Anti insect compositions, methods for producing same, and use of same

Abstract

The present invention provides steroidal glycoalkaloids (SGA), and compositions including same. Further provided arc methods for synthesizing SGA, and using same.

Description

ANTI INSECT COMPOSITIONS, METHODS FOR PRODUCING SAME, AND USEOF SAMEREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (YEDA-PLANCK-P-048-PCT.xml; size: 30,021 bytes; and date of creation: May 5, 2025) is herein incorporated by reference in its entirety.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of Israel Patent Application No. 312663 titled “ANTI INSECT COMPOSITIONS, METHODS FOR PRODUCING SAME, AND USE OF SAME” filed 6 May 2024, and of U.S. Provisional Patent Application No. 63 / 689,728, titled “ANTI INSECT COMPOSITIONS, METHODS FOR PRODUCING SAME, AND USE OF SAME” filed 1 September 2024. The contents of both applications are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0003] The present invention relates to anti-insect compositions, methods for preparing same, and methods of using same.BACKGROUND

[0004] Solanaceae plants, which include some of man’s favorite food crops (tomato, potato and eggplant), have evolved to synthesize cholesterol, which is converted into a wide variety of specialized metabolites. Black nightshade (Solatium nigrum), a medicinal plant belonging to the Solanaceae family, produces two classes of specialized metabolites from this common cholesterol precursor: steroidal saponins in leaves, and steroidal glycoalkaloids in berries. Steroidal glycoalkaloids are toxic anti-nutrients (e.g., a-tomatine in tomato, a-solanine and a-chaconine in potato) with well-characterized roles in plant defense. Steroidal saponins exhibit detergent-like properties and hold significant interest for pharmaceutical, cosmetic and food industries. However, the ecological roles of steroidal saponins remain unknown.

[0005] Although many of the genes required for the biosynthesis of steroidal glycoalkaloids (SGAs) have been identified in tomato and potato, efforts to heterologously reconstitute the biosynthesis of steroidal metabolites have never been successful. Here the inventors reportthe discovery of a non-catalytic protein, GLYCOALKALOID METABOLISM15 (GAME 15) that is essential for the production of cholesterol derived steroidal saponins and glycoalkaloids. Knock-out of game15 in 5. nigrum led to leaves completely lacking steroidal saponins, which in turn revealed the long undiscovered role that steroidal saponins play in plant defense. Steroidal saponin-deficient 5. nigrum plants were remarkably susceptible to a natural herbivore of nightshade, leafhopper Empoasca decipiens, as well as the agricultural ‘super-pest’, Colorado potato beetle (Leptinotarsa decemlineata Say). Furthermore, SGAs typically produced in the berries of 5. nigrum were not detected in game! 5 mutant lines, indicating that this scaffold protein is likely required for both saponin and SGA biosynthesis. The discovery of a non-catalytic protein crucial for both steroidal saponin and glycoalkaloid production opens new opportunities for metabolic engineering of these important classes of natural products that have medicinal, and as shown here, a potential role as a new target in crop protection.

[0006] There is still a great need for insecticide compositions, such as including uttroside B, as well as methods of preparing / producing same, and use of same for controlling, e.g., preventing and / or treating a phytopathogenic infection in a plant.SUMMARY

[0007] According to the first aspect, there is provided a composition comprising an effective, and an agriculturally acceptable carrier, wherein the composition being an insecticide.

[0008] According to another aspect, there is provided a method for controlling an insect infection in a plant, the method comprising contacting the plant with an effective amount of a compound represented by Formula I.

[0009] According to another aspect, there is provided a method for synthesizing a compound represented by Formula I, the method comprising the steps: (a) providing a cell comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 2, a third polynucleotide comprising the nucleic acidsequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and (b) culturing the cell from step (a) such that at least one protein being encoded by any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula I.

[0010] According to another aspect, there is provided an extract obtained according to the method of the invention.

[0011] According to another aspect, there is provided an artificial DNA molecule comprising: (a) a first nucleic acid sequence encoding the enzyme GLYCOALKALOID METABOLISM 6 (GAMES) comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) a second nucleic acid sequence encoding the enzyme GAMES comprising the amino acid sequence set forth in SEQ ID NO: 11; (c) a third nucleic acid sequence encoding the enzyme GAME11 comprising the amino acid sequence set forth in SEQ ID NO: 12; and (d) a fourth nucleic acid sequence encoding the enzyme GAME15 comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0012] According to another aspect, there is provided a plasmid or an expression vector comprising the artificial DNA molecule of the invention.

