Parthenocarpic plants comprising a loss of function mutation in MBP3 and at least one of AGL6 and MBP22 and methods of producing same

Abstract

A Solanaceous plant exhibiting a parthenocarpy and down-regulation in activity or expression of MBP3 and at least one of AGL6 and MBP22, as compared to a control plant, wherein the control plant is of the same genetic background and developmental stage. Also provided are methods of producing the plant and uses thereof.

Description

[0001] PARTHENOCARPIC PLANTS COMPRISING A LOSS OF FUNCTION MUTATION IN MBPS AND AT LEAST ONE OF AGL6 AND MBP22 AND METHODS OF PRODUCING SAME

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 714,208 filed on October 31, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING STATEMENT

[0005] The XML file, entitled 105577. xml, created on October 30, 2025, comprising 126,976 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to parthenocarpic plants comprising a loss of function mutation in MBPS and at least one of AGL6 and MBP22 and methods of producing same.

[0008] In flowering plants, fruit development is critical for the completion of their life cycle because the biological function of fruiting is the production and dissemination of seeds. Fruits develop from various floral tissue(s) that expand, and "true" fruits, such as tomato (Solatium ly coper sicum), originate from the flower ovary that contains one or more ovules (seed precursors) (Gillaspy et al., 1993). The ovules are the site for sexual plant reproduction where megagametogenesis, fertilization and embryogenesis take place. At anthesis, the mature tomato ovule consists of a female gametophyte (embryo sac) that is enclosed by a single maternal integument containing 6-7 cell layers of which, the innermost one is differentiated into a typical endothelium. The funiculus, a stalk-like maternal structure, anchors the ovule to the placenta (Cooper, 1931). In tomato, the unpollinated ovary, which develops in concert with the rest of the floral organs, enters a temporary growth-arrest phase 1-2 days before anthesis (Gillaspy et al., 1993). It is well established that fruit set, the developmental switch that turns a quiescent ovary into a rapidly growing fruit, depends on successful flower pollination and ovule fertilization (Seymour et al., 2013). In contrast, in the absence of fertilization, ethylene is produced, promoting ovary senescence and flower abscission (Llop-Tous et al., 2000; Carbonell-Bejerano et al., 2010). This scenario implies that a fertilized ovule emits a signal that induces the quiescent ovary to set a fruit.

[0009] Among the phytohormones identified as playing a role in the transition from arrested ovary to a growing fruit, several lines of evidence singled out auxin and gibberellins (GAs) as prominent positive regulators of fruit set, with auxin operating both independent and upstream to GA (Serrani et al., 2008; Vriezen et al., 2008; de Jong et al., 2009; Molesini et al., 2020; Fenn and Giovannoni, 2021). In tomato, following double fertilization, auxin levels increase in the ovules and in the ovary inducing active GA accumulation in the ovary. The increased auxin and GA levels in the ovary activate corresponding signaling pathways that in turn trigger ovary growth (z.e., fruit set) by regulating cell division and cell expansion (Fenn and Giovannoni, 2021). Based on the demonstrated ability of Auxin Response Factors (ARFs), Auxin / Indole-3 -Acetic Acid (Aux / IAA) and DELLA proteins to physically interact, a cross-talk between the auxin and GA pathways has been suggested (Hu et al., 2018). This crosstalk was proposed to involve the gaseous hormone ethylene acting to prevent GA perception and tomato fruit set (Shinozaki et al., 2018a). However, the exact regulatory mechanisms translating these hormonal signals to tomato ovary arrest before anthesis, and its release in response to fertilization or auxin application remain unclear (Joldersma and Liu, 2018; Fenn and Giovannoni, 2021).

[0010] However, fruit set sometimes occurs in an alternative, fertilization-independent pathway referred to as parthenocarpy, which means 'virgin fruiting’ in Greek (Joldersma and Liu, 2018). Previously it was shown that the tomato AGAMOUS-Like6 (SIAGL6) MADS-box transcription factor (TF) loss-of-function mutant produces seeded as well as parthenocarpic fruits with wildtype characteristics (Klap et al., 2017). And a similar phenotype was later reported for the agl6 / Pat-k mutant (Takisawa et al., 2018). The expression of S1AGL6 peaks in the anthesis flower ovary and declines in the set fruit (Klap et al., 2017), as well as, in preanthesis ovaries triggered to set fruit either by auxin and GA application (Tang et al., 2015; Hu et al., 2018) or by silencing their signaling inhibitors AUX / IAA9 (Wang et al., 2009) and S1DELLA (Shinozaki et al., 2020), respectively. Within the developing ovary, S1AGL6 is predominantly expressed in the immature ovule integument, and upon ovule maturation, its expression shifts to the endothelium. Consistent with this, unfertilized slagl6CR'sglmutant ovules are enlarged due to integument over-proliferation and lack a characteristic endothelium, the integument’s innermost layer. RNA-Seq analysis of unfertilized slagl6CR'sglovules transcriptome indicated that it profoundly differs from that of wildtype ovules and underwent vast reprogramming that mimics the transcriptional changes occurring in wild-type ovules following fertilization. In particular, genes that were found to preferentially express in the mature unfertilized ovule were downregulated and those that were preferentially express in 4 days post anthesis (DPA) fertilized ovule integument and funiculus were upregulated (Gupta et al., 2021). These studies suggest that S1AGL6 acts from within the ovule integument as a switch that, unless turned off, prevents fruit set by suppressing fertilization-associated transcriptional reprogramming.

[0011] The SIAGL6 gene encodes a MIKCctype II MADS-box TF, and as such it is formed by the MADS (M), intervening (I), keratin-like (K) and C-terminal domains (Kaufmann et al., 2005a). Seed plants MIKCcgroup proteins function as master developmental regulators by repressing or activating target genes (Smaczniak et al., 2012a; Schilling et al., 2018). This is accomplished by binding through the highly conserved MADS domain, as homo- or heterodimers, to the DNA- binding motif CArG-box (CC(A / T)6GG) (Aerts et al., 2018), or as tetramers formed by complexing of two dimers attached to two spaced CArG-box motifs (TheiBen et al., 2016). The K domain acts as a determinant of oligomerization strength and specificity, playing roles in both dimerization and tetramerization (Kaufmann et al., 2005a). It was suggested that the petunia AGL6, which is highly similar to S1AGL6, has a SEPALLATA (SEP)-like function in floral patterning (Rijpkema et al., 2009). Namely it functions like an E-class protein which, according to the ABCDE and the floral quartet models, participates and serves as central protein- protein interaction hub in the various tetrameric complexes determining floral organ identity (Kaufmann et al., 2005b; Smaczniak et al., 2012b; TheiBen et al., 2016). Indeed, AGL6 homologs from petunia and other plants showed a conserved and broad spectrum of interactions with other MIKCcMADS-box proteins (Immink et al., 2003; Rijpkema et al., 2009; Dreni and Zhang, 2016a).

[0012] Additional related background art:

[0013] Li et al. 10.21203 / rs.3.rs-519249 / v 1;

[0014] Li et al. (2020) Plant Science 301: 110672;

[0015] WO2017 / 125931;

[0016] Nesi et al. 2002 The Plant Cell 14: 2463-2479. de Folter et al. 2006. The Plant Journal 47: 934-946

[0017] Zhang et al. 2019 Journal of Experimental Botany 70: 909-924.

[0018] Huang et al. 2021 Nature Communications 12: 6892.

[0019] SUMMARY OF THE INVENTION

[0020] According to an aspect of some embodiments of the present invention there is provided a Solanaceous plant exhibiting a parthenocarpy and down-regulation in activity or expression of MBP3 and at least one of AGL6 and MBP22, as compared to a control plant, wherein the control plant is of the same genetic background and developmental stage. According to some embodiments of the invention, the down-regulation is a result of a loss- of function mutations in MBP3 and AGL6.

[0021] According to some embodiments of the invention, the down-regulation is a result of a loss- of function mutations in MBP3 and MBP22.

[0022] According to some embodiments of the invention, the down-regulation is a result of a loss- of function mutations in MBP3, AGL6 and MBP22.

[0023] According to some embodiments of the invention, the plant exhibits fruit yield defined as an average fruit weight that is at least the same as that of the control plant.

[0024] According to some embodiments of the invention, the plant exhibits fruit yield defined as an average number of fruits of the plant per growing season which is at least the same as that of the control plant.

[0025] According to some embodiments of the invention, the plant exhibits fruit yield defined as an average number of fruits of the plant per growing season which is at least 2-fold higher than that of the control plant.

[0026] According to some embodiments of the invention, the plant exhibits fruit yield defined as an average number of fruits of the plant per growing season which is at least 4-fold higher than that of the control plant.

[0027] According to some embodiments of the invention, the plant exhibits fruit yield defined as an average number of fruits of the plant per growing season which is at about 2 to 5 -fold higher than that of the control plant.

[0028] According to an aspect of some embodiments of the present invention there is provided a Solanaceous plant exhibiting a parthenocarpy and down-regulation in activity of AGL6, MBP3 and / or MBP22, as compared to the control Solanaceous plant, wherein the control plant is of the same genetic background and developmental stage, wherein the down-regulation in activity is manifested by aberrant interaction between either AGL6-MBP3, AGL6-MBP22, MBP3-MBP22, and wherein the plant is not a tomato plant which comprise a C to t substitution at exon 3 that generate a stop codon after amino acid residue Tyr89.

[0029] According to some embodiments of the invention, the down-regulation is a result of a loss of function mutation in any of MBP3, AGL6 and / or MBP22.

[0030] According to some embodiments of the invention, the loss-of-function mutation is in a K domain of AGL6or MBP22 and optionally MBP3.

[0031] According to some embodiments of the invention, the loss-of-function mutation is a deletion in at least an a2 helix of the K domain, optionally wherein the loss-of-function mutation results in a peptide comprising a MADS domain, an I domain and / or a C domain. According to some embodiments of the invention, the loss-of-function mutation results in a peptide, which comprises an a2 helix but is devoid of any or all of MADS domain, an I domain and a C domain, the peptide serving as a dominant negative peptide.

[0032] According to some embodiments of the invention, the plant is a tomato.

[0033] According to some embodiments of the invention, the plant is a tomato, eggplant or pepper.

[0034] According to some embodiments of the invention, fruit of the tomato comprises a jelly fill.

[0035] According to some embodiments of the invention, the plant is a processing tomato.

[0036] According to some embodiments of the invention, the plant is a determinate tomato.

[0037] According to some embodiments of the invention, the plant is an indeterminate tomato.

[0038] According to some embodiments of the invention, the plant is a semi-determinate tomato.

[0039] According to some embodiments of the invention, the plant is of an elite line.

[0040] According to some embodiments of the invention, the plant is transgenic.

[0041] According to some embodiments of the invention, the tomato is of a species selected from the group consisting of Lycopersicon esculentum, Lycopersicon cerasiforme, Lycopersicon pimpinellifolium, Lycopersicon cheesmanii, Lycopersicon parviflorum, Lycopersicon chmielewskii, Lycopersicon hirsutum, Lycopersicon penellii, Lycopersicon peruvianum, Lycopersicon chilense and Solanum lycopersicoides.

[0042] According to some embodiments of the invention, the tomato is selected from the group consisting of a single fruit per truss, branched tomato and cherry tomato.

[0043] According to some embodiments of the invention, the parthenocarpy is facultative parthenocarpy optionally manifested under heat or cold stress.

[0044] According to some embodiments of the invention, the plant is homozygous for a mutation or mutations conferring the down-regulation in activity or expression of MBP3 and at least one of AGL6 and MBP22.

[0045] According to some embodiments of the invention, the plant is heterozygous for a mutation or mutations conferring the down-regulation in activity or expression of MBP3 and / or MBP22.

[0046] According to some embodiments of the invention, the plant is AGL6 / MBP3+ or AGL6 / MBP22+.

[0047] According to some embodiments of the invention, the plant is an inbred.

[0048] According to some embodiments of the invention, the plant comprises a silencing agent for down-regulation in activity or expression of MBP3 and at least one of AGL6 and MBP22.

[0049] According to some embodiments of the invention, the plant exogenously expresses a nuclease selected from the group consisting of a meganuclease, an RNA-guided DNA endonuclease, a zinc-finger nuclease and a TALEN. According to an aspect of some embodiments of the present invention there is provided a fruit of the plant as described herein.

[0050] According to an aspect of some embodiments of the present invention there is provided a seed of the plant as described herein.

[0051] According to an aspect of some embodiments of the present invention there is provided a hybrid seed produced of the plant as described herein.

[0052] According to an aspect of some embodiments of the present invention there is provided an edible processed product of the plant or fruit as described herein.

[0053] According to some embodiments of the invention, the processed product is selected from the group consisting of a tomato paste, a ketchup, a tomato sauce a tomato soup, a tomato juice, a tomato powder, a tomato dice, a crushed tomato, a chopped tomato and a tomato concentrate.

[0054] According to an aspect of some embodiments of the present invention there is provided a method of producing a processed product, the method comprising processing the fruit as described herein to produce an edible processed product.

[0055] According to an aspect of some embodiments of the present invention there is provided a method of producing the plant as described herein, the method comprising down-regulating expression or activity of MBP3 gene and at least one of AGL6 and MBP22 in the plant.

[0056] According to some embodiments of the invention, the down-regulating MBP3 gene and at least one of AGL6 and MBP22 is in the same plant.

[0057] According to some embodiments of the invention, the down-regulating MBP3 gene and at least one of AGL6 and MBP22 is in different plants and further crossing to result in a plant where MBP3 gene and at least one of AGL6 and MBP22 are down-regulated.

[0058] According to some embodiments of the invention, the down-regulating is effected by treating the plant with an DNA editing agent.

[0059] According to an aspect of some embodiments of the present invention there is provided a method of breeding comprising selfing or crossing the plant as described herein.

