was filtered on a 100pm nylon mesh, the epidermal sheets were collected and mixed with 600 pL of the opening solution, and 300 mL of the mixture was transferred to a microscope slide.

[0159] Leaf samples were collected two hours after sunrise and measured under a bright- field microscope (Nikon Eclipse E 200) every two hours. Samples were taken and measured six hours after sunrise for the control, opening solution, and ABA treatments. For the ABA treatments, after blending and filtering, the plant material was incubated with 60 pL of 10 mM ABA for 120 minutes at room temperature in the light before imaging. Photos of at least 50 stomata from at least five epidermal fragments were taken and analyzed in the ImageJ program (Schneider C A et al., 2012. Nat. Methods, 9, 671- 675). The ratios between the pore's width and the guard cells' length were calculated for each stoma and processed using ImageJ, as reported in the literature (Legnaioli T et al., 2009. EMBO Journal, 28, 3745-3757). rate

[0160] Transpiration rate measurements were taken using a high-throughput, telemetric, gravimetric -based phenotyping system (Plantarry 3.0 system; Plant-DiTech, Israel) in the greenhouse of the LCORE Center for Functional Phenotyping (plantscience.agri.huji.ac.il / icore-center), as described in the literature (Gosa S C et al., 2022. Plant Sci., 315:111122. doi:10.1016 / j.plantsci.202L 111122). 16 plants from each genotype, M82, ASnRK2.3, and ASnRK2.4 were taken and grown in the greenhouse for 28 days. Each plant was placed on a highly sensitive and temperature-controlled load cell, used as weighing lysimeters. The data for the transpiration rate was measured and analyzed using the Plantarray system and SPAC- analytics (Plant-Ditech) program. The transpiration rate was calculated as previously described (Attia Z et al., 2015. J Exp Bot., 66, 4373-4381.; Halperin O et al., 2017. The Plant Journal, 89, 839-850).

[0161] Data presentation and statistical analysis

[0162] For statistical analyses, JMP® ver. 14 statistical packages (SAS Institute, Cary, NC, USA) was used. The variance between the genotypes was examined using Tukey HSD. Each analysis used a set significance level of P < 0.05.

[0163] Pairwise Pearson correlations between traits under greenhouse conditions and the yield and its components measured in the open field, i.e., plant vegetative weight, red fruit yield, green fruit yield, Brix of fruit, and total yield, were calculated using the genotype's mean performance.

[0164] The roles of SnRK2 kinases in plants' physiological processes were tested by knocking down / knocking out the SnRK2 genes using the CRISPR / cas9 technique. Each gene was mutated separately, and transgenic homozygous plants were obtained in F2 plants.

[0165] Of these mutated genes, the mutations in the SnRK2.3 kinase gene Solyc01gl08280 (Fig. 3) were selected for further analysis.

[0166] To this end, CRISPR-Cas9 technology was used. Two single-guide RNAs (sgRNAs) targeting the seventh exon of SlSnRK2.3 (Solyc01gl08280) in the M82 line were designed. The Cas9 construct was transfected into M82 using Agrobacterium- mediated transformation. After screening tomato transformants, two mutant alleles in the M82 background were isolated, each of the mutants harboring a single bp insertion: ASnRK2.3-C on guide-2 and ASnRK2.3-D on guide-1 at the target sequence (Table 4). These mutations caused an early stop codon in the translated protein and thus created inactive SnRK2.3 proteins (Figure 4). Table 4: SnRK2.3 mutations

[0167] A series of crosses and screening was performed to isolate plants with homozygous mutations in the SnRK2.3 gene that do not contain the T-DNA sequence with the CRISPR / Cas9 constructs in the genome. Transformed plants of the To generation were identified by sequencing a PCR-amplified DNA of the SnRK2.3. T1 plants bearing the mutation were crossed with the wild-type line, M82. Plants from the F2 generation were screened to identify homozygous SnRK2.3 mutated gene plants that lost the transgenic T-DNA sequence and carrying the Cas9-gRNA construct.

[0168] The identification of these plants (F2 generation) was performed using PCR sequencing of the Cas9 sequence and the endogenous gene SnRK2. Plants in the F3 generation that did not contain the transgenic CDS of SpCas9, kanamycin resistance gene, and the right border region, but carried various mutations in the SnRK2 gene were cultivated in the field in Acre, Israel.