[0013] According to another aspect, there is provided a transgenic cell comprising any one of: (a) the artificial DNA molecule of the invention; (b) the plasmid or expression vector of the invention; or (c) both (a) and (b).

[0014] In some embodiments, the compound represented by the Formula I is biosynthetically produced.

[0015] In some embodiments, biosynthetically produced is by a recombinant or a transgenic cell.

[0016] In some embodiments, the recombinant or transgenic cell is any one of: a unicellular organism, a cell of a multicellular organism, and a cell in a culture.

[0017] In some embodiments, the unicellular organism comprises a fungus or a bacterium.

[0018] In some embodiments, the fungus is a yeast cell.

[0019] In some embodiments, the compound represented by the Formula I is present in the composition as a purified extract of a Solanum plant.

[0020] In some embodiments, the Solatium plant is Solarium nigrum.

[0021] In some embodiments, the controlling comprises treating and / or preventing.

[0022] In some embodiments, the controlling comprises increasing resistance of the plant to the insect.

[0023] In some embodiments, the controlling comprises: repelling the insect from the plant, reducing feeding rate of the insect on the plant, reducing number of eggs laid by the insect on the plant, or any combination thereof.

[0024] In some embodiments, the insect is any one of: Empoasca decipiens, Leptinotarsa decemlineata, and both.

[0025] In some embodiments, the artificial DNA molecule further comprises a fifth polynucleotide encoding a protein being a glucose transferring (GT) enzyme.

[0026] In some embodiments, the at least one protein comprises an amino acid sequence set forth in any one of SEQ ID Nos: 10-13.

[0027] In some embodiments, the culturing comprises supplementing the cell with an effective amount of cholesterol.

[0028] In some embodiments, the artificial DNA molecule is an expression vector.

[0029] In some embodiments, the cell is a prokaryote cell or a eukaryote cell.

[0030] In some embodiments, the cell is a transgenic cell or a cell transfected with the artificial DNA molecule.

[0031] In some embodiments, the method further comprises a step before step (a), comprising introducing or transfecting the cell with the artificial DNA molecule.

[0032] In some embodiments, the method further comprises a step comprising isolating, extracting, purifying, or any combination thereof, the compound represented by Formula I.

[0033] 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.

[0034] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0035] Figs. 1A-1B include a biosynthetic pathway and a diagram showing a proposed steroidal saponins and glycoalkaloids (SGAs) biosynthesis in black nightshade (Solatium nigrum) and discovery of GAME 15 through co-expression network analysis. (1A) Simplified biosynthetic pathway for uttroside B, a major steroidal saponin in leaves and SGAs (a-solasonine, a-solamargine, malonyl-solamargine) in green berries of 5. nigrum, starting from cholesterol precursor. Different color shades represent the common and tissue specific steps in biosynthetic pathways. The earlier hydroxylation steps catalyzed by GAME6, GAME8 and GAME 11 are highlighted in red on cholesterol backbone. Dashed arrows represent multiple biosynthetic reactions whereas solid arrows represent a single step. GAME: GLYCOALKALOID METABOLISM; Glc: Glucose; Gal: Galactose; Xyl: Xylose; Rha: Rhamnose; DOX: 2-oxoglutarate dependent dioxygenase; UGT: UDP- glycosyltransferase. (IB) Co-expression network analysis using GAME6, GAME8 and GAME 11 as baits in 5. nigrum. Red dots represent the baits (GAME6, GAME8 and GAME 11). The circle of black dots in the middle represents genes co-expressed with all the baits (r-value > 0.80; 363 genes in total). GAME15 and four UGTs (UGT1-4) co-expressed with all baits are shown in enlarged yellow and orange dots respectively.