[0060] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0061] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0062] In the drawings:

[0063] FIGs. 1A-F show complementation of the slagl6CR'sglmutant. (A) Schematic of the S1AGL6 genomic region (top) and the binary constructs introduced into the slagl6CR'sglmutant (bottom). The length of the S1AGL6 promoter (pS!AGL6) fragment used in each construct is specified. The full-length SIAGL6 gene (gSlAGL6). SIAGL6 coding sequence (SIAGL6) and pDefH9 promoter used were modified for GoldenBraid cloning. Light grey and dark grey boxes represent the S1AGL6 and Nos terminators, respectively. (B) PCR validation of slagl6CR~sglpSlAGL6(4Kb)::gSlAGL6 (pSlAGL6(4Kb)), slagl6CR-sglpSlAGL6(6Kb)::gSlAGL6 (pSlAGL6(6Kb)) and slagl6CR-sglpDefH9::SlAGL6-5.4 (pDefH9 -5.4) transgenic plants using specific primers targeting gSlAGL6 (1), SIAGL6 (2, 7),PSIAGL6 (4, 5), pDefH9 (6), and pNOS (3), as shown in (A). Wild-type MP-1 and slagl6CR~sglserved as controls. (C) Images of flowers one day post-anthesis, alongside scanning electron micrographs of the adaxial side of their petals (bottom). Scale bars: 1 cm (flowers), 20 pm (SEM). (D) Cross-sections of representative ovules from stage 18 ovaries. Scale bars = 20 pm. ov, ovule; et, endothelium; es, embryo sac. (E) Images of red fruits and their cross-sections (bottom). Only seedless fruit is displayed for the slagl6CR'sglmutant. (F) Quantitation of transgenic S1AGL6 expression in slagl6CR~sglpDefH9: :SlAGL6-5.4 (pDefH9 -5.4) stage 18 ovaries (Ovary), isolated ovules (Ovules) and ovary tissues excluding ovules (Non-ovule) by RT-qPCR. Error bars represent ± SE over three biological replicates, each containing eight whole ovaries or the indicated ovary tissues. The P-values were calculated using a Student’s t-test.

[0064] FIGs. 2A-D show the characterization of S1AGL6 Y2H interactors. (A) Schemes of S1AGL6 and the proteins identified as its category A interactors in the Y2H screen. Domains are color-coded: MADS-box (yellow), Intermediate (blue), K domain (pink), and C-terminal (black line). Protein lengths and Y2H clone extents are shown on the right and with white dotted lines, respectively. The number of Y2H positive clones for each interactor are indicated in parentheses. (B-C) Expression profiles of indicated interactor gene in anthesis ovary tissues of S. pimpinellifolium (B) and S. lycopersicum cv. M82 (C). Expression data was retrieved from the Tomato Expression Atlas (Shinozaki et al., 2018b). Tissue labels: ov, ovule; pl, placenta; sp, septum; p, pericarp; c, columella; It, locular tissue; tp, total pericarp. (D) Expression levels in 2 days ahead of anthesis (2DAA) Micro-Tom ovaries, 4 days post their treatment at anthesis by artificial pollination (4DPAP), exogenous application of 2,4-D (4DPAT) or GA3 (4DPGT), or 6 days post flower emasculation (6DPE). Expression data was retrieved from (Tang et al., 2015).

[0065] FIGs. 3A-D show validation of S1AGL6 Y2H interactors in planta. (A) BiFC confirmation of protein interactions among S1AGL6 and its Y2H interactors. Fusion proteins of S1AGE6 and its Y2H interactors with either the N (nYFP) or C (cYFP) -terminal halves of eYFP were pairwise- expressed in N. benthamiana leaves. Nuclear localized mCherry (mCherry-NES) was used to locate the nucleus and the split YFP fused nuclear localized transcription factor mSlNAM2 served as a negative control. Images of fluorescent signals alone (as indicated) and merged with visible light (Merged) are shown. Scale bars = 20 pm. (B-C) FRET-FEIM analysis of S1AGE6 with its Y2H interactors. The SlAGE6-mCrFP fusion protein alone or with the eYFP fused Y2H interactor protein were transiently expressed in N. benthamiana leaves. The nuclear localized mCrFP-NES protein alone was transiently expressed to determine the fluorescence lifetime of free mCrFP and the eYFP fused nuclear localized transcription factor mSlNAM2 was used as a negative control. Microscopy was performed 36 h after agro-infiltration using a Eeica Stellaris 8 Falcon FEIM confocal microscope with a white laser. The fluorescence lifetime values of expressed mCrFP proteins (B) were quantified in multiple different cells (n = 10), allowing for the calculation of respective average FRET efficiency (C). Statistical significance was calculated using one way ANOVA, ***P < 0.001. Mean values are indicated by +. (D) BiFC confirmation of protein interactions among indicated proteins. BiFC experiments were conducted by transiently expressing indicated proteins as fusion proteins with either the N- (nYFP) or C- (eYFP) terminal halves of eYFP along with the nuclear marker mCherry-NES, in N. benthamiana leaves. Images of fluorescent signals alone (as indicated) and merged with visible light (Merged) are shown. Scale bars = 20 pm.

[0066] Figures 4A-H. S1AGE6 facilitates S1MBP22 and S1MBP3 multimerization via its K domain a2 helix. Schemes of full length (A) and truncated (E) S1AGE6 domains architectures. The MADS -box (M), I domain (I), K domain (K) and the C domain (C) are shown and last amino acid is indicated. In (A) the names and locations of K domain a helices are shown. An arrow marks the border between a2 N-terminal part (a2N) and a2 C-terminal part (a2C). (B, F) BiFC confirmation of protein interactions among S1MBP3 and S1MBP22 in the presence of S1AGE6 (B) or truncated versions of S1AGE6 (F). Fusion proteins of S1MBP3 and S1MBP22 themselves and each other with either the N (nYFP) or C (eYFP) -terminal halves of eYFP, as indicated, and SlAGE6-mCrFP or indicated truncated SlAGE6-mCrFP were coexpressed in N. benthamiana leaves. Nuclear localized mCherry (mCherry-NES) was used to locate the nucleus and nuclear localized mCrFP (mCrFP-NLS) was used as a negative control. Images of fluorescent signals alone (as indicated) and merged with visible light (Merged) are shown. Scale bars = 20 pm. (C-D, G-H) FRET-FLIM analysis of S1AGL6 or truncated S1AGL6 with S1MBP3 and S1MBP22. The SlAGL6-mCrFP (C-D) or indicated truncated S1AGL6 (G-H) fusion proteins alone or with the eYFP fused S1MBP3, S1MBP22 or both were transiently expressed in N. benthamiana leaves. The nuclear localized mCrFP-NLS protein alone was transiently expressed to determine the fluorescence lifetime of free mCrFP. Microscopy was performed 36 h after agro-infiltration using a Leica Stellaris 8 Falcon FLIM confocal microscope with a white laser. The fluorescence lifetime values of expressed mCrFP proteins (C, G) were quantified in multiple different cells (n = 10), allowing for the calculation of respective average FRET efficiency (D, H). Statistical significance was calculated using one way ANOVA, ***P < 0.001. Mean values are indicated by +.

[0067] FIGs. 4LJ show that the S1AGL6 is an MIKCCMADS-domain transcription factor (MTF). (I) S1AGL6 protein sequence with indicated domains. The predicted K-domain al and a2 helices amino acid residues are highlighted in red and purple, respectively. (J) Predicted 3D structure of S1AGL6 by AlphaFold (accession number: AF-A0A3Q7EKL1-F1). The protein domains and the first residue in each are labeled. The K-domain al and a2 helices and their boundaries are indicated. The a-helices are depicted as spring-like structures, while P-sheets are shown as arrows. The plane axis and color-coded model confidence are also displayed.

[0068] FIGs. 5A-C show CRISPR / Cas9-mediated knockout of SIMBP3 and SIMBP22. (A) Schematic representation of the SIMBP3 and SIMBP22 gene structures and gRNAs target sequences. Black boxes represent exons, lines indicate introns, and red sequences mark protospacer adjacent motifs (PAMs). Deleted gene regions are marked by brackets. (B) Aligned nucleotide sequences of the CRISPR mutant alleles slmbp22CR'20and slmbp3CR'20with their corresponding wild-type sequences. The gRNA target sequences are highlighted in red, and indicates omitted nucleotides. A denotes the size of deletions. (C) Schematic of SIAGL6, SIMBP22, and SIMBP3 gene regions showing CRISPR / Cas9-induced deletions (top; marked by brackets) alongside representative PCR genotyping of the mutants (bottom). For genotyping, two primers were used for slagl6CR'sgland three primers for slmbp22CR'20and slmbp3CR'20. Wild-type (MP-1), heterozygous, and homozygous alleles appear as a single upper band, double bands, and a single lower band, respectively. Half-arrowheads indicate primer locations and numbers correspond to their sequences in Table 1. M, DNA marker; NTC, non-template control.

[0069] FIGs. 6A-F show the characterization of SIAGL6, SIMBP22 and SIMBP3 CRISPR single and double mutants fruits. (A) Images of representative isolated anthesis flower pistils of slagl6CR~sgl(6), slmbp22CR~20(22), slmbp3CR'20(3) and their double mutant combinations. Scale bars = 5 mm. (B) Images of seeded (MP-1, 6 / +, 22 / +, 3 / +, 3 6 / +, 3, 3 221+) and seedless (other genotypes) fruits and their cross-sections (C). For each parthenocarpic genotype except 3 22, cross sections of representative seedless (top) and seeded (bottom) fruits are shown. For the 322 genotype crosssections of seedless fruits are shown. Asterisks mark typical seeds. Scale bars = 2 cm. (D-F) Mean rate (%) of seedless fruits per plant (D), number of fruits per plant (E) and fruit weight (F) of indicated genotypes. Error bars indicate + SE over seven independent plants. Different letters indicate statistically significant differences as determined by Tukey-Kramer multiple comparison test (P<0.01).

[0070] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0071] The present invention, in some embodiments thereof, relates to parthenocarpic tomato comprising a loss of function mutation in MBP3 and at least one of AGL6 and MBP22 and methods of producing same.

[0072] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0073] Fruit set, the transition from an arrested ovary to a developing fruit, is initiated by ovule fertilization, yet, how the ovary growth arrest is achieved remains poorly understood. Previously, it has been demonstrated that a loss-of-function mutant of the AGAMOUS-LIKE6 (AGL6) gene [e.g., slagl6-2012 loss-of-function mutant slagl6CR~sg (Klap et al., 2017)] develop normally in tomatoes until anthesis, but instead of arresting, they can set normal fruits independent of fertilization (parthenocarpy).

[0074] Whilst conceiving embodiments of the invention and reducing them to practice, the present inventors have uncovered that the S1AGL6, S1MBP22, and S1MBP3 proteins are subunits of floral quartet-like complexes that operate within the ovules to repress ovary growth and prevent parthenocarpy. It is therefore suggested that down-regulation of these proteins will result in synergistic parthenocarpy. In fact the present inventors have shown that down-regulation in activity or expression of MBP3 and at least one of AGL6 and MBP22 results in unprecedented levels of parthenocarpy and enhanced yield.

[0075] As is illustrated hereinbelow and in the Examples section which follows, the present inventors demonstrate that complementing the previously described S1AGL6 loss-of-function mutant slagl6CR'sgl), employing a heterologous ovule- specific promoter, eliminates the parthenocarpic phenotype, confirming that the S1AGL6 MTF suppresses parthenocarpy by acting from within the ovules of the arrested ovary. In order to understand the molecular mechanism underlying this phenomenon, the present inventors screened a yeast two-hybrid library of arrested ovaries with the S1AGL6 protein identified several MTFs as potential interactors. Similar to S1AGL6 and known ovary growth repressors, the expression of the interactors MADS-BOX PROTEIN 22 (S1MBP22) and S1MBP3 peaks in the ovules of the arrested ovary and diminishes in the set fruit. In planta, S1AGL6 forms heterodimers with S1MBP22 or S1MBP3 and remarkably serves as a scaffold allowing them to interact within multimeric complexes. This is the first time where genetic evidence is provided for the contribution of these and of unknown SlMBP3-based complex / es not involving S1AGL6 in parthenocarpy suppression, by showing that differential reduction in S1MBP3, S1MBP22 and S1AGL6 activities, which allows the assembly of all, some or no complexes, leading to variable rates of parthenocarpy. Specifically, although SIMBP3 knockout alone did not induce parthenocarpy, its knockout in slmbp22CR'20and slagl6CR'sglbackgrounds nearly eliminated parthenocarpy suppression, raising the parthenocarpy rate to nearly 100%. This suggests that S1MBP3 functions synergistically with each of S1MBP22 and S1AGL6 in parthenocarpy suppression, and that the S1AGL6, S1MBP3 and S1MBP22 play major roles in this process, as subunits of floral quartet-like complexes that operate within the ovules to repress ovary growth and prevent parthenocarpy.

[0076] These findings can be harnessed towards the development of parthenocarpic Solanaceous plants.

[0077] Thus, according to an aspect of the invention, there is provided a Solanaceous plant exhibiting a parthenocarpy and down-regulation in activity or expression of MBP3 and at least one of AGL6 and MBP22, as compared to a control plant, wherein said control plant is of the same genetic background and developmental stage.

[0078] The term '"plant" as used herein encompasses whole plants, a grafted plant, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots, rootstock, scion, cuttings and plant cells, tissues and organs. The plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.

[0079] According to a specific embodiment, the plant is in a form of a cutting.

[0080] According to another specific embodiment, the plant part is a seed (e.g., a hybrid seed). According to another specific embodiment, the plant part is a fruit.

[0081] According to another specific embodiment, the plant is a plantlet.

[0082] As used herein a “solanaceous plant” refers to a crop plant (domesticated) of this family, including tomatoes, potatoes, eggplant, bell, and peppers. According to a specific embodiment the plant is a tomato plant.

[0083] The tomato plant can be of a cultivated genetic background or a wild (e.g., tomato) genetic background.

[0084] As used herein, the term "tomato" refers to a plant, line or population within the species Solanum lycopersicum (synonyms are Lycopersicon lycopersicum or Lycopersicon esculentum) or formerly known under the genus name of Lycopersicon including but not limited to L. cerasiforme, L. cheesmanii, L. chilense, L. chmielewskii, L. esculentum (now S. pennellii), L. hirsutum, L. parviborum, L. pennellii, L. peruvianum, L. pimpinellifolium, or S. lycopersicoides. The newly proposed scientific name for L. esculentum is S. pennellii. Similarly, the names of the wild species may be altered. L. pennellii has become S. pennellii, L. hirsutum may become S. habrochaites, L. peruvianum may be split into S. 'N peruvianum' and S. 'Callejon de Hueyles', S. peruvianum, and S. comeliomuelleri, L. parviflorum may become S. neorickii, L. chmielewskii may become S. chmielewskii, L. chilense may become S. chilense, L. cheesmaniae may become S. cheesmaniae or S. galapagense, and L. pimpinellifolium may become S. pimpinellifolium.

[0085] Generally a cultivated tomato refers to tomato which is suitable for consumption and meets the requirements for commercial cultivation, e.g. typically classified as Solanum lycopersicum. In addition to the tomato plants themselves, and the parts thereof suitable for consumption, such as the fruit, the invention comprises parts or derivatives of the plant suitable for propagation. Examples of parts suitable for propagation are organ tissues, such as leaves, stems, roots, shoots and the like, protoplasts, somatic embryos, anthers, petioles, cells in culture and the like. Derivatives suitable for propagation are for instance seeds. The plants according to the invention can be cultivated or propagated in the conventional manner but also by means of tissue culture techniques from plant parts.