[0169] The field tests were performed over three seasons. Detailed quantitative trait analysis was conducted in the field: M82 and ASnRK2.3 mutants were grown in Acre and Newe Ya' ar (Israel) fields under irrigation or semi-dry conditions. Field-grown plants at the 80% fruit ripening stage were harvested, and their overall biomass, average fruit size, total fruit yield per plant, number of seeds, and Brix, were measured. Samples of red fruit pericarp were taken for carotenoid analysis using HPLC-DAD.

[0170] Example 2: Effect of the SnRK2.3 mutant gene on water-stress related phenomena

[0171] The ability of the ASnRK2.3 plants to cope with stress conditions was tested by the effectiveness of stomatai closure in detached leaves. This test records the subsequent weight loss rate due to transpiration. The leaves from most SnRK2 knockdown mutants exhibited weight loss at a rate similar to the rate observed for leaves from the wild type (M82) plants. As present in Figure 5, leaves from the SnRK2.3 mutant exhibited significantly faster weight loss than the other mutants (after one hour, SnRK2.3 lost ten percent more weight than the M82 plant). Moreover, the SnRK2.3 mutant lost weight faster than the hp3 mutant line. The reduced stomatai closure in hp3 leaflets is a characteristic of ABA deficiency. Without wishing to be bound by any specific theory or mechanism of action, the weight loss in SnRK2.3 mutants may be attributed to the lack / reduction of SnRK2.3 kinase activity in ABA signal transduction.

[0172] Leaf Relative Water content (RWC) is a reliable indicator of plant water status, reflecting the balance between water supply to the leaf tissue and transpiration rate. In this test, WT and mutant plants were grown under two conditions: a dry state in which the plants were not watered for 15 days and a normal state in which the plants were grown under normal irrigation conditions. Under regular irrigation, all the plants (WT and mutant) maintained constant values throughout the experiment (RWC value between 80- 95%) (Figure 6, right panel). After four days under dry conditions, it can be seen that ASnRK2.2 and ASnRK2.4 mutants exhibit the highest RWC and maintain a stable water balance, while ASnRK2.3 exhibits the lowest RWC (Figure 6 left panel). Without wishing to be bound by ant specific theory or mechanism of action, these results may indicate that SnRK2.3 is the primary kinase involved in drought tolerance.

[0173] Example 3: SnRK2.3 regulation of stomatai closure

[0174] The involvement of SnRK2.3 in stomatai closure was tested in WT, ASnRK2.3-D, and other mutants impaired in ABA signaling. Sitiens leaves were used to test the effect of exogenic ABA, since this tomato mutant lacks ABA. Leaves were harvested two hours after sunrise. Stomata opening was measured immediately and treated with an “opening solution” (see Materials and Methods hereinabove) that enhances opening, or with ABA to induce closure. After treatment with opening solution alone, there was no significant difference in stomatai aperture among sitiens, ASnRK2.3-D, 2sSnRK2.4, and wild type (M82). Treatment with ABA showed a significant response (*p<0.005, Student's T-test) on M82, sitiens and 2sSnRK2.4 plants. By contrast, ASnRK2.3-D leaves showed no response to ABA treatment (Figure 7).

[0175] The involvement of the SnRK2.3 kinase in stomatai closure was also measured by Stomatai conductance (gsw) in the leaves of plants grown under different conditions in the field using the Li-600 Porometer / Fluorometer. For plants grown in the irrigated, wet part of the field, no difference between the WT (M82) plants and ASnRK2.3-D mutant plants was observed, while in plants grown in the non-irrigated, dry field, where there is a lack of water, the Stomatai conductance (gsw) in WT leaves was reduced, meaning the plants preferred to close the stomata (Figure 8). On the other hand, in dry conditions, the stomatai conductance (gsw) that was measured on ASnRK2.3-D leaves was higher than the WT leaves. Without wishing to be bound by ant specific theory or mechanism of action, this result may show that SnRK2.3 kinase is responsible for the stomata closure in guard cells.

[0176] Water transpiration from leaves is a function of stomatai opening. Transpiration rate was measured as described in Materials and Methods hereinabove. As shown in Figure 9, the morning transpiration is low and increases in mid-day. The results indicate that the transpiration in ASnRK2.3-D leaves is higher compared to the transpiration from WT (M82) and ASnRK2.4 plants, particularly at mid-day.

[0177] Example 4: Fruit yield in SnRK2.3 mutations (alleles C and D)

[0178] Detailed quantitative trait analyses were performed in field-grown plants. WT and mutants ASnRK2.3-C and ASnRK2.3-D plants were grown in Acre (Akko) and Newe Ya' ar under full or semi-dry conditions (irrigation during the first three weeks after planting only). Fruit were harvested when the ripening rate was >80%.