[0036] Figs. 2A-2F include sequences, chemical structure, graphs, and fluorescent micrographs showing that ER- localized GAME 15 is indispensable for the production of steroidal saponins and SGAs in S. nigrum. (2A) Schematic representation of GAME15 gene (SEQ ID NO: 4)with location of guide RNAs and game15ko mutant sequences. Three independent game15ko mutant lines were generated using sgRNAl (SEQ ID NO: 5), sgRNA2 (SEQ ID NO: 6) or sgRNA3 (SEQ ID NO: 7). Protospacer adjacent motifs (PAMs) and inserted nucleotides are marked in bold and red respectively, while deleted nucleotides are replaced by dashes. (2B-2C) Levels of uttroside B in leaves (2B) and SGAs (a- solamargine, a-solasonine and malonyl-solamargine) in leaves and green berries (2C) of wild-type (WT) and game15ko mutant lines as determined by LC-MS. The values indicate means of biological replicates ± standard error (n=3). nd, not detected. (2D-2E) Levels of cholesterol in leaves (2D) and green berries (2E) of WT and gamel5ko mutants as determined by GC-MS. The values indicate means of biological replicates ± standard error (n=3). (2F) Confocal images showing the sub-cellular localization of GAME15. Enhanced green fluorescent protein (eGFP) was used as a reporter and fused to the C-terminus ofGAME 15 and co-infiltrated along with an endoplasmic reticulum cellular compartment marker (AtWAK2:mCHERRY-HDEL), in 3-week-old N. benthamiana leaves. Each panel (upper and lower) contains images of mCHERRY, eGFP, both channel merged and bright field. Scale bars: upper panels 20 pm, lower panels 10 pm.

[0037] Figs. 3A-3G include photographs, a chromatogram, and graphs showing that GAME 15 knockout reveals that steroidal saponins play a key role in plant defense. (3A) Greenhouse observation of WT, game15ko#l and game15ko#2 S. nigrum plants. (3B) LC- MS trace of uttroside B in WT and gamel5ko#l leaves. (3C) Empoasca choice assay. WT detached leaves were paired with either game!5ko#l or game15ko#2 leaf (n=10), and one individual Empoasca. Damage and position of Empoasca was recorded one week later (P < 0.0001). (3D) leafhopper of the genus Empoasca on a S. nigrum leaf. (3E) Colorado potato beetle (CPB) on a S. nigrum leaf. (3F) “Forced-feeding” experiment with CPB. Single detached leaves of either WT, game15ko#l or game15ko#2 were placed individually together with one beetle (n=10). Damage was recorded after 6 hours (P < 0.001 and P < 0.0001). (3G) CPB choice assay, with detached leaves of WT paired with either game15ko#l or game15ko#2 leaves, together with one beetle (n=20) (P <0.001). P values were determined for the choice experiments by unpaired two-tailed t tests and by one-way analysis of variance (ANOVA) for the “forced feeding” experiment. *P < 0.05; **P < 0.01; ***P< 0.001; ****P < 0.0001.

[0038] Figs. 4A-4B include a chromatogram and a heatmap showing uttroside B levels and GAME genes expression in S. nigrum tissue types. (4A) Aligned chromatograms (LC-MS) showing uttroside B levels in young leaves, old leaves, stem and roots of S nigrum plants. (4B) Expression of GAME genes in different tissues (young leaves, old leaves, stem and roots) of S. nigrum plants (RNA seq expression data). Normalized fragments per kilobase of transcript per million mapped reads (FPKM) values were used to infer the expression profile.

[0039] Fig. 5 includes a non-limiting scheme and sequences showing GAME 15 knockout (game 15 ko) displayed mutations. Three independent (game 15 ko#l , game 15 ko#2, and game 15 ko# 3) game 15 mutant lines were generated in .S', nigrum. Three biological replicates (n=3) from each line (e.g., for line game 15 ko#l ; game 15 ko# 1.1, game 15 ko# 1.2, and game 15 ko# 1.3 are three biological replicates collected from three independent plants) were used for mutational analysis Protospacer adjacent motifs (PAMs) and inserted nucleotides are marked in bold and red respectively, while deleted nucleotides are replaced by dashes. For game 15ko#2.2 and game 15ko#2.3 an insertion of +93 bp and +86 bp, respectively, was noted between sgRNA#l and sgRNA#2 during DNA sequencing analysis.

[0040] Fig. 6 includes a photograph showing that knockout of GAME 15 (game 15 ko) does not affect the normal growth and development of edited 5. nigrum plants compared to wild type (WT). Representative image is shown for ~2-months old game 15ko and WT 5. nigrum plants.