[0086] The present invention is aimed at using any cultivars (Solanaceous), such as of domestic use or industrial use, which can be for industrial growth but also included are heirloom varieties.

[0087] Specifically, the present invention is aimed at using any tomato cultivars, such as of domestic use, fresh market tomatoes and processing tomatoes.

[0088] As used herein “open-field tomato” refers to tomato grown by commercial growers in direct seeded, large fields - sprawled for machine harvesting and at times artificial ripening off the vine.

[0089] The choice of the variety depends on market demand, regional adaptability, disease resistance and the end use of the product. Exemplary segments for fresh market tomatoes include, but are not limited to, Beef (fruit weight of about 220-400 gr), Standard (fruit weight of about 160- 220 gr) and Cluster (uniform fruit weight of about 120-180 gr). Such varieties are available from major seed companies e.g., Grodena, Macarena, Estatio, Zouk, Climbo and Climstar, all available from Syngenta. Other varieties can be proprietary or available from other vendors, including but not limited to, Cherry-micro (up to 5 gr) round cherry, mini round cherry (7.5-15 gr), mini plum elongated cherry (10-25 gr). Examples for these varieties are: Creative (Clause), Batico (Nirit seeds), Shiren (Hazera Genetics). Cocktail round and elongated (25-40 gr): Romanita, Cherry and Cocktail with red, yellow, orange, pink, zebra, chocolate background. Examples include, but are not limited to, Summer sun (Hazera Genetics), Black pearl (Burpee) Tyty (Tomodori). Roma determinate and indeterminate. 120-200gr. Examples for the intermediate marker include, but are not limited to, lancelot (Vilomorin) and Parsifal (Vilomorin). Pink tomato divided to beef (220- 400), standard (160-220) and cluster (120-180). Example: Momotaro type, Cor di bue tomato, (150-350 gr), Pinton (250-300 gr), open field tomato- determinate or semi-determinate (180- 400gr).

[0090] Exemplary cultivars of processing tomatoes include, but are not limited to, Roma, SUN 6366, AB 2, Heinz 9780, Heinz 9557, Halley 3155 and Hypeel 303.

[0091] There are two major types of tomato growth; determinate and indeterminate. Determinate growth produces "bush" tomatoes and which are bred for compactness. The entire plant stops growing once the terminal fruit ripens, the remainder of the fruit all ripen nearly simultaneously, and then the plant dies. Indeterminate growth produces tomatoes that can grow up to 10 feet in height (so-called "vining" tomatoes) and will only stop growing when killed (e.g. by frost). Their fruits ripen sequentially. In a typical plant, all growth arises from the reiteration of modular sympodial units that each produce three leaves and a multiflowered inflorescence. Most field- grown varieties of tomato, including MP-1, are determinate plants whose shoots produce an average of six sympodial units, each harboring a single inflorescence, within which leaf number gradually decreases before a precocious termination of growth. In general, determinate tomatoes are suitable for open field production. Semi-determinate and indeterminate "cultivated" varieties are suitable for staked cultivation in the open field or protected nets and for glasshouse cultivation.

[0092] According to an embodiment of the invention the tomato plant is a determinate tomato.

[0093] According to an embodiment of the invention the tomato plant is an indeterminate tomato.

[0094] According to an embodiment of the invention the tomato plant is a semi-determinate tomato.

[0095] According to an embodiment, the tomato is selected from the group consisting of a single fruit per truss, branched tomato and cherry tomato.

[0096] As used herein "pepper" refers to the cultivated species "Capsicum (hereinafter, referred to as "C") annuum", or wild species "C. pubescens", "C. baccatum", "C. chinense", and "C. frutescens”. Moreover, "pepper" is a concept that encompasses plants called by names other than "pepper", e.g., horticultural crops called "piment", "paprika", and "sweet pepper".

[0097] As used herein “eggplant" refers to the cultivated species "Solanum (hereinafter, referred to as "S") melongena" or wild species "S. incanum", "S. torvum", "S. nigrum", "S. aethiopicum", "S. macrocarpon", and "S. quitoense”.

[0098] It will be appreciated that the terms "parthenocarpy", "parthenocarpic fruit formation" "seedlessness" and "fertilization-independent fruit formation" are used interchangeably herein.

[0099] As used herein “parthenocarpy” refers to fruit production in the absence of fertilization. Hence, the parthenocarpic fruits according to some embodiments are characterized by no or less than 5 seeds per fruit.

[0100] Maintenance of sexual reproduction capability is evident upon the production of seedbearing fruit.

[0101] For example, in tomato, seed-bearing fruits are considered as at least 1 seed per fruit.

[0102] According to a specific embodiment, parthenocarpy is considered facultative when the parthenocarpy rate is less than 100 %.

[0103] As used herein “parthenocarpy rate” refers to the proportion or percentage of fruits that develop without fertilization in a given crop.

[0104] According to a specific embodiment, the parthenocarpy rate is at least 60 %, 70 %, 75 %, 80 %, 90 %, 95 % 98 %, 99 %.

[0105] According to a specific embodiment, the parthenocarpy rate is at least 70 %. For example, the present inventors have shown that mutant slagl6 slmbp3 / + resulted in 75.43 % parthenocarpy rate, where the (+) indicates a wild type allele, i.e., heterozygous for a loss of function mutation in SIMBP3.

[0106] According to another specific embodiment the parthenocarpy rate is at least 90 %, e.g., at least 95 %. For example, the present inventors have shown that the double homozygous mutant slmbp3 slmbp22 resulted in 95.86 % parthenocarpy rate. In addition, the double homozygous mutant slmbp3 slmbp6 resulted in 94.71 % parthenocarpy rate.

[0107] As used herein “MADS-BOX PROTEIN 3 (MBPS)” refers to the genomic sequence, or encoded RNA or protein of the MBP3ge,n&.

[0108] When the Solanaceous plant is tomato then the MBP3 gene is SXMBP3 gene i.e., Solyc06g064840.

[0109] When the Solanaceous plant is an eggplant then the MBP3 gene relate to all isoforms, e.g.,:

[0110] MBP3a: SMEL4.1_12g007250.1.01

[0111] MBP3b: SMEL4.1_06g018550.1.01 When the Solanaceous plant is pepper then the AfBP3gene is for example:

[0112] Capsicum annuum zunla Capana01g002457

[0113] Capsicum annuum glabriusculum

[0114] Capang01g002169

[0115] Capsicum annuum cv CM334 CA01g22590.

[0116] As used herein “MADS-BOX PROTEIN 22 (MBP22y'>refers to the genomic sequence, or encoded RNA or protein of the MBP22 gene.

[0117] When the Solanaceous plant is tomato then the MBP22 gene is S1MBP22 gene i.e., Solycl lg005120, SEQ ID No: 105.

[0118] When the Solanaceous plant is eggplant then the MBP22 gene is SMEL4.1_04g011260.1. When the Solanaceous plant is pepper then the MBP22 gene is for example, CAOOgl881O + CAOOg 18820 + CAOOg 18830,

[0119] Capsicum annuum zunla Capana04g001527

[0120] Capsicum annuum glabriusculum Capang04g001434

[0121] As used herein “AGL6” refers to a transcription factor which is a key regulator gene of the transition between the state of ‘ovary arrest’ imposed towards anthesis and the fertilization- triggered fruit set.

[0122] When the Solanaceous plant is tomato then the AGL6 gene is S1AG6 gene i.e., Solyc01g093960 coding sequence, e.g., SEQ ID NO: 115).

[0123] When the Solanaceous plant is eggplant then the AGL6 gene is Sme2.5_06058.1.

[0124] When the Solanaceous plant is pepper then the AGL6 gene is Capana01g001334 (ChrOl- 44476983-44483323).

[0125] The skilled artisan would know how to uncover sequence information for different cultivars within the cultivated species.

[0126] As mentioned, the plant (e.g., tomato) exhibits down-regulation in activity or expression of MBP3 and at least one of AGL6 and MBP22.

[0127] As used herein “control” refers to a plant of the same genetic background and growth stage, subjected to the same growth conditions as the plant of the present invention, however it carries both wild type alleles of MBP3 and optionally MBP22 and / or AGL6.

[0128] As used herein “down-regulation in activity or expression” means that the level of activity or expression of MBP3, MBP22 and / or AGL6 in suppressing parthenocarpy is reduced as compared to control.

[0129] As used herein “expression” refers to mRNA and ultimately protein expression. As used herein “reduced” or “decreased” refers to a reduction of at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 % 70 %, 80 % 90 % or more, even 100 % reduction as compared to control.

[0130] Down-regulation of MBP3 and optionally MBP22 and / or AGL6 can be achieved by downregulation the expression of the mRNA or protein (which ultimately leads to down-regulation of the activity of the protein),

[0131] Thus, downregulating a protein (e.g. MBP3 and at least one of MBP22 and AGL6) can be effected at the genomic (e.g. homologous recombination and site specific endonucleases) and / or the transcript level using a variety of molecules which interfere with transcription and / or translation (e.g., RNA silencing agents) or on the protein level (e.g., aptamers, small molecules and inhibitory peptides / dominant negative peptides).

[0132] According a specific embodiment, down-regulation is achieved by at least one loss-of- function mutation in MBP3 gene and at least one of MBP22 gene and AGL6 gene.

[0133] As used herein, the phrase “loss-of-function mutation” or “loss-of-function alteration” refers to any mutation in the DNA sequence of a gene (in this case MBP3 and optionally MBP22 and / or AGL6), which results in downregulation of the expression level and / or activity of the expressed product, i.e., the mRNA transcript and / or the translated protein. Non-limiting examples of such loss-of-function alterations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the regulatory activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the regulatory activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the regulatory activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non-functional protein, devoid of the regulatory activity of the non-mutated polypeptide; a readthrough mutation due to a frame- shift mutation or a modified stop codon mutation (i.e., when the stop codon is mutated into an amino acid codon), with an abolished regulatory activity; a promoter mutation, i.e., a mutation in a promoter sequence, usually 5' to the transcription start site of a gene, which results in downregulation of a specific gene product; a regulatory mutation, i.e., a mutation in a region upstream or downstream, or within a gene, which affects the expression of the gene product; a deletion mutation, i.e., a mutation which deletes coding nucleic acids in a gene sequence and which may result in a frame-shift mutation or an in-frame mutation (within the coding sequence, deletion of one or more amino acid codons); an insertion mutation, i.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence, and which may result in a frame- shift mutation or an in-frame insertion of one or more amino acid codons; an inversion, z.e., a mutation which results in an inverted coding or non-coding sequence; a splice mutation z.e., a mutation which results in abnormal splicing or poor splicing; and a duplication mutation, i.e., a mutation which results in a duplicated coding or non-coding sequence, which can be in-frame or can cause a frame-shift.

[0134] According to a specific embodiment, the loss-of-function mutation is in the K-domain of the gene.

[0135] The K domain in a MADS-box protein is a conserved protein domain that plays a crucial role in protein-protein interactions, particularly in the formation of dimers or higher-order complexes. The K domain is typically located downstream of the MADS (MCM1, AGAMOUS, DEFICIENS, and SRF) domain and the I domain. K domains typically comprises around 90-100 amino acids, forming two amphipathic a-helices (sub-domains: KI (al), K2, and K3 (a2)) that facilitate interactions with other proteins.

[0136] Domain architecture predicted according to AlphaFold [Jumper et al. Nature, 596(7873), 583- 589]:

[0137] For SlMBP3:

[0138] MADS domain aa 1-66

[0139] I domain aa 67 - 86

[0140] K domain aa 87-186

[0141] For S1MBP22:

[0142] MADS domain aa 1-63

[0143] I domain aa 64 - 83

[0144] K domain aa 84-178

[0145] For S1AGE6:

[0146] MADS domain aa 1-58

[0147] I domain aa 59 - 80

[0148] K domain aa 81-176

[0149] According to a specific embodiment, the loss-of-function mutation is in the K domain of AGE6, MBP3 and / or MBP22.

[0150] According to a specific embodiment, the loss-of-function mutation is a deletion in at least an a2 helix of the K domain of AGE6, MBP3 and / or MBP22.

[0151] According to a specific embodiment, the loss-of-function mutation result in no mRNA product. According to a specific embodiment, the loss-of-function mutation result in no protein product.

[0152] According to a specific embodiment, the loss-of-function mutation result in an aberrant protein product (not wild-type).

[0153] According to a specific embodiment, the loss-of-function mutation results in a peptide comprising a MADS domain, an I domain and / or a C domain but no K domain or subdomains thereof (e.g., a2).

[0154] According to a specific embodiment, the loss-of-function mutation results in a peptide, which comprises an a2 helix but is devoid of any or all of MADS domain, an I domain and a C domain, said peptide serving as a dominant negative peptide.

[0155] As used herein “dominant negative peptide” refers to an inhibitory peptide which competes with the wild-type protein by binding to the same target molecules but failing to exert the biological function, in this case suppression of parthenocarpy. Accordingly, In this case, a dominant negative peptide would be a fragment of the protein which is responsible for the interaction between AGL6, MBP3 and MBP22. Thus, it may comprise the K domain or at least the a2 helix thereof but typically devoid of a MADS domain, I domain and / or even other subdomains of the K domain e.g., al helix or the c terminal portion of the a2 domain.

[0156] According to a specific embodiment, the loss of function mutation is in a coding sequence of the gene and / or in a regulatory sequence which is upstream or downstream of the coding region.

[0157] According to a specific embodiment, the loss of function mutation is in exon 1 of the MBP3 gene (e.g., SIMBP3).

[0158] According to a specific embodiment, the loss of function mutation generates a deletion in exon 1 optionally including the start codon, that disrupts the functions of respective proteins (e.g., this is exemplified in the Examples section for slmbp3CR'20and slmbp22CR'20').

[0159] According to a specific embodiment, the mutation in MBP3 is in exon 1 (as shown in Figure 5 a).

[0160] According to a specific embodiment, the loss of function mutation is in exon 1 of the MBP22 gene (e.g., SIMBP22). According to a specific embodiment, the loss of function mutation is a deletion which deletes at least a portion or all of exon 1 or exon 1 and sequences upstream and / or downstream thereto. For example, , the deletion encompasses the entire 1stexon, including the start codon and 141 bp upstream to it. Alternatively, the slMBP22CR'20mutant allele harbors a 391 bp deletion that eliminates 46 bp upstream of the 1stexon, the entire 1stexon and 61 bp of the 1stintron. Such mutants are expected to produce truncated, non-functional mutant proteins, which would lack the MADS-box domain encoded by their 1stexon, and probably other domains, due to introduction of in-frame premature stop codons. This structure of the two alleles strongly suggests that they represent null alleles.