[0179] As is clearly demonstrated in Figures 10-12, unexpectedly, fruit yield of tomato plants harboring the SnRK2.3 mutant was higher compared to wild type plants, with ASnRK2.3-D plants showing the highest yield over all seasons examined (2019, 2021 and 2023). Figure 12 further shows that the high-yield phenomenon is more pronounced when the plants are grown under regular irrigation (wet) conditions.

[0180] Example 5: Snrk2.3 protein and encoding sequences (Table 5)

[0181] Table 5

[0182] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

CLAIMS1. A crop plant comprising at least one genetically engineered cell having a reduced expression and / or activity of an SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 compared to the protein expression and / or activity in a corresponding nonengineered cell, wherein the yield of the crop plant is higher compared to the yield of a control plant grown under the same conditions.

2. The crop plant of claim 1, wherein the SnRK2.3 protein is encoded by SnRK2.3 gene, the SnRK2.3 gene comprising a nucleic acid sequence having at least 70% identity to the nucleic acid sequence set forth in SEQ ID NO:2.

3. The crop plant of any one of the preceding claims wherein the SnRK2.3 protein is encoded by a nucleic acid sequence having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:3 or a part thereof.

4. The crop plant of any one of the preceding claims wherein the SnRK2.3 protein is encoded by a nucleic acid sequence having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:4.

5. The crop plant of any one of the preceding claims, wherein the genetically engineered cell comprises within its genome homozygous mutant alleles of the SnRK2.3 encoding gene, wherein the mutant allele encodes an SnRK2.3 mutant protein exhibiting reduced activity.

6. The crop plant of claim 5, wherein the mutant allele comprises at least one mutation in the seventh exon of the SnRK2.3 encoding gene.

7. The crop plant of claim 6, wherein the at least one mutation results in a premature stop codon, thereby forming a mutant allele encoding a truncated SnRK2.3 protein.

8. The crop plant of claim 7, wherein the encoded truncated protein is lacking the last 100 to 199 amino acid residues at the carboxyl-terminus of SEQ ID NO:1.

9. The crop plant of any one of claims 7-8, wherein the mutant allele comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO:5, and having the nucleic acid sequence AGAATAGTGA at positions 1811-1820 of SEQ IDN0:5.

10. The crop plant of any one of claims 7-9, wherein the mutant allele is transcribed to a mRNA comprising a nucleic acid sequence having at least 75% identity to SEQ ID NO:6 and having the nucleic acid sequence AGTGACGGAA at positions 961-970 of SEQ ID NO:6.

11. The crop plant of any one of claims 7-10, wherein the encoded truncated SnRK2.3 protein comprises an amino acid sequence having at least 90% identity to amino acids 1-194 of SEQ ID NO:1, wherein said truncated protein is lacking amino acids 195-362 of SEQ ID NO:1.

12. The crop plant of claim 11, wherein the encoded truncated SnRK2.3 mutant protein comprises the amino acid sequence set forth in SEQ ID NO:9.

13. The crop plant of any one of claims 7-8, wherein the mutant allele comprises a nucleic acid sequence having at least 70% identity to SEQ ID NO:7, and having the nucleic acid sequence TTGTTGCATT at positions 1741-1750 of SEQ ID NO:7.

14. The crop plant of claim 13, wherein the mutant allele is transcribed to mRNA comprising a nucleic acid sequence having at least 75% identity to SEQ ID NO:8 and having the nucleic acid sequence CTCGGTTGTT at positions 881-890 of SEQ ID NO:8.

15. The crop plant of any one of claims 13-14, wherein the encoded truncated SnRK2.3 protein comprises an amino acid sequence having at least 90% identity to amino acids 1-170 of SEQ ID NO:1, wherein said truncated protein is lacking amino acids 176-362 of SEQ ID NO:1.

16. The crop plant of claim 15, wherein the encoded truncated SnRK2.3 protein comprises the amino acid sequence set forth in SEQ ID NO: 10.

17. The crop plant of any one of claims 5-16, wherein the mutation is a site-specific mutation.

18. The crop plant of claim 17, wherein the site-specific mutation is inserted by a gene-editing method using at least one artificially engineered nuclease.

19. The crop plant of claim 18, wherein said plant is produced by inserting a mutation within the at least one allele of the SnRK2.3 encoding gene using CRISPR / Cas system, CRISPR / Cas homologous system, or a modified CRISPR / Cas system.

20. The crop plant of any one of the preceding claims, wherein the yield of said crop plant is at least 5% higher compared to the yield of the control plant.

21. The crop plant of any one of the preceding claims, wherein the control plant does not comprise the at least one genetically engineered cell and is of said crop plant species.