[0041] Figs. 7A-7B include chromatograms showing that steroidal saponins and SGAs are lost in game! 5 mutant S. nigrum plants. (7A) Aligned chromatograms (LC-MS) of uttroside B, major steroidal saponin in leaves of wild type (WT) and game15ko mutant plants. (7B) Aligned LC-MS chromatograms showing levels of main SGAs ( α-solasonine, α-solasonine and malonyl- solamargine) in green unripe berries of WT and game15ko plants. Three independent (game15ko# 1, game15ko# 2, and game15ko# 5) game 15 mutant lines were generated in S. nigrum. Three replicates collected from each independent mutant line are presented. Uttroside B and malonyl-solamargine standards shown here were isolated from leaves and green berries of S nigrum plants and their structures were confirmed by NMR experiments (Grzech et al., Lucier et al., in revision), m / z mass to charge.

[0042] Figs. 8A-8B include fluorescent micrographs showing that GAME 15 does not localize to the plasma membrane or the nucleus. (8A-8B) Confocal images of the sub-cellular localization of GAME 15 together with either mCHERRY plasma membrane (8A) or mCHERRYmucleus markers (8B) in transiently expressed N. benthamiana leaves. Each panel contains images of mCHERRY, eGFP, both channel merged and bright field. Scale bar: (8A) 5 pm for the upper panels, and 2 pm for the lower panels; (8B) 20 pm for the upper panels, and 10 pm for the lower panels.

[0043] Figs. 9A-9B include fluorescent micrographs showing that GAME6 co-localizes with GAME15 in ER membrane. (9A-9B) Confocal images of the sub-cellular localization of GAME6 fused to mCERULEAN marker together with mCHERRY:ER and GAME15:eGFP (9A) and only with mCHERRY:ER markers (9B) in N. benthamiana leaves. Each panel contains images of mCerulean, mCHERRY eGFP both channel merged and bright field. Scale bar 10 pm.

[0044] Figs. 10A-10B include fluorescent micrographs showing that GAME8 co-localizes with GAME 15 in ER membrane. (10A-10B) Confocal images of the sub-cellular localization of GAMES fused to mCERULEAN marker using mCHERRY:ER and GAME15:eGFP (10A) and exclusively mCHERRY:ER markers (10B) in transiently expressed N. benthamiana leaves. Each panel contains images of mCERULEAN, mCHERRY, eGFP, both channel merged and bright field. Scale bar 5 pm upper panels, and 10 pm lower panels.

[0045] Fig. 11 includes chemiluminescent photographs showing that a split luciferase complementation assay in N. benthamiana leaves suggests interactions between GAME 15 and other GAMEs (GAME6, GAME8, and GAME11). GAME15 was fused to the N- terminus of the luciferase fragment and co-infiltrated with the C-terminus of the luciferase fragment fused to the potential partners (GAME6, GAME8, and GAME 11). Pictures of N. benthamiana leaves (n=3, three biological replicates collected from three independently infiltrated N. benthamiana plants) were taken 48 h post Agro -infiltration. Four different combinations of cLUC and nLUC vectors (shown as numbers 1, 2, 3, and 4) were infiltrated each time into single leaf of N. benthamiana. Three combinations 1, 2, and 4 served as a negative control (NC) in the split luciferase assay experiments. EV, empty vector. The experiment was repeated twice.

[0046] Fig. 12 includes chemiluminescent photographs showing that GAME 15 does not interact with SGA pathway specific GAME4 and GAME 12 proteins as well as with AtCYP 90B1 steroidal saponin / SGA pathway unrelated protein. Split luciferase complementation assay in N. benthamiana leaves (n=3 biological replicates) to test the interaction between GAME15 and other potential partner proteins (GAME4, GAME12, and AtCYP90Bl). GAME 15 was fused to the N-terminus of the luciferase fragment and co infiltrated with the C-terminus of the luciferase fragment fused to the potential partners GAME4, GAME 12, and AtCYP90B 1). Pictures of N. benthamiana leaves were taken 48 h post Agro-infiltration. Four different combinations of cLUC and nLUC vectors (shown as numbers 1, 2, 3, and 4) were infiltrated each time into single leaf of N. benthamiana. Three combinations 1, 2, and 4 were used as a negative control (NC) in the split luciferase assay experiments EV, empty vector. The experiment was repeated twice.

[0047] Fig. 13 includes chromatograms, and chemical structures showing heterologous reconstitution of steroidal saponins scaffold in N. benthamiana leaves. Aligned chromatograms LC-MS showing diosgenin (steroidal saponin aglycone) production in the leaves of N. benthamiana following transient expression of GAME 15 and appropriate GAME genes combination. For simplicity, GAME6 GAME8, and GAME11 genes combination was annotated as ‘scaffold genes’, m / z mass to charge.