[0161] According to a specific embodiment, the S1AGL6 loss-of-function mutation is as described in Klap et al., 2017, whereby single nucleotide substitutes C->T in exon 3 position 3635 (the 2012 mutation); or Sgl which is a 178 bp deletion that completely deletes exon 2 and surrounding sequences.

[0162] According to a specific embodiment, the S1AGL6 loss-of-function mutation is not as described in Klap et al., 2017.

[0163] According to specific embodiments loss-of-function alteration of a gene may comprise at least one allele of the gene.

[0164] The term "allele" as used herein, refers to any of one or more alternative forms of a gene locus, all of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

[0165] According to other specific embodiments, a loss-of-function alteration of a gene comprises both alleles (or all alleles in an induced polyploid plant) of the gene. In such instances the e.g. MBP3 and at least one of MBP22 and AGL6 (as in a double mutant or triple mutant) may be in a homozygous form or in a heterozygous form. According to this embodiment, homozygosity is a condition where both alleles at the e.g. MBP3, MBP22 and / or AGL6 loci are characterized by the same nucleotide sequence. Heterozygosity refers to a condition where alleles of the gene at the e.g. MBP3, MBP22 and / or AGL6 loci display different sequence, especially at the site of the loss- of-function mutation(s).

[0166] According to a specific embodiment the loss of function mutation is in a homozygous or heterozygous form yet both encode for dis-functioning products. An almost full parthenocarpic phenotype as described herewith is evident when both alleles comprise a loss of function mutation. Heterozygous forms of MBP3 and MBP22 typically result in facultative parthenocarpy which is higher than that obtained in the presence of a homozygous loss-of-function mutation in AGL6 alone.

[0167] According to a specific embodiment, the loss-of-function mutation is heterozygous, e.g., of SXMBP3 and the resultant fruit in case of tomato comprises a jelly fill. According to a specific embodiment, the loss-of-function mutation is heterozygous, e.g., of MBP3 and homozygous for a loss-of-function in either MBP22 or AGL6 or both the resultant fruit in case of tomato comprises a jelly fill.

[0168] According to a specific embodiment, the down-regulation is a result of a loss-of function mutations in MBP3 and AGL6. According to a specific embodiment, the down-regulation is a result of a loss-of function mutations in MBP3 and MBP22.

[0169] According to a specific embodiment, the down-regulation is a result of a loss-of function mutations in MBP3, AGL6 and MBP22.

[0170] According to a specific embodiment, the loss-of-function mutation in each or all of said MBP3, AGL6 and MBP22 is homozygous, e.g., of S\MBP3 and the resultant fruit in case of tomato is full flesh.

[0171] According to a specific embodiment, the loss of function mutation is a deletion e.g., exon 1 of e.g., S1MBP3 and / or S1MBP22.

[0172] According to a specific embodiment, the loss of function mutation causes a premature stop codon.

[0173] According to some embodiments, the parthenocarpic plant (as used herein “the plant”) of the invention exhibits at least one of: fruit yield defined as an average fruit weight that is at least the same as that of the control plant; fruit yield defined as an average number of fruits of the plant per growing season which is at least the same as that of the control plant; fruit yield defined as an average number of fruits of the plant per growing season which is at least 2-fold (e.g., at least 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold) higher than that of the control plant; fruit yield defined as an average number of fruits of the plant per growing season which is at least 4-fold higher than that of the control plant; fruit yield defined as an average number of fruits of the plant per growing season which is at about 2 to 5-fold (2-3 fold, 2-4 fold) higher than that of the control plant.

[0174] According to other embodiments, the parthenocarpic plant (as used herein “the plant”) of the invention exhibits at least one of: fruit yield defined as an average number of fruits of the plant per growing season / growth area which is at least the same as that of the control plant; fruit yield defined as an average number of fruits of the plant per growing season / growth area which is at least 2-fold (e.g., at least 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold) higher than that of the control plant; fruit yield defined as an average number of fruits of the plant per growing season / growth area which is at least 4-fold higher than that of the control plant; fruit yield defined as an average number of fruits of the plant per growing season / growth area which is at about 2 to 5-fold (2-3 fold, 2-4 fold) higher than that of the control plant.

[0175] According to some embodiments, the characteristic of the plant can be attained from studies on a plurality of plants (open field or greenhouse) and the values provided herein relate to an average.

[0176] According to a specific embodiment, a growth area is an acre.

[0177] As used herein “about the same” refers to ± 10-20 %, 10 % or 20 %, at the same developmental stage and under the same conditions.

[0178] According to other embodiments “fruit yield” refers to the total weight of the marketable harvested fruit, which is the product of the number of fruits per plant multiplied by the average weight of the harvested fruits.

[0179] The “same genetic background” refers to at least 95 %, 96 %, 97 %, 98 %, 99 % or 99.9 % of the genome is shared between the plant and the non-parthenocarpic plant.

[0180] As used herein “jelly fill” refers to the fluid to semi-fluid filling in the locular cavity of the fruit.

[0181] In some embodiments, the tomato fruit (e.g., homozygous mutants in MBP3 and MBP22) exhibit a slightly pointy blossom end however, while other morphological phenotypes seem normal and matching those of control plants.

[0182] Thus, according to a specific embodiment, the plant or portions thereof (e.g., fruit) is devoid of homeotic aberrations.

[0183] As used herein “homeotic aberrations” refers to developmental aberrations in the anatomic structure of the plant, e.g., floral or fruit structures, which deviate from the normal flower shape with regards to whorls number or shape of the organs comprising the wild type (WT) flower, or fruit shape, size and internal structure clearly different from that of seeded fruit, besides the lack of normal seeds, which is inherent to parthenocarpy.

[0184] As used herein “enlarged ovules” refers to the small pseudo-seeds observed in mature seedless fruits.

[0185] The enlarged ovules are accompanied by the absence of a characteristic endothelium layer which is present in the wild type ovary (e.g. as in Figure ID).

[0186] However, with respect to eggplant it should be noted that the fruits are harvestable at different sizes. According to a specific embodiment, when the plant is a determinate tomato plant the fruit yield is a yield concentration where fruits along the 4-6 stages ripe almost simultaneously.

[0187] According to a specific embodiment, the plant is of an elite line. Examples of tomato elite lines are known in the art and some of which are listed herein.

[0188] Examples of Elite pepper cultivars: include, but are not limited to, Bastille, Rampart, Bayonet, Cutlass, Lafayette, Crusader, Pageant, Rising Sun, Trifecta (Syngenta); Atir, Gilad, Serenada, Vilmorin: E5661 Fl, RIFLESSI, Lussac, Vivaldi, Tyson (Hazera Genetics), Razer, E20B 10015 (Enza Zaden), Alma Paprika Peppe, and others.

[0189] Examples of Eggplant elite cultivars include, but are not limited to, Hybrid cultivars: Classic, Dancer, Dusky, Fairy Tale, Ghostbuster, Nadia, Purple Rain,

[0190] According to a specific embodiment, the plant is a transgenic plant (e.g., for a genome editing agent or for an RNA silencing agent, as described herein below).

[0191] According to a specific embodiment, the plant may be a transgenic plant but the transgene may not be associated with (i.e., not the cause for) parthenocarpy, as described herein. For example, the transgene may function to improve biotic stress resistance, pesticide resistance or abiotic stress resistance.

[0192] According to a specific embodiment, the plant comprises a diploid genome.

[0193] According to a specific embodiment, the plant is an inbred.

[0194] According to a specific embodiment, the plant is a hybrid plant or the seed is a hybrid seed, where e.g., each of the parental lines is homozygous for a loss-of-function mutation in MBP3 and at least one of MBP22 and AGL6 as described herein.

[0195] Methods of producing the plant as described herein may rely on the use of mutagens e.g., EMS or genetic engineering which is naturally a more directed method and therefore involves less breeding steps.

[0196] Thus, according to an aspect of the invention there is provided a method of producing the plant as described herein, the method comprising down-regulating expression or activity of MBP3 and at least one of MBP22 and AGL6 in the plant.

[0197] Following is a non-limiting description of methods of inducing loss-of-function mutation(s) in the MBP22 gene and optionally AGL6 gene which can be used to produce the plant.

[0198] Thus, according to some embodiments of the invention, down-regulating MBP3 and at least one of MBP22 and AGL6 is effected by treating the plant or a regenerative portion thereof with a mutagen. In such a case the plant is non-genetically modified with an agent for inducing downregulation of MBPS and at least one of MBP22 and AGL6. Alternatively or additionally, occurrence of the genetic event responsible for the facultative parthenocarpic trait may be achieved by exposing a plant (i.e., tomato, pepper, eggplant) or part thereof to a chemical or physical mutagen (as described in the Examples section). Examples of chemical mutagens include, but are not limited to nitrous acid, alkylating agents such as ethyl methanesulfonate (EMS), methyl methane sulfonate (MMS), diethylsulfate (DES), and base analogs such as 5-bromo-deoxyuridine (5BU). Physical mutagens include radiation (e.g. fast neutron, gamma radiation).

[0199] Initial exposure is typically followed by additional steps of selfing, selection, crossing and selfing or combinations thereof, where any step can be repeated more than once, as long as the loss-of-function in MBP3 and at least one of MBP22 and AGL6 is in a homozygous or heterozygous form. Selection can be phenotypic or using marker-assisted breeding as further described hereinbelow.

[0200] According to another specific embodiment, the non-genetically modified plant of the invention results from a spontaneous genetic event incurred by multiple crossings / selfings.

[0201] Below is a description of platform technologies for effecting knock-out (also referred to as “genome editing”) and transcriptional silencing in plants.

[0202] Methods of introducing nucleic acid alterations to a gene of interest (in this case MBP22 and optionally AGL6)are well known in the art [see for example Menke D. Genesis (2013) 51: - 618; Capecchi, Science (1989) 244: 1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; US Patent Nos. 8771945, 8586526, 6774279 and UP Patent Application Publication Nos. 20030232410, 20050026157, US20060014264; the contents of which are incorporated by reference in their entireties] and include targeted homologous recombination, site specific recombinases, PB transposases and genome editing by engineered nucleases. Agents for introducing nucleic acid alterations to a gene of interest can be designed publically available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences.

[0203] Following is a description of various exemplary methods used to introduce nucleic acid alterations to a gene of interest and agents for implementing same that can be used according to specific embodiments of the present invention.

[0204] Any of the below methods can be directed to any part of the MBP3 and at least one of MBP22 and AGL6 genes as long as a loss-of-function is achieved. When needed further steps of selfing are effected in order to achieve a homozygous form of the mutation.

[0205] As used herein ’’target sequence” refers to the MBP3 and at least one of MBP22 an<3AGL6 DNA coding or RNA transcript or regulatory sequences of the genes. It will be appreciated that MBP3 and at least one of MBP22 and AGL6 can also be down-regulated at the protein level using a dominant negative peptide or a DNA sequence encoding same (over-expression). Although this option is not discussed here at length, it is still considered an embodiment for producing the plant.

[0206] Genome Editing using engineered endonucleases - this approach refers to a reverse genetics method using artificially engineered nucleases to cut and create specific double- stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDR) and non-homologous end-joining (NHEJ). NHEJ directly joins the DNA ends in a double-stranded break, while HDR utilizes a homologous sequence as a template for regenerating the missing DNA sequence at the break point. In order to introduce specific nucleotide modifications to the genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location. To overcome this challenge and create site-specific single- or doublestranded breaks, several distinct classes of nucleases have been discovered and bioengineered to date. These include the meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR / Cas system.

[0207] Meganucleases - Meganucleases are commonly grouped into four families: the LAGLID ADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLID ADG family are characterized by having either one or two copies of the conserved LAGLID ADG motif. The four families of meganucleases are widely separated from one another with respect to conserved structural elements and, consequently, DNA recognition sequence specificity and catalytic activity. Meganucleases are found commonly in microbial species and have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific for cutting at a desired location. This can be exploited to make site-specific double- stranded breaks in genome editing. One of skill in the art can use these naturally occurring meganucleases, however the number of such naturally occurring meganucleases is limited. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to create hybrid enzymes that recognize a new sequence. Alternatively, DNA interacting amino acids of the meganuclease can be altered to design sequence specific meganucleases (see e.g., US Patent 8,021,867). Meganucleases can be designed using the methods described in e.g., Certo, MT et al. Nature Methods (2012) 9:073-975; U.S. Patent Nos. 8,304,222; 8,021,867; 8, 119,381; 8, 124,369; 8, 129,134; 8,133,697; 8,143,015; 8,143,016; 8, 148,098; or 8, 163,514, the contents of each are incorporated herein by reference in their entirety. Alternatively, meganucleases with site specific cutting characteristics can be obtained using commercially available technologies e.g., Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

[0208] ZFNs and TALENs - Two distinct classes of engineered nucleases, zinc-finger nucleases (ZFNs) and transcription activator- like effector nucleases (TALENs), have both proven to be effective at producing targeted double- stranded breaks (Christian et al., 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).

[0209] Basically, ZFNs and TALENs restriction endonuclease technology utilizes a non-specific DNA cutting enzyme which is linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically, a restriction enzyme whose DNA recognition site and cleaving site are separate from each other is selected. The cleaving portion is separated and then linked to a DNA binding domain, thereby yielding an endonuclease with very high specificity for a desired sequence. An exemplary restriction enzyme with such properties is Fokl. Additionally Fokl has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity. The heterodimer functioning nucleases avoid the possibility of unwanted homodimer activity and thus increase specificity of the doublestranded break.

[0210] Thus, for example to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind adjacent sequences at the targeted site. Upon transient expression in cells, the nucleases bind to their target sites and the Fokl domains heterodimerize to create a double-stranded break. Repair of these double- stranded breaks through the nonhomologous end-joining (NHEJ) pathway most often results in small deletions or small sequence insertions. Since each repair made by NHEJ is unique, the use of a single nuclease pair can produce an allelic series with a range of different deletions at the target site. The deletions typically range anywhere from a few base pairs to a few hundred base pairs in length, but larger deletions have successfully been generated in cell culture by using two pairs of nucleases simultaneously (Carlson et al., 2012; Lee et al., 2010). In addition, when a fragment of DNA with homology to the targeted region is introduced in conjunction with the nuclease pair, the double- stranded break can be repaired via homology directed repair to generate specific modifications (Li et al., 2011; Miller et al., 2010; Umov et al., 2005).