22. The crop plant of any one of the preceding claims wherein said plant and the control plant are grown under irrigation conditions.

23. The crop plant of any one of the preceding claims, wherein said crop plant is selected from the group consisting of a field crop plant and an ornamental plant.

24. The crop plant of claim 23, wherein said crop plant is of the family Solanaceae.

25. The crop plant of claim 24, wherein said plant is selected from the group consisting of tomato (Solanum lycopersicum), eggplant (Solarium melongend), potato (Solarium tuberosum) tobacco (Nicotiana tabacum), pepper (Capsicum annum), and tomatillo (Physalis philadelphica and Physalis ixocarpa).

26. The crop plant of any one of the preceding claims, wherein said crop plant is tomato (Solanum lycopersicum).

1. The crop plant of claim 26, wherein the tomato (Solanum lycopersicum) plant exhibits a determinate growth habit.

28. The crop plant of any one of claims 26-27, wherein said crop plant yield is fruit yield.

29. A seed of the crop plant of any one of the preceding claims, wherein a plant grown from the seed comprises at least one genetically engineered cell having a reduced expression and / or activity of an SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1 compared to the protein expression and / or activity in a correspondingnon-engineered cell, wherein the yield of the grown plant is higher compared to the yield of a control plant grown under the same conditions.

30. A tissue culture comprising at least one genetically engineered cell of the crop plant of any one of claims 1-28, wherein a plant regenerated from the tissue culture comprises at least one genetically engineered cell having a reduced expression and / or activity of an SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 compared to the protein expression and / or activity in a corresponding nonengineered cell, wherein the yield of the regenerated plant is higher compared to the yield of a control plant grown under the same conditions.

31. A plant grown from the seed of claim 29 or regenerated from the tissue culture of claim 30.

32. A method for producing a crop plant having an enhanced yield production, the method comprises genetically engineering at least one cell of the crop plant to have reduced expression and / or activity of SnRK2.3 protein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1 compared to a non-engineered cell, thereby producing a crop plant having an enhanced yield production compared to a control plant.

33. The method of claim 32, wherein the SnRK2.3 protein is encoded by SnRK2.3 gene or a part thereof, the SnRK2.3 gene comprising a nucleic acid sequence having at least 70% identity to the nucleic acid sequence set forth in SEQ ID NO:2.

34. The method of any one of claims 32-33, wherein the SnRK2.3 protein is encoded by a nucleic acid sequence having at least 75% identity to the nucleic acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

35. The method of any one of claims 32-34, wherein said method comprises introducing at least one mutation in the plant endogenous allele of the SnRK2.3 encoding gene, wherein the mutation confers reduced function of the encoded SnRK2.3 protein.

36. The method of claim 35, wherein the at least one mutation is selected from thegroup consisting of an insertion, a deletion, and a combination thereof.

37. The method of any one of claims 35-36, wherein the mutation is inserted into the seventh exon of the SnRK2.3 encoding gene.

38. The method of any one of claims 35-37, wherein the mutation is a site-specific mutation.

39. The method of any one of claims 35-38, wherein said method comprises introducing the mutation by genome editing using at least one artificially engineered nuclease.

40. The method of claim 39, wherein the artificially engineered nuclease is selected from the group consisting of meganuclease, Zinc finger nuclease (ZFN), transcription-activator like effector nuclease (TALEN), CRISPR / Cas system, CRISPR / Cas homologous system and CRISPR / Cas modified system.

41. The method of claim 40, wherein the mutation is introduced by genome editing using a CRISPR / Cas system.

42. The method of any one of claims 35-41, wherein the control plant is of the same species and does not comprise the at least one genetically engineered cell.

43. A crop plant produced by the method of any one of claims 35-42, wherein said crop plant produces at least 5% higher yield compared to the control plant.

44. The crop plant of claim 43, wherein said plant is selected from the group consisting of a field crop plant and an ornamental plant.

45. The crop plant of claim 44, wherein said plant is of the family Solanaceae.

46. The crop plant of claim 45, wherein said plant is selected from the group consisting of tomato (Solanum lycopersicum), eggplant (Solarium melongend), potato (Solarium tuberosum) tobacco (Nicotiana tabacum), pepper (Capsicum annum), and tomatillo (Physalis philadelphica and Physalis ixocarpa).Al. The crop plant of claim 46, wherein said plant is tomato (Solanum lycopersicum).

48. The crop plant of claim 47, wherein the tomato (Solanum lycopersicum) plant exhibits a determinate growth habit.

Images