[0048] Fig. 14 includes photographs showing 3 month old leaves comparison between game15ko and wild type (WT) S. nigrum plants. Leaves from mutant lines show more susceptibility, evident by visible leaf damage due to pest insects naturally occurring in the greenhouse facility (wherein the current study was conducted) compared to the WT plants.

[0049] Fig. 15 includes photographs of a “Forced feeding” bioassay of Colorado potato beetle. Colorado potato beetles feeding bioassay with detached leaves of wild type (WT) and game15ko (#1 and #2) mutant 5. nigrum plants for 6 hours. Note that almost no visible or very little damage is recorded in WT detached leaves.

[0050] Fig. 16 includes photographs showing a choice and feeding bioassay of Colorado potato beetle with saponin deficient game! 5 mutant S. nigrum plants. Adult beetles were placed in a glass jar together with detached leaves of wild type (WT) paired with either game15ko#l or game15ko#2 mutant leaves. Pictures were taken 3 h after the beginning of the experiment. A Representative image is shown. Note that almost no visible or very little damage is recorded in WT detached leaves.

[0051] Figs. 17A-17D include a biosynthetic pathway, chemical structures, and chromatograms showing steroidal saponin pathway engineering in A. benthamiana. (17A) A schematic representation of the saponin (e.g., uttroside B) pathway reconstitution approach in N. benthamiana. Pathway divergent in SGAs and saponins is represented in the dashed line rectangle. Specific stereochemistry displayed by 5. lycopersicum GAME8 (S1GAME8, 25S) and S. melongena GAME8 (SmGAME8, 25R) is shown on structures. Production of saponins (e.g., Uttroside B, highlighted in yellow) was observed upon the transient expression of tomato genes (15 / 6 / 1 l / 4 / 12 / l / 17 / 18 / 2 / 25 / S5<zR2) with SmGAME8 and S1GAME8. S1GAME25 and 5-alpha-reductase (C5 double bond reduction) reaction step marked in the blue color rectangle shape. C5 double bond and single bond on structure is highlighted with oval shape green and blue color, respectively. Proposed tomato and eggplant specific saponin (e.g., Uttroside B) biosynthetic steps highlighted in red and purple color arrow, respectively. Gene combination (e.g. CoreTom- S1GAME 15 / 6 / 11 / 1 / 17 / 18 / 2 / S1GAME8) expressed in N. benthamiana is shown in the solid line rectangle box. (17B) Chemical structures of uttroside B and dehydrouttroside B shown with detected m / z in positive mode run corresponding to [M-HiO+H]4". (17C-17D) Extracted ion chromatogram (EIC) showing the products detected in N. benthamiana and S. nigrum leaf extract. EIC of N. benthamiana leaves infiltrated with an empty vector (EV) represented as a control. EICs were obtained by LC-MS analysis (40 min run). GAME: GLYCOALKALOID METABOLISM; Glc: Glucose; Gal: Galactose; Xyl: Xylose.

[0052] Figs. 18A-18B include chemical structures and chromatograms showing the MS2spectra of saponin products observed in pathway reconstitution in N. benthamiana. (18A) MS2spectrum of the uttroside B from S. nigrum displaying the fragments of the ion of m / z 1197.5918 corresponding to [M-HiO+H]"1". Matching fragments in 5. nigrum uttroside B andproducts (e.g., uttroside B) detected in the pathway reconstitution in N. benthamiana. (18B) MS2spectrum of the dehydrouttroside B from S. nigrum displaying the major fragments of the ion of m / z 1195.5762 corresponding to [M-H2O+H]+. Mass Fragment matching between S. nigrum dehydrouttroside B and products (e.g., dehydrouttroside B) detected in pathway reconstitution in N. benthamiana. GAME: GLYCOALKALOID METABOLISM; Glc: Glucose; Gal: Galactose; Xyl: Xylose; Hex: Hexose, Pent: Pentose. Gene combinations: CoreTom-SIGAME 15 / 6 / 11 / 1 / 17 / 18 / 2 / S1GAME8, CoreEgg-SlGAME15 / 6 / ll / l / 17 / 18 / 2 / SmGAME8.