[0211] Although the nuclease portions of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases is in their DNA recognition peptide. ZFNs rely on Cys2- His2 zinc fingers and TALENs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically found in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs. Because both zinc fingers and TALEs happen in repeated patterns, different combinations can be tried to create a wide variety of sequence specificities. Approaches for making site-specific zinc finger endonucleases include, e.g., modular assembly (where Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence), OPEN (low- stringency selection of peptide domains vs. triplet nucleotides followed by high-stringency selections of peptide combination vs. the final target in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries, among others. ZFNs can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA).

[0212] Method for designing and obtaining TALENs are described in e.g. Reyon et al. Nature Biotechnology 2012 May;30(5):460-5; Miller et al. Nat Biotechnol. (2011) 29: 143-148; Cermak et al. Nucleic Acids Research (2011) 39 (12): e82 and Zhang et al. Nature Biotechnology (2011) 29 (2): 149-53. A recently developed web-based program named Mojo Hand was introduced by Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (can be accessed through www(dot)talendesign(dot)org). TALEN can also be designed and obtained commercially from e.g., Sangamo Biosciences™ (Richmond, CA).

[0213] Another agent capable of downregulating MBP3 and at least one of MBP22 and AGL6 is a RNA-guided endonuclease technology e.g. CRISPR system (that is exemplified in great details in the Examples section which follows).

[0214] As used herein, the term "CRISPR system" also known as Clustered Regularly Interspaced Short Palindromic Repeats refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated genes, including sequences encoding a Cas9 gene (e.g. CRISPR-associated endonuclease 9), a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat) or a guide sequence (also referred to as a "spacer") including but not limited to a crRNA sequence (i.e. an endogenous bacterial RNA that confers target specificity yet requires tracrRNA to bind to Cas) or a sgRNA sequence (i.e. single guide RNA, see Table 1 of the Examples section which follows, that is deemed to be part of the specification). It will be appreciated that due to the high sequence homology the same gRNA can be used for the indicated Solaneceous plants.

[0215] In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system (e.g. Cas) is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes, Neisseria meningitides, Streptococcus thermophilus or Treponema denticola.

[0216] In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).

[0217] In the context of formation of a CRISPR complex, "target sequence" in this case MBP3 and at least one of MBP22 and AGL6 refers to a sequence to which a guide sequence (i.e. guide RNA e.g. sgRNA or crRNA) is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. Thus, according to some embodiments, global homology to the target sequence may be of 50 %, 60 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 % or 99 %. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.

[0218] Thus, the CRISPR system comprises two distinct components, a guide RNA (gRNA) that hybridizes with the target sequence, and a nuclease (e.g. Type-II Cas9 protein), wherein the gRNA targets the target sequence and the nuclease (e.g. Cas9 protein) cleaves the target sequence. The guide RNA may comprise a combination of an endogenous bacterial crRNA and tracrRNA, i.e. the gRNA combines the targeting specificity of the crRNA with the scaffolding properties of the tracrRNA (required for Cas9 binding). Alternatively, the guide RNA may be a single guide RNA capable of directly binding Cas.

[0219] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence.

[0220] In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, a complete complementarity is not needed, provided there is sufficient to be functional. In some embodiments, the tracr sequence has at least 50 %, 60 %, 70 %, 80 %, 90 %, 95 % or 99 % of sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0221] Introducing CRISPR / Cas into a cell may be effected using one or more vectors driving expression of one or more elements of a CRISPR system such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. A single promoter may drive expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron).

[0222] “Hit and run” or “in-out” - involves a two-step recombination procedure. In the first step, an insertion-type vector containing a dual positive / negative selectable marker cassette is used to introduce the desired sequence alteration. The insertion vector contains a single continuous region of homology to the targeted locus and is modified to carry the mutation of interest. This targeting construct is linearized with a restriction enzyme at a one site within the region of homology, transformed into the cells, and positive selection is performed to isolate homologous recombinants. These homologous recombinants contain a local duplication that is separated by intervening vector sequence, including the selection cassette. In the second step, targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the duplicated sequences. The local recombination event removes the duplication and, depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without the retention of any exogenous sequences.

[0223] The “double-replacement” or “tag and exchange” strategy - involves a two-step selection procedure similar to the hit and run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homology arms is used to insert a dual positive / negative selectable cassette near the location where the mutation is to be introduced. After transformation and positive selection, homologously targeted clones are identified. Next, a second targeting vector that contains a region of homology with the desired mutation is transformed into targeted clones, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating unwanted exogenous sequences.

[0224] Site-Specific Recombinases - The Cre recombinase derived from the Pl bacteriophage and Flp recombinase derived from the yeast Saccharomyces cerevisiae are site-specific DNA recombinases each recognizing a unique 34 base pair DNA sequence (termed “Lox” and “FRT”, respectively) and sequences that are flanked with either Lox sites or FRT sites can be readily removed via site-specific recombination upon expression of Cre or Flp recombinase, respectively. For example, the Lox sequence is composed of an asymmetric eight base pair spacer region flanked by 13 base pair inverted repeats. Cre recombines the 34 base pair lox DNA sequence by binding to the 13 base pair inverted repeats and catalyzing strand cleavage and religation within the spacer region. The staggered DNA cuts made by Cre in the spacer region are separated by 6 base pairs to give an overlap region that acts as a homology sensor to ensure that only recombination sites having the same overlap region recombine.

[0225] Basically, the site specific recombinase system offers means for the removal of selection cassettes after homologous recombination. This system also allows for the generation of conditional altered alleles that can be inactivated or activated in a temporal or tissue-specific manner. Of note, the Cre and Flp recombinases leave behind a Lox or FRT “scar” of 34 base pairs. The Lox or FRT sites that remain are typically left behind in an intron or 3' UTR of the modified locus, and current evidence suggests that these sites usually do not interfere significantly with gene function.

[0226] Thus, Cre / Lox and Flp / FRT recombination involves introduction of a targeting vector with 3' and 5' homology arms containing the mutation of interest, two Lox or FRT sequences and typically a selectable cassette placed between the two Lox or FRT sequences. Positive selection is applied and homologous recombinants that contain targeted mutation are identified. Transient expression of Cre or Flp in conjunction with negative selection results in the excision of the selection cassette and selects for cells where the cassette has been lost. The final targeted allele contains the Lox or FRT scar of exogenous sequences.

[0227] Silencing MBP3 and at least one of MBP22 and AGL6 transcript (RNA) level can be effected using the below exemplary platforms.

[0228] As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0229] As used herein, the term "RNA silencing agent" refers to an RNA which is capable of specifically inhibiting or "silencing" the expression of a target gene (MBP3 and at least one of MBP22 and AGL6). In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and / or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.

[0230] In one embodiment, the RNA silencing agent is capable of inducing RNA interference.

[0231] In another embodiment, the RNA silencing agent is capable of mediating translational repression.

[0232] According to an embodiment of the invention, the RNA silencing agent is specific to the target RNA and does not cross inhibit or silence other targets or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene; as determined by PCR, Western blot, Immunohistochemistry and / or flow cytometry.

[0233] RNA interference refers to the process of sequence- specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).

[0234] Following is a detailed description on RNA silencing agents that can be used according to specific embodiments of the present invention. DsRNA, siRNA and shRNA - The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.

[0235] Accordingly, some embodiments of the invention contemplate use of dsRNA to downregulate protein expression from mRNA.

[0236] According to one embodiment dsRNA longer than 30 bp are used. Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects - see for example [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res. Protoc. 2004;13: 115- 125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P.J., et al., Proc. Natl Acad. Sci. USA. 2002;99: 1443-1448; Tran N., et al., FEBS Lett. 2004;573: 127-134],

[0237] According to some embodiments of the invention, dsRNA is provided in cells where the interferon pathway is not activated, see for example Billy et al., PNAS 2001, Vol 98, pages 14428- 14433. and Diallo et al, Oligonucleotides, October 1, 2003, 13(5): 381-392. doi: 10.1089 / 154545703322617069.

[0238] According to an embodiment of the invention, the long dsRNA are specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression. For example, Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5'-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.

[0239] Another method of evading the interferon and PKR pathways in mammalian systems is by introduction of small inhibitory RNAs (siRNAs) either via transfection or endogenous expression.

[0240] The term "siRNA" refers to small inhibitory RNA duplexes (generally between 18-30 base pairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3'-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is suggested to result from providing Dicer with a substrate (27mer) instead of a product (21mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.

[0241] It has been found that position of the 3'-overhang influences potency of a siRNA and asymmetric duplexes having a 3 '-overhang on the antisense strand are generally more potent than those with the 3'-overhang on the sense strand (Rose et al., 2005). This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.

[0242] The strands of a double- stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem- loop structure (e.g., an shRNA). Thus, as mentioned, the RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).

[0243] The term "shRNA", as used herein, refers to an RNA agent having a stem- loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5'-CAAGAGA-3' and 5’-UUACAA-3’ (International Patent Application Nos. WO2013126963 and WO2014107763). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem- loop or hairpin structure comprising a doublestranded region capable of interacting with the RNAi machinery.

[0244] Synthesis of RNA silencing agents suitable for use with some embodiments of the invention can be effected as follows. First, the MBP3 and at least one of MBP22 an<2AGL6 mRNA sequences are scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3’ adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and / or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5’ UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level (www(dot)ambion(dot)com / techlib / tn / 91 / 912(dot)html).

[0245] Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov / BLAST / ). Putative target sites which exhibit significant homology to other coding sequences are filtered out.

[0246] Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G / C content as these have proven to be more effective in mediating gene silencing as compared to those with G / C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.

[0247] Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The coding sequence constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The genetic construct can be an expression vector wherein the nucleic acid sequence is operably linked to one or more regulatory sequences allowing expression in the plant cells.

[0248] Plant cells may be transformed stably or transiently with the nucleic acid constructs of the present invention. In stable transformation, the nucleic acid molecule of the present invention is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the nucleic acid molecule is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.

[0249] There are various methods of introducing foreign genes into both monocotyledonous and dicotyledonous plants (Po try kus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205- 225; Shimamoto et al., Nature (1989) 338:274-276).

[0250] The principle methods of causing stable integration of exogenous DNA into plant genomic DNA include two main approaches:

[0251] (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Amtzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.

[0252] (ii) direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6: 1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio / Technology (1988) 6:559-563; McCabe et al. Bio / Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217;

[0253] Glass fibers or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, U.S. Pat. No. 5,464,765 or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

[0254] The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.

[0255] There are various methods of direct DNA transfer into plant cells. In electroporation, the protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.

[0256] Following stable transformation plant propagation is exercised. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since seeds are produced by plants according to the genetic variances governed by Mendelian rules. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferred that the transformed plant be produced such that the regenerated plant has the identical traits and characteristics of the parent transgenic plant. Therefore, it is preferred that the transformed plant be regenerated by micropropagation which provides a rapid, consistent reproduction of the transformed plants.

[0257] However other methods of production are also contemplated including sexual reproduction (and selection for the phenotype whether morphologically or using molecular markers as described herein), tissue culture and more.

[0258] Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar. This process permits the mass reproduction of plants having the preferred tissue expressing the fusion protein. The new generated plants which are produced are genetically identical to, and have all of the characteristics of, the original plant. Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.

[0259] Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening. During stage one, initial tissue culturing, the tissue culture is established and certified contaminant-free. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet gradually increased so that it can be grown in the natural environment.

[0260] Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV, TRV and BV. Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, is described in WO 87 / 06261.

[0261] Regardless of the method used to produce the Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) of some embodiments of the invention, once plants or any reproductive material is at hand, it is selected for the parthenocarpic trait. Thus, according to an aspect of the invention there is provided a method of selecting a Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plant being parthenocarpic, the method comprising detecting in a genome of a Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plant a loss of function mutation in MBP3 and at least one of MBP22 and AGL6, wherein presence of the mutation is indicative of a Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) having being parthenocarpic.

[0262] Many methods are known in the art for analyzing for mutations including for example single base extension (SBE), allele- specific primer extension sequencing (ASPE), DNA sequencing, RNA sequencing, microarray-based analyses, universal PCR, Melting Curve SNP method, allele specific extension, hybridization, mass spectrometry, ligation, extension-ligation, Flap Endonuclease-mediated assays, restriction fragment length polymorphism (RFLP), electrophoresis, sequence alignment, allelic specific oligonucleotide hybridization (ASO) and random amplified polymorphic DNA (RAPD).

[0263] Thus, the present invention contemplates oligonucleotides (e.g. Primers) that can be used to distinguish between the mutated and non-mutated forms of MBP3 and at least one of MBP22 and AGL6.

[0264] Thus, once a plant carrying the loss of function genetic alteration is identified it is considered as being parthenocarpic. This plant material can be used as a breeding material in the development of Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) varieties having agriculturally desired traits.

[0265] According to a specific embodiment, down-regulating MBP3 gene and at least one of AGL6 and MBP22 is in the same plant. The resultant plant can be subjected to further breeding programs to improve agriculturally desirable traits as known in the art.

[0266] Alternatively, down-regulating MBP3 gene and at least one of AGL6 and MBP22 is done in different plants that may be crossed to result in a plant where MBP3 gene and at least one of AGL6 and MBP22 are down-regulated. The resultant plant can be subjected to further breeding programs to improve agriculturally desirable traits as known in the art.

[0267] According to one embodiment, the plants of the present invention are of a hybrid variety - i.e. are generated following the crossing (i.e. mating) of two non-isogenic plants both being homozygous for a loss of function mutation in MBP3 gene and at least one of AGL6 and MBP22 genes. The hybrid may be an Fi Hybrid.

[0268] An "Fi Hybrid" as used herein, refers to first generation progeny of the cross of two non- isogenic plants. The development of Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) hybrids of the present invention requires the development of stable parental lines. In breeding programs desirable traits from two or more germplasm sources or gene pools are combined to develop superior breeding varieties. Desirable inbred or parent lines are developed by continuous self-pollinations and / or backcrosses and selection of the best breeding lines, sometimes utilizing molecular markers to speed up the selection process.

[0269] Once the parental lines that give the best hybrid performance have been identified e.g., both carrying the loss of function mutation as described above, the hybrid seed can be produced indefinitely, as long as the homozygosity of the parents are maintained. According to one embodiment the Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plants of the present invention are stable parent plant lines (carrying the loss of function mutation in a heterozygous form or a homozygous form).

[0270] As defined herein, the phrase "stable parental lines" refers to open pollinated, inbred lines, stable for the desired plants over cycles of self-pollination and planting. According to a specific embodiment, 95% of the genome is in a homozygous form in the parental lines of the present invention.

[0271] A common practice in plant breeding is using the method of backcrossing to develop new varieties by single trait conversion.

[0272] The phrase "single trait conversion" as used herein refers to the incorporation of new single gene into a parent line wherein essentially all of the desired morphological and physiological characteristics of the parent lines are recovered in addition to the single gene transferred.

[0273] The term "backcrossing" as used herein refers to the repeated crossing of a hybrid progeny back to one of the parental Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plants. The parental Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plant which contributes the gene for the desired characteristic is termed the non-recurrent or donor parent. This terminology refers to the fact that the non-recurrent parent is used one time in the backcross protocol and therefore does not recur. The parental Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) plant to which the gene from the non-recurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol.

[0274] In a typical backcross protocol, a plant from the original varieties of interest (recurrent parent) is crossed to a plant selected from second varieties (non-recurrent parent) that carries the single gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a Solanaceous plant (i.e., eggplant, tomato and pepper e.g., tomato) is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred gene from the non-recurrent parent.

[0275] Thus, near-isogenic lines (NIL) may be created by many backcrosses to produce an array of individuals that are nearly identical in genetic composition except for the trait or genomic region under interrogation in this case loss of function genetic alteration e.g., in the MBP3 gene and at least one of AGL6 and MBP22.

[0276] Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the parent lines. Marker assisted breeding (selection) as described above can be used in this method.

[0277] According to a specific embodiment, the plant or the plant seed is an inbred.

[0278] According to a specific embodiment, the plant is a hybrid plant or the seed is a hybrid seed.

[0279] The invention also relates to progeny of the tomato, eggplant, pepper plants of the invention. Such progeny can be produced by sexual or vegetative reproduction of a plant of the invention or a progeny plant thereof. The regenerated progeny plant grows fruits independent of fertilization in the same or a similar way as the parthenocarpic parent. In addition to this, the progeny plant may be modified in one or more other characteristics. Such additional modifications are for example effected by mutagenesis or by transformation with a transgene.

[0280] As used herein the word "progeny" is intended to mean the offspring or the first and all further descendants from a cross with a plant of the invention that shows fertilization independent fruit formation. Progeny of the invention are descendants of any cross with a plant of the invention that carries the mutation (in a homozygous form) trait that leads to fertilization independent fruit formation.

[0281] "Progeny" also encompasses plants that carry the trait of the invention which are obtained from other plants of the invention by vegetative propagation or multiplication.

[0282] As mentioned, embodiments described herein, furthermore, relate to hybrid seed and to a method of producing hybrid seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed. In this case the trait is recessive, therefore both parent plants need to be homozygous for the fertilization independent fruit formation trait in order for all of the hybrid seed to carry the trait of the invention. They need not necessarily be uniform for other traits.

[0283] Embodiments described herein also relate to the germplasm of the plants. The germplasm is constituted by all inherited characteristics of an organism and according to the invention encompasses at least the facultative fertilization independent fruit formation trait of the invention. Embodiments described herein also relate to cells of the plants that show the facultative fertilization independent fruit formation trait. Each cell of such plants carries the genetic information (i.e., loss of function mutation in MBP3 gene and at least one of AGL6 and MBP22) that leads to the facultative parthenocarpy. The cell may be an individual cell or be part of a plant or plant part, such as the fruit.

[0284] The present teachings further relate to consumed products which comprise the genomic (DNA) information (i.e., loss of function mutation in MBP3 gene and at least one of AGL6 and MBP22) that leads to the parthenocarpy.

[0285] Fruits of any of the plants described herein may be selected or qualified for fruit color, Brix, pH, sugars, organic acids and defect levels (insect damage, mold, etc.) at ripening or post harvest. For example, tomatoes are typically transported to a large processing facility, where they are collected and where they may subsequently be washed, typically using chlorinated water and rinsed using tap water and further selected to remove those that present defects (e.g., inadequate ripening, disease damage, molds etc.). Tomatoes may be stored (especially those exhibiting improved shelflife as described above) or immediately sent to the consumer (fresh-market tomatoes). Processing tomatoes may be processed into a wide variety of products.

[0286] For juice or pulp production, the tomatoes may be subject to oven dehydration and are comminuted and macerated (disintegrated and broken) to obtain a pumpable mass. As will be clear to the skilled person these operations per se are known and common in the field of tomato processing and any adjustments to the method can be made in this regard without departing from the scope.

[0287] Methods for processing tomatoes and / or producing tomato-based compositions are well known in the art, see generally U.S. Patent No. 6,924,420. Also reported are specific methods for preparing, for example, paste (U.S. Patent No. 7,074,451), sterile paste (U.S. Patent No. 4,206,239), puree (U.S. Patent No. 4,556,576), sauce (U.S. Patent No. 7,122,217), solidified sauce (U.S. Patent No. 4,038,424), barbecue sauce (U.S. Patent No. 6,869,634), salsa (U.S. Patent No. 5,914,146), ketchup (U.S. Patent No. 6,689,279), tomato fiber composition (U.S. Patent No. 7,166,315) and dehydrated tomato-product (U.S. Patent No. 5,035,909). Methods of modifying the texture and consistency of tomato paste, pulp, and puree has also been reported, see, for example, U.S. Patent No. 6,720,019.

[0288] Also provided is an edible processed tomato product comprising the tomato or an edible portion thereof (e.g., fruit or an edible part thereof).

[0289] Also provided is a tomato paste generated according to the present teachings. Examples of such edible products include, but are not limited to, canned tomatoes (whole), a tomato paste, a ketchup, a tomato sauce a tomato soup, a dehydrated tomato, a tomato juice, a tomato powder, a tomato dice, a crushed tomato, a chopped tomato and a tomato concentrate.

[0290] Pepper products that can benefit from seedlessness include: varieties for fresh consumption, as well as for processed and preserved pepper. Cultivars grown for spices (paprika) production consist of dried, ground pods of Capsicum annuum L., sweet red pepper. Sweet paprika spice processing includes removal of the seeds before grinding of the pericarp, which is otherwise of reduced quality. Other products are made from paprika oleoresin, an oil-soluble extract from the fruits of Capsicum annuum which is primarily used as a coloring and / or flavouring in food products. It is also used to color cosmetics products including bath and beauty products and moisturizing lipstick.

[0291] Seedless eggplant are in great demand by the consumers as the seeds add bitterness and are associated with fruit flesh browning. Eggplant is consumed usually following cooking, backing, frying, or roasting. It is also consumed pickled or as dried, and dried baby eggplant skins serve for stuffing. It is also consumed as processed products like frozen entrees and specialty dips.

[0292] According to some embodiments, the products comprise the DNA (carrying a loss of function mutation in MBP3 gene and at least one of AGL6 and MBP22causing the parthenocarpic phenotype) of the tomato, pepper or eggplant (e.g., paste, dried fruit, juice and the like)

[0293] As used herein the term “about” refers to ± 10 %.

[0294] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0295] The term “consisting of’ means “including and limited to”.

[0296] 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.

[0297] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0298] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0299] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0300] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0301] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0302] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0303] It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.

[0304] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0305] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0306] EXAMPLES

[0307] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0308] MATERIALS AND METHODS

[0309] Plant material and growth conditions

[0310] All tomato genotypes described were in the MP- 1 background, which is referred to as wildtype. The MP-1 cultivar and MP-1 mutant slagl6CR'sglwere previously described in (Barg et al., 1997) and Klap et al., (2017), respectively. The slagl6CR'sgland slmbp22CR'20mutants were grown from their respective seeds, while slmbp3CR'20was obtained following PCR genotyping of the Fl progeny from a cross between slmbp3CR'20(c ) x slmbp3CR'20 / + ($) seeds. PCR-confirmed homozygous slagl6CR'sgl, slmbp22CR'20and slmbp3CR'20mutants were crossbred to attain all described double mutant combinations, as determined by PCR genotyping of their F2 progeny. Tomato and Nicotiana benthamiana seed germination and seedling growth were conducted in a growth chamber under a 16-hour light / 8-hour dark photoperiod at a constant 24 °C. Approximately one-month post-germination, tomato seedlings were transferred to a greenhouse. Here, they were cultivated in 4-liter pots filled with a nutrient-enriched tuff-peat mixture. Meanwhile, N. benthamiana seedlings remained in the growth chamber until required. For the genetic interaction study, seven independent plants from each mutant genotype and MP-1 were grown in an alternating arrangement in a controlled greenhouse.

[0311] Parthenocarpic fruit assay

[0312] For testing parthenocarpic fruit set, flowers at stage 18 (-2 days post-anthesis) (Gupta et al., 2021) from indicated genotype underwent emasculation. These flowers were then allowed to set fruit without any pollination. To qualify as a valid parthenocarpic fruit, three criteria had to be met: the fruit must be seedless, weigh over 15 grams, and exhibit a morphology similar to the MP- 1 wild type.

[0313] Yeast 2-hybrid (Y2H) screening

[0314] Y2H "prey" library construction, screening and classification was performed by HYBRiGENiCS SERVICES (https: / / www(dot)hybrigenics-services(dot)com). For "prey" library construction, mRNA was purified from ~1 mg of total RNA extracted from tomato cv. MP-1 ovaries at stages 15 (-4 DPA) to stage 20 (0 DPA) and at 2-4 DPA. The cDNAs were ligated into the pP6 vector containing the LEU 3 selectable marker. For "bait" construction, the sequence corresponding to the full-length S1AGL6 ORF was codon optimized for yeast expression using the GeneUniversal service (GeneUniversal, USA) and cloned into the gateway pBD-GAL4 vector containing the TRP1 selectable marker. "Bait" and "prey" constructs were transformed into the yeast strain CG1945 and brought together by mating. Positive colonies were selected on dropout media lacking tryptophan, leucine, histidine and supplemented with 0.5 mM 3-amino- 1,2,4- triazole (3-AT), to eliminate weak self-activation background by S1AGL6. Around 66 million clones were screened. The recovered preys were amplified by PCR, sequenced, annotated and attributed a predicted biological score (PBS®) to classify each interaction into categories from very high confidence (A) to lower confidence (D).

[0315] Plasmids construction

[0316] For the CRISPR / Cas9 mutagenesis, two gene-specific gRNAs targeting SIMBP22 and SIMBP3 coding sequence were designed (Table 1). Each gRNA was incorporated in silica into sgRNA consisting of itself followed by a 76-bp generic scaffold and a 7xT Polymerase III terminator sequence. Then, a construct delimited by 5’ -Mini and 3' -H in dill and containing respective sgRNAs in tandem, each under the control of the synthetic Arabidopsis U6 promoter, was artificially synthesized and cloned into the pUC57 plasmid (GENEWIZ, USA). The pUC57- sgRNAs plasmid was digested with Mini and Hindlll, and the released U6::sgRNAl-U6::sgRNA2 fragment was ligated into the compatible sites of the pRCS binary vector alongside the plant codon-optimized version of Cas9 expressed under the constitutive CaMV 35S promoter.

[0317] For expression of SIAGL6 via its native promoter, the 4 kb (SEQ ID NO: 106) and 6 kb (SEQ ID NO: 105) long SIAGL6 promoter fragments (pSlAGL6) and terminator tSlAGL6) (SEQ ID NO: 107) were PCR amplified from MP-1 genomic DNA. The SIAGL6 gene IgSlAGL6) was domesticated through overlapping PCR amplification for GoldenBraid cloning system from MP- 1 genomic DNA. Once amplified, pSlAGL6, tSlAGL6 and domesticated gSlAGL6 were cloned individually into the level 0 pUPD2 plasmid (Sarrion-Perdigones et al., 2011). Using GoldenBraid cloning, domesticated gSlAGL6 was assembled with pSlAGL6 and tSlAGL6 with additional NOS terminator (tNOS, SEQ ID NO: 108) in level 1 binary plasmid pDGB3-Alpha2. Subsequently, the pSlAGL6::gSlAGL6::tSlAGL6::tNOS and pNOS::nptII::tNOS plant kanamycin resistance cassette (in level 1 binary plasmid pDGB3-AlphalR) were assembled in the level 2 binary plasmid pDGB3_Omegal to generate the pDGB3-Omegal-pSlAGL6::gSlAGL6::tSlAGL6::tNOS binary plasmid. For ovule-specific expression of SIAGL6, Snapdragon ovule- specific DefH9 promoter (p efH ) (Spena et al., 2002) and SIAGL6 coding sequence were both domesticated via overlapping PCR amplification from Snapdragon genomic DNA and MP-1 ovule cDNA, respectively, and subsequently each cloned into the GoldenBraid pUPD2 plasmid. Using GoldenBraid cloning, domesticated pDefH9 and S1AGL6 were first assembled with NOS terminator (tNOS) in pDGB3-Alpha2 plasmid. Subsequently, the pDefH9::SlAGL6::tNOS and pNOS::nptII::tNOS were assembled in pDGB3_Omegal plasmid to generate the pDGB-Omegal- pDefH9::SlAGL6::tNOS binary plasmid.

[0318] For BiFC and FRET-FLIM assays, The SIAGL6, SIMBP22, SIMBP3, SIAGL11, SITM5 and SITM29 coding sequences were each PCR amplified from MP-1 ovule cDNA (SEQ ID NOs: 103, 110, 109, 112, 111, respectively). The CaMV 35S (35S) promoter sequence, nuclear localized mCherry (mCherry-NLS), eYFP, mSlNAM2 (Hendelman et al., 2013), domesticated mCerulean (mCrFP), domesticated nuclear localized mCerulean (mCrFP-NLS), domesticated S1AGL6, its truncated versions S1AGL6MI(SEQ ID NO: 113), S1AGL6MIK119(SEQ ID NO: 114), S1AGL6MIK143(SEQ ID NO: 115) and S1AGL6MIK177(SEQ ID NO: 116), S1MBP22, S1MBP3, S1AGL11, S1TM5 and S1TM29 coding sequences were cloned into level 0 pUPD2 plasmid between Bsal restriction sites. The split eYFP partsnYFP (residues l-154)-linker andCYFP (residues 155-240)-linker coding sequences (Table 1) were artificially synthesized (TWIST Bioscience, Israel) and cloned into level 0 pTwist plasmid between Bsal restriction sites. Using GoldenBraid cloning, mCrFP- NLS and mCherry-NLS were assembled with 35S promoter and NOS terminator (tNOS) in pDGB3-Alphal plasmid. mSlNAM2, SIAGL6, SIMBP22, SIMBP3, SIAGL11, SITM5 and SITM29 were assembled with 35S promoter, tNOS and eithernYFP,CYFP and whole eYFP to generate N- terminal translational fusions in pDGB 3 -Alpha 1 plasmid. S1AGL6, S1AGL6MI, S1AGL6MIK119, S1AGL6MIK143and S1AGL6MIK177were assembled with 35S promoter, mCrFP and tNOS to generate C-terminal translational fusions in pDGB3-Alphal plasmid.

[0319] The sequences of all cloned genes were validated through sequencing of corresponding binary plasmids used for transformation into Agrobacterium. All gRNA and primer sequences used are listed in Table 1.

[0320] BiFC assays

[0321] The BiFC constructs were mobilized into Agrobacterium tumefaciens GV 3101 through electroporation. Following PCR validation, transformants were grown in 1 ml LB overnight at 28°C with shaking. Cultures were pelleted by centrifugation next day, subsequently resuspended in 5 ml infiltration buffer (10 mM MgC12, 10 mM MES pH 5.6, 150 pM acetosyringone). The ODeoo of each culture was measured and was diluted up to a specified concentration with infiltration buffer to achieve a final working ODeoo of 0.17, after mixing of indicated cultures for co-infiltration. Mixed cultures were then gently rocked while incubating at 28°C for 2 hours. For transient expression, each culture mixture was infiltrated into young (3rdfrom top) Nicotiana benthamiana leaves of five independent plantlets. The Agroinfiltrated leaves were analyzed 36 hrs post infiltration using a Leica SP8 laser scanning confocal microscope (Leica, Wetzlar, Germany). Imaging of YFP, mCrFP and mCherry-NLS signal was done using the 514 nm, 448 nm and 552 nm laser line with 0.4% laser intensity and the emission was detected in a range of 520-580 nm, 456-500 nm and 560-640 nm, respectively.

[0322] FLIM and FRET analysis

[0323] Donor and acceptor proteins were transiently expressed in N. benthamiana leaves as described for the BiFC assays. Images were acquired by the Leica Stellaris 8 Falcon confocal microscope containing the white laser WLL / supercon. Image acquisition parameters were as follows; resolution 512 X 512, excitation 440 nm, emission 450-510 nm, 20% laser power, line accumulation X8, laser pulse frequency 80Mh (12.5 ns), objective HC PL APO CS2 63X 1.2 water. The fluorescence life time of SlAGL6-mCrFP, SlAGL6MI-mCrFP, SlAGL6MIK119-mCrFP, SlAGL6MIK143-mCrFP, SlAGL6MIK177-mCrFP and mCrFP-NLS donors was measured alone (D) or in the presence of the acceptor (DA). For each treatment ten measurements were performed from ten different cells. Fluorescence measurements were acquired from the whole nucleus. Fluorescence life time was extracted from pixels clusters on a Phasor plot, with mCrFP only behaving as a mono exponential component. FRET was calculated by the equation E=l-(tDA / tD). t is mCrFP fluorescence life time, D - donor, DA - donor in the presence of the acceptor.

[0324] Tomato transformations and mutants isolation

[0325] The binary plasmids pRCS-35S::Cas9-U6::SlMBP22-sgRNAs and pRCS-35S::Cas9- U6::SlMBP3-sgRNAs were transformed into tomato cultivar MP-1 as described previously (Barg et al., 1997). The binary plasmids pDGB3-Omegal-pSlAGL6::gSlAGL6::tSlAGL6::tNOS and pDGB-Omegal-pDefH9::SlAGL6::tNOS, were transformed into the MP-1 mutant slagl6CR'sglin a similar way. Regenerated explants were selected on 100 mg / 1 kanamycin-containing media and primary (To) transformants were identified among them by PCR with respective primer pair (Table 1).

[0326] To isolate SIMBP22 and SIMBP3 CRISPR mutants, TO plants were screened by PCR with specific primers flanking the gRNAs targeted sequences (Table 1) for amplicons containing gel- visible indels. Positive TO plants were backcrossed to MP-1, followed by PCR genotyping of the Fl and F2 progeny to isolate the slmbp22CR'20and slmbp3CR'20homozygous mutants (Figure 5C), which were sequenced and further characterized. Ovules isolation

[0327] Tomato ovules and non-ovule tissues were isolated from slagl6CR'sglpDefH9::SlAGL6-5.4 ovaries (8 per biological replicate) as described in Gupta et al., (2021). Total RNA extraction and reverse transcription quantitative PCR (RT-qPCR)

[0328] Total RNA was extracted from tomato tissues using Bio-Tri RNA reagent (Bio-Lab, Israel) following the manufacturer’ s protocol. First- strand cDNA was prepared from 1.5 pg of total RNA with a Maxima first strand cDNA synthesis kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Real-time quantification of gene expression was performed with at least three independent biological replicates for each sample, and quantification was performed in triplicate. QPCR was performed in StepOnePlus (Thermo Fisher Scientific) with specific primers (Table 1). Relative expression levels were calculated by the comparative delta delta Ct method and normalized to SITIP41. Table 1 - List of primers, gRNAs and split YFP sequences.

[0329]

[0330]

[0331]

[0332]

[0333] EXAMPLE 1

[0334] Ovule-specific expression of SIAGL6 is sufficient to abolish slagl6CR'sglparthenocarpy

[0335] The slagl6CR~sglmutant is facultative parthenocarpic and this phenotype is associated with narrow pale flower petals and ovules missing a distinct endothelium layer (Klap et al., 2017; Gupta et al., 2021). To confirm that lack of S1AGL6 activity is responsible for these mutant phenotypes, the present inventors reintroduced the full-length S1AGL6 gene driven by either its 4 kb or 6 kb putative promoter into the slagl6CR'sglmutant plants (Figures 1A-B), and analyzed the phenotypes of the slagl6CR'sglpSlAGL6::gSlAGL6 (pSlAGL6::gSlAGL6) plants. Examination of slagl6CR'sglpale flower petals revealed the conversion of the adaxial conical petal epidermal cells into flatter and more jigsaw-shaped cells. In contrast, the pSlAGL6::gSlAGL6 plants flower petals exhibited bright yellow color and accordingly their epidermis displayed conical cell morphology, similar to MP-1 flowers (Figure 1C). These observations indicate that S1AGL6 is involved in petal identity, consistent with AGL6 role in determining of floral organ identity in other plant species (Rijpkema et al., 2009; Hsu et al., 2015; Kong et al., 2022). Consistent with their restored flower petal phenotype, histology of pSlAGL6::gSlAGL6 plants ovaries indicated that their ovules contain a typical endothelium layer similar to MP-1 ovules (Figure ID). Analysis of pSlAGL6: : gSlAGL6 plants over two generations showed that their progeny consistently produced only seeded fruits (Tl: (pSlAGL6(4Kb)::gSlAGL6 4 plants 58 fruits; pSlAGL6(6Kb)::gSlAGL6 4 plants 36 fruits), T2: (pSlAGL6(4Kb)::gSlAGL6 2 plants 30 fruits; pSlAGL6(6Kb)::gSlAGL6 4 plants 43 fruits)) (Figure IE). Together, these results unequivocally demonstrate that the observed parthenocarpy in slagl6CR~sgl, along with the associated flower and ovule phenotypes, is a direct consequence of the absence of S1AGL6 activity. In anthesis ovaries, approximately half of S1AGL6 mRNA accumulates in ovules, with the rest in the placenta, columella, and septa (Figure 2B). To test whether S1AGL6 functions from the ovules to suppress parthenocarpy, the present inventors attempted to complement slagl6CR~sglparthenocarpy by expressing S1AGL6 under the Snapdragon DEFICIENS E10M0E0G 9 DefEl9) promoter, known for its specific activity in tomato mature ovules (Rotino et al., 1997; Spena et al., 2002) (Figure 1A). Nine slagl6CR'sglpDefH9::SlAGL6 (pDefH' 9::SlAGL6) transgenic TO plants were obtained and analyzed. Of them, plant #17 did not produce fruits, plants #2 and #18 produced abnormally large and heart shaped, respectively, parthenocarpic fruits, and the rest of the TO plants produced normal looking fruits with variable rates of parthenocarpy (Table 2), suggesting that none was fully complemented.

[0336] Table 2 - Summary of obtained slagl6CR~sglPDEFH9::SIAGE6 TO plants and their fruit phenotypes. N / A; not applicable.

[0337] Surprisingly, among all the T1 progeny of plant #5, the present inventors identified 4 pDefH9: :SIAGE6 plants that produced between 0 % - 20 % parthenocarpy. The pDefEl9::SlAGE6- 5.4 plant that produced exclusively seeded fruits was further examined (Figures IB and IE). It was found that its loss-of-parthenocarpy persisted through T2 (2 plants, 0 / 35 seedless / seeded fruits) and T3 (10 plants, 0 / 64 seedless / seeded fruits ) generations, indicating the stability of this trait. In line with the ovule- specificity of the DefH9 promoter, the pDefH9: :SlAGL6-5.4 flowers retained slagl6CR'sglfloral traits, namely, narrow pale flower petals that their adaxial epidermis exhibits atypical flat cells (Figure 1C). In contrast, histological examination of pDefH9::SlAGL6- 5.4 ovules revealed a characteristic endothelium layer, mirroring wild-type MP-1 (Figure ID). Quantitation of S1AGL6 transgenic transcripts within pDefH9::SlAGL6-5.4 whole stage 18 ovaries and their isolated tissues demonstrated that nearly 97.3 % of all transcripts accumulated within the ovules (Figure IF), supporting targeted expression. These results suggest that SIAGL6 expression in slagl6CR'sglovules is sufficient to recover their abnormal endothelium phenotype. In addition, they reinforce the hypothesis that slagl6CR'sglparthenocarpy stems from a lack of functional S1AGL6 in its mutant ovules.

[0338] EXAMPLE 2

[0339] Identification of potential interactors of S1AGL6 in the ovules

[0340] To identify partner proteins that may interact with S1AGL6 in ovules and might play a role in parthenocarpy suppression, the full-length S1AGL6 protein was used as 'bait' (Figure 2A) in a Y2H screen. This screen employed a 'prey' library constructed from mRNAs in arrested and fruitsetting ovaries. Out of 66 million tested clones, 373 yielded positive interactions, identified as interacting open reading frames (ORFs) (Table 3). The majority of them (282) were classified as category A (very high confidence in the interaction) and 16 as category B (high confidence in the interaction), all encoding MIKCcMTFs. The remaining 75 positive clones were classified as the less confident interaction categories (C, D, and N / A) and corresponded to 41 different proteins each represented by only one or few clones (Table 3). The 282 category A clones corresponded to six MTFs (Figure 2A and Table 3). Expression analysis of the category A interactors in anthesis ovary tissues revealed significant expression in ovules for all except SlAGL42-like, suggesting their potential interactions with S1AGL6 in ovule cells (Figures 2B-C).

[0341] Table 3- Summary of putative protein interactors of S1AGL6 identified in the Y2H screen. *Global

[0342] PBS score from HYBRiGENiCS SERVICES. N / A - not applicable.

[0343] To validate the physical interaction between S1AGL6 and the ovule-expressed category A interactors in planta, the present inventors first employed BiFC assays to test their co-localization in Nicotiana benthamiana epidermal cells. A reconstituted YFP signal was observed in the leaf epidermis nuclei when either S1MBP3, S1AGL11, TM5, S1MBP22 and TM29 were co-expressed with S1AGL6, but not when the non-relevant nuclear localized mSlNAM2 transcription factor (Hendelman et al., 2013) was co-expressed with S1AGL6 (Figure 3A). The fluorescence lifetime of mCerulean (mCrFP) fused to S1AGL6 was then measured and a FRET-FLIM analysis was performed to detect dimerization of S1AGL6 and its interactors in planta. The present inventors found that the fluorescence lifetime of mCrFP fused to S1AGL6 in the presence of enhanced Yellow fluorescent protein (eYFP) fused to non-interacting S1NAM2 was not different from that of nuclear localized free mCrFP indicating that FRET was not taking place. Conversely, when SlAGL6-mCrFP was co-expressed with eYFP fused to either S1MBP3, TM5, S1MBP22, S1AGL11 and TM29, fluorescence lifetime was significantly shortened indicating the occurrence of FRET (Figures 3B-C). Taken together the present results corroborate the Y2H results and suggest that S1AGL6 can heterodimerize in the nucleus with tested category A interactors in planta.

[0344] BiFC assays were further utilized to investigate pairwise interactions among the category A interactors and assess their homodimerization potential, including that of S1AGL6. A clear nuclear-localized YFP signal was observed when TM29 was coexpressed with S1MBP22 and TM5, as well as when TM5 was coexpressed with S1MBP22, indicating co-localization of the split YFP fusion proteins and suggesting potential interactions. Additionally, a nuclear-localized YFP signal was detected when nYFP-SlAGL6 was coexpressed with CYFP-S1AGL6, demonstrating that S1AGL6 can homodimerize in planta. No YFP signal was observed for other tested proteins (Figure 3D), indicating a lack of direct interaction between them.

[0345] EXAMPLE 3

[0346] S1AGL6 forms multimeric protein complexes with S1MBP3 and S1MBP22 through its K domain

[0347] The present results indicate that S1AGL6 can heterodimerize with S1MBP3, TM5, S1MBP22, S1AGL11, and TM29 (Figure 3 A), none of which interact with themselves or each other, except for TM5 and TM29, both of which interact with each other and S1MBP22 (Figure 3D). In several plant species, AGL6 exerts its functions by interacting with other MTFs in multimeric complexes (Hsu et al., 2015; Dreni and Zhang, 2016b; Kong et al., 2022).

[0348] The present inventors therefore asked whether S1AGL6 (Figure 4A), may act as a 'bridge' between the non-interacting S1MBP22 and S1MBP3 to form multimeric protein complexes. To answer that, initially, the present inventors performed BiFC assays in N. benthamiana leaves to investigate S1MBP22 and S1MBP3 localization in the absence and presence of S1AGL6. Coexpression of mCrFP-NLS with the split YFP fused S1MBP3 and S1MBP22 pair or either of them with SlAGL6-mCrFP, did not yield a reconstituted YFP signal. Conversely, when mCrFP- S1AGL6 was co-expressed with the split YFP fused S1MBP3, S1MBP22 and both, clear reconstituted YFP signals were observed in the nuclei of the epidermal cells (Figure 4B).

[0349] These results imply that S1AGL6 expression enables the colocalization of S1MBP3 and S1MBP22 individually, and S1MBP3 and S1MBP22 together. The present inventors then measured the fluorescence lifetime of mCrFP and performed FRET-FLIM analysis to find out if S1AGL6, S1MBP3 and S1MBP22 were in molecular proximity to form a genuine protein complex in planta. It was found that the fluorescence lifetime of SlAGL6-mCrFP was significantly shortened (Figure 4C), indicating the occurrence of FRET (Figure 4D), only when it was coexpressed with the split YFP fused pairs of S1MBP3, S1MBP22 and both. These results indicate that S1AGL6 mediates the interaction between S1MBP3, S1MBP22 and both in vivo, and suggest that they form the protein complexes S1MBP3-S1AGL6-S1MBP3, S1MBP22-S1AGL6-S1MBP22 and S1MBP3-S1AGL6-S1MBP22.

[0350] MIKCc-type protein dimerization and tetramerization are primarily mediated by the K- domain, which in SEP3 consist of two a-helices separated by a kink (Rumpier et al., 2018). AlphaFold prediction of the 3D structure of S1AGL6 protein sequence (Figure 41), suggest that its K-domain (Gly81-Phe176) has a similar structure comprising al (Gly81-Leu109), a kink (Leu110- Ser118) and a2 (Val119-Phe176) (Figure 4J). The present inventors thus asked if the S1AGL6 K- domain is responsible for forming the S1MBP3-S1AGL6-S1MBP22 protein complex. To answer that, truncated versions of S1AGL6 that lack the C-terminal domain (Glu177- Leu252) and only comprises MADS-, I- domains and variable parts of the K-domain were produced, as follows: no K domain (S1AGL6MI), the K domain al helix (S1AGL6MIK119), the K-domain al helix and the N- terminal part of a2 helix (S1AGL6MIK143) and the complete K-domain (S1AGL6MIK177) (Figure 4E). Notably, the S1AGL6MIversion is identical to the truncated protein predicted to be translated in the slagl6-2012 loss-of-function mutant (Klap et al., 2017). Thereafter, S1AGL6 ability to mediate interactions between split YFP-fused S1MBP3 and S1MBP22 using BiFC combined with FRET- FLIM assays was determined. These analyses showed that S1MBP3 and S1MBP22 co-localized, as indicated by reconstituted YFP (Figure 4F), and interact, as indicated by FRET (Figures 4G- H), in the presence of S1AGL6MIK177and S1AGL6MIK143, but not in the presence of S1AGL6MIor S1AGL6MIK119. These results corroborate the specificity of the interaction between S1AGL6, S1MBP3 and S1MBP22 proteins and indicate that it is mediated by the a2 helix of the SIAGL6 K- domain, independent of the C-terminal domain. Additionally, the inability of SIMBP3 and SIMBP22 to interact in the presence of S1AGL6MIprovides a potential explanation for the nonfunctionality of the slagl6-2012 mutant (Klap et al., 2017). EXAMPLE 4

[0351] SIMBP3 plays a synergistic role with SIAGL6 and SIMBP22 in suppression of parthenocarpy

[0352] To investigate whether S1MBP22 and S1MBP3 play a role in the SlAGL6-parthenocarpy pathway, the present inventors first generated CRISPR mutants for each gene (Figure 5A). The resulting slmbp3CR'20and slmbp22CR'20mutant alleles bore large deletions spanning their entire first exon, including the start codon, that disrupted the functions of respective proteins (Figures 5B). The relatively large deletions in slmbp3CR'20and slmbp22CR'20enabled the genotyping of homozygous and heterozygous mutant plants by PCR of their genomic DNA (Figure 5C). To evaluate their phenotypes, the homozygous single mutants were grown in an alternating arrangement alongside the MP-1 parental line, slagl6CR'sgl, and their heterozygous and homozygous double mutants in a controlled greenhouse under fertilization-permissive conditions. The slmbp3CR'20and slmbp22CR'20homozygous mutants developed normal looking flowers and the morphology of their ovaries resembled those of slagl6CR'sgland MP-1 ovaries (Figure 6A), indicating that ovary growth was not initiated before anthesis. In line with that, the fruits of slmbp3CR'20and slmbp22CR'20homozygous mutants, like slagl6CR'sglfruits, displayed MP-1 morphology (Figure 6B). While slmbp22CR'20fruits exhibited characteristic jelly fill, slmbp3CR'20fruits exhibited a fully fleshed phenotype (Figure 6C), consistent with the reported phenotypes of slmbp3 loss-of-function mutants (Zhang et al., 2019; Huang et al., 2021). As for seed bearing, under pollination permissive conditions, slmbp3CR'20plants rarely produced a seedless fruit, like MP-1. In contrast, approximately 11% of slmbp22CR'20fruits were seedless. This seedless rate was significantly more than MP-1 (p = 0.0067 t-test), but approximately 3.6 fold less than the mean rate of seedless fruits in slagl6CR'sgl(42 %; Figure 6D). To confirm slmbp22CR'20parthenocarpy, fruit set was assayed after stage 18 flower emasculation. In the absence of pollination, none (n = 32) of the emasculated MP-1 flowers produced fruits, but 15 % (n = 40) of slmbp22CR'20emasculated flowers did set fruits in the absence of fertilization thus confirming that the seedless fruits produced by slmbp22CR~20are parthenocarpic.

[0353] To investigate functional redundancy in parthenocarpy suppression between S1AGL6, S1MBP22 and S1MBP3, the present inventors studied their genetic interactions by co-inactivating them. S1AGL6, SIMBP22 and SIMBP3 double homozygous and heterozygous mutants were generated by crossing slagl6CR'sgl, slmbp3CR'20and slmbp22CR'20to each other and screening the F2 progeny for respective mutants by PCR genotyping (Figure 5C). At anthesis, the size of the ovaries of all double mutants were not significantly larger than those of their respective single mutants (Figure 6A). The fruits of all generated mutants displayed MP-1 morphology, whereas the slmbp3CR'20slmbp22CR'20fruits exhibited a slightly pointy blossom end (Figure 6B). The fruits of all generated mutants exhibited MP- 1 -like jelly fill, except those homozygous to slmbp3CR'20that exhibited a fully fleshed phenotype (Figure 6C). Compared with the no and poor parthenocarpy of slmbp3CR'20and slmbp22CR'20single mutants, respectively, the rate of seedless fruits produced by their homozygous double mutant (96 %) was much higher. Interestingly, a dramatic increase in the rate of seedless fruits was also observed in the slmbp3CR'20slagl6CR'sgldouble mutant (94 %) compared to its respective single mutants (Figure 6D). The enhanced parthenocarpic phenotypes of slagl6CR~sglslmbp3CR~20and slmbp22CR~20slmbp3CR~20double mutants compared to their respective single mutants suggest that SIMBP3 plays redundant roles with SIAGL6 and SIMBP22 in suppression of parthenocarpy. Conversely, the mean rate of seedless fruits production in slmbp22CR~20slagl6CR~sgl(40 %) was not different that of slagl6CR~sgl(Figure 6D), suggesting that the SIAGL6 gene has an epistatic effect over the SIMBP22 gene. The parthenocarpy of double mutants was further confirmed by flower emasculation. In the absence of pollination, fruit production was observed in 69 % (n = 26) of slmbp22CR-20slagl6CR-sgl, 89% (n = 35) of slmbp3CR~20slagl6CR~sgland 91% (n = 35) of slmbp22CR~20slmbp3CR~20emasculated flowers. In addition, it was observed that the heterozygous double mutants that contain homozygous slagl6CR'sglor homozygous slmbp22CR'20but not homozygous slmbp3CR'20display an increase in seedless fruit production compared to respective single mutants (Figure 6D).

[0354] These results suggest that the genetic interaction of the SIMBP22 gene is do sage- sensitive to the levels of SIMBP3 and S1AGL6, and the genetic interaction of S1AGL6 is dosage-sensitive to the levels of SIMBP3 and SIMBP22. In addition, the increase in seedless fruit production in slagl6CR~sglslmbp22CR~20 / + compared to slagl6CR~sglslmbp22CR'20(Figure 6D) suggest that complete loss of SIMBP22 activity enhance the suppression of parthenocarpy.

[0355] Analysis of fruit numbers per plant across various genotypes revealed a positive correlation between parthenocarpy rate and fruit yield. The parthenocarpic genotypes slmbp3CR~20slagl6CR~sgland slmbp3CR'20slmbp22CR'20, which exhibited nearly 100% parthenocarpy, produced approximately 2.9- and 2.5-fold more fruits, respectively, than MP-1. Similarly, the slagl6CR'sglslmbp22CR'20 / + and slagl6CR'sglslmbp3CR'20 / + heterozygous double mutants, which exhibited approximately 72 % parthenocarpic fruits, yielded approximately 2.5 times more fruits than MP- 1. The slagl6CR~sgl, slmbp22CR'20slagl6CR~sgl, slmbp22CR'20slagl6CR~sgl1+ double mutants, which exhibited about 36 % parthenocarpic fruits, produced about 1.8-fold more fruits than MP-1. In contrast, the slmbp22CR'20slmbp3CR'20l+ and slmbp22CR'20weak parthenocarpic mutants and the non-parthenocarpic genotypes did not significantly differ in fruit yield compared to MP- 1 (Figure 6E). Fruit weight analysis revealed around 15 % decrease in genotypes containing homozygous slmbp22CR'20, except for slmbp3CR'20slmbp22CR'20(Figure 6F). Surprisingly, other homozygous and heterozygous double mutants with increased fruit yield (Figure 6D), including the high- yielding slmbp3CR'20slagl6CR'sgl, produced fruits with an average weight comparable to MP-1 (Figure 6F).

[0356] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0357] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

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Claims

WHAT IS CLAIMED IS:

1. A Solanaceous plant exhibiting a parthenocarpy and down-regulation in activity or expression of MBP3 and at least one of AGL6 and MBP22, as compared to a control plant, wherein said control plant is of the same genetic background and developmental stage.

2. The plant of claim 1, wherein said down-regulation is a result of a loss-of function mutations in MBP3 and AGL6.

3. The plant of claim 1, wherein said down-regulation is a result of a loss-of function mutations in MBP3 and MBP22.

4. The plant of claim 1, wherein said down-regulation is a result of a loss-of function mutations in MBP3, AGL6 and MBP22.

5. The plant of any one of claims 1-4, exhibiting fruit yield defined as an average fruit weight that is at least the same as that of said control plant.

6. The plant of any one of claims 1-5, exhibiting fruit yield defined as an average number of fruits of the plant per growing season which is at least the same as that of said control plant.

7. The plant of any one of claims 1-5, exhibiting fruit yield defined as an average number of fruits of the plant per growing season which is at least 2-fold higher than that of said control plant.

8. The plant of any one of claims 1-5, exhibiting fruit yield defined as an average number of fruits of the plant per growing season which is at least 4-fold higher than that of said control plant.

9. The plant of any one of claims 1-5, exhibiting fruit yield defined as an average number of fruits of the plant per growing season which is at about 2 to 5-fold higher than that of said control plant.

10. A Solanaceous plant exhibiting a parthenocarpy and down-regulation in activity of AGL6, MBP3 and / or MBP22, as compared to said control Solanaceous plant, wherein said control plant is of the same genetic background and developmental stage, wherein said down-regulation in activity is manifested by aberrant interaction between either AGL6-MBP3, AGL6-MBP22, MBP3- MBP22, and wherein the plant is not a tomato plant which comprise a C to t substitution at exon 3 that generate a stop codon after amino acid residue Tyr89.

11. The plant of any one of claims 1-10, wherein said down-regulation is a result of a loss of function mutation in any of MBP3, AGL6 and / or MBP22.

12. The plant of claim 11, wherein said loss-of-function mutation is in a K domain of AGL6or MBP22 and optionally MBP3.

13. The plant of claim 12, wherein said loss-of-function mutation is a deletion in at least an a2 helix of said K domain, optionally wherein said loss-of-function mutation results in a peptide comprising a MADS domain, an I domain and / or a C domain.

14. The plant of claim 12, wherein said loss-of-function mutation results in a peptide, which comprises an a2 helix but is devoid of any or all of MADS domain, an I domain and a C domain, said peptide serving as a dominant negative peptide.

15. The plant of any one of claims 1-14, being a tomato.

16. The plant of any one of claims 1-14, being a tomato, eggplant or pepper.

17. The plant of claim 15, wherein fruit of said tomato comprises a jelly fill.

18. The plant of claim 15 or 17, being a processing tomato.

19. The plant of claim 15 or 17, being a determinate tomato.

20. The plant of claim 15 or 17, being an indeterminate tomato.

21. The plant of claim 15 or 17, being a semi-determinate tomato.

22. The plant of any one of claims 1-21, being of an elite line.

23. The plant of any one of claims 1-22, being transgenic.

24. The plant of any one of claims 15-22, wherein said tomato is of a species selected from the group consisting of Lycopersicon esculentum, Lycopersicon cerasiforme, Lycopersicon pimpinellifolium, Lycopersicon cheesmanii, Lycopersicon parviflorum, Lycopersicon chmielewskii, Lycopersicon hirsutum, Lycopersicon penellii, Lycopersicon peruvianum, Lycopersicon chilense and Solanum lycopersicoides.

25. The plant of any one of claims 18-22 and 24, wherein said tomato is selected from the group consisting of a single fruit per truss, branched tomato and cherry tomato.

26. The plant of any one of claims 1-25, wherein said parthenocarpy is facultative parthenocarpy optionally manifested under heat or cold stress.

27. The plant of any one of claims 1-25, being homozygous for a mutation or mutations conferring said down-regulation in activity or expression of MBP3 and at least one of AGL6 and MBP22.

28. The plant of any one of claims 1-25, being heterozygous for a mutation or mutations conferring said down-regulation in activity or expression of MBP3 and / or MBP22.

29. The plant of claim 28, wherein said plant is AGL6 / MBP3+ or AGL6 / MBP22+.

30. The plant of any one of claims 1-26 being an inbred.

31. The plant of any one of claims 1-30, comprising a silencing agent for downregulation in activity or expression of MBP3 and at least one of AGL6 and MBP22.

32. The plant of any one of claims 1-30, exogenously expressing a nuclease selected from the group consisting of a meganuclease, an RNA-guided DNA endonuclease, a zinc-finger nuclease and a TALEN.

33. A fruit of the plant of any one of claims 1-32.

34. A seed of the plant of any one of claims 1-32.

35. A hybrid seed produced of the plant of claim 1.

36. An edible processed product of the plant or fruit of any one of claims 1-33.

37. The processed product of claim 36 selected from the group consisting of a tomato paste, a ketchup, a tomato sauce a tomato soup, a tomato juice, a tomato powder, a tomato dice, a crushed tomato, a chopped tomato and a tomato concentrate.

38. A method of producing a processed product, the method comprising processing the fruit of claim 33 to produce an edible processed product.

39. A method of producing the plant of any one of claims 1-32, the method comprising down-regulating expression or activity of MBP3 gene and at least one of AGL6 and MBP22 in the plant.

40. The method of claim 39, wherein said down-regulating MBP3 gene and at least one of AGL6 and MBP22 is in the same plant.

41. The method of claim 39, wherein said down-regulating MBP3 gene and at least one of AGL6 and MBP22 is in different plants and further crossing to result in a plant where MBP3 gene and at least one of AGL6 and MBP22 are down-regulated.

42. The method of any one of claims 39-41, wherein said down-regulating is effected by treating the plant with an DNA editing agent.

43. A method of breeding comprising selfing or crossing the plant of any one of claims 1-18.

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