[0053] Figs 19A-19E include chromatograms and graphs. (19A) LC-MS extracted ion chromatograms (EICs) depicting accumulation of uttroside B (25R ) and c / L-uttrosidc B (25S) in leaf, stem and root of Solanum nigrum. (19B) EICs illustrating solasodine (25S) and tomatidenol (25R ) production by GAME8-1 and GAME8-2 enzymes from .S', nigrum in N. benthamiana. (19C) qPCR analysis of relative expression levels of GAME8-1 and GAME8-2 in S. nigrum tissues. (19D) EICs showing accumulation of uttroside B and 25-epi-uttroside B in WT and SIGAME8 expressing Solanum nigrum leaves. (19E) Fraction of 25-epi-uttroside B in WT and SIGAME8 expressing 5. nigrum. Asterisks indicate statistically significant differences as compared to control or WT determined by Student’s t-test (**** - P<0.0001).DETAILED DESCRIPTIONComposition

[0054] According to one aspect, there is provided an insecticide comprising a compound represented by Formula I:

[0055] In some embodiments, a compound represented by Formula I is also termed herein "Uttroside B".

[0056] In some embodiments, the insecticide comprises a compound represented by Formula I in an effective amount. In some embodiments, an effective amount comprises an amount reducing fitness, wellbeing, survival, of an insect. In some embodiments, aneffective amount comprises an amount reducing fitness, wellbeing, survival, of 50% or more of a population of insects.

[0057] In some embodiments, the insecticide further comprises an agriculturally acceptable carrier.

[0058] As used herein, the term “insecticide” refers to any compound characterized by being capable of reducing fitness, wellbeing, survival, or any combination thereof, of an insect. In some embodiments, an insecticide kills or promotes death of an insect. In some embodiments, an insecticide inhibits or reduces reproduction of an insect. In some embodiments, an insecticide reduces egg hatching (or rate thereof) of an insect. In some embodiments, an insecticide reduces the number of eggs being laid by an insect. In some embodiments, an insecticide induces, promotes, enhances, propagates, or any combination thereof, genetic, genomic, phenotypic, or any combination thereof, developmental aberrations, abnormalities, or defects in a progeny of an insect contacted therewith.

[0059] According to another aspect, there is provided a composition comprising an effective amount of a compound represented by Formula I, and an agriculturally acceptable carrier. In some embodiments, the composition is an insecticide composition.

[0060] In some embodiments, a compound represented by Formula I is biosynthetically produced. In some embodiments, biosynthetically produced is by a recombinant or a transgenic cell.

[0061] In some embodiments, a recombinant or a transgenic cell comprises: a unicellular organism, a cell of a multicellular organism, or a cell in a culture.

[0062] In some embodiments, a unicellular organism comprises a fungus or a bacterium.

[0063] In some embodiments, a fungus comprises a yeast cell.

[0064] In some embodiments, a compound represented by Formula I is present in a composition of the invention as purified extract obtained or derived from a Solarium plant. In some embodiments, a compound represented by Formula I is present in a composition of the invention as purified extract of a Solatium plant.

[0065] In some embodiments, a Solarium plant comprises Solarium nigrum.

[0066] In some embodiments, the composition of the invention comprises at least one saponin. In some embodiments, the composition of the invention comprises one or more saponins. In some embodiments, the composition of the invention comprises a plurality ofsaponins. In some embodiments, a plurality comprises any integer being equal to or greater than 2. In some embodiments, a saponin comprises or is a steroidal saponin.

[0067] In some embodiments, the saponin or the saponin content of the composition of the invention consist essentially of the compound represented by Formula I.

[0068] As used herein, the phrase "consisting essentially of" denotes that a given compound or substance, e.g., compound represented by Formula I, constitutes the vast majority of the active ingredient's portion or fraction of the composition.

[0069] In some embodiments, consisting essentially of means that the compound represented by Formula I constitutes at least 95%, at least 98%, at least 99%, or at least 99.9% by weight, of the active ingredient(s) of the composition, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0070] In some embodiments, consisting essentially of means that the compound represented by Formula I constitutes at least 95%, at least 98%, at least 99%, or at least 99.9% by weight, of the saponin(s) of the composition, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0071] In some embodiments, the active ingredient(s) of the composition comprise or consist of the compound represented by Formula I. In some embodiments, the active ingredient(s) of the composition comprise or consists essentially of the compound represented by Formula I.

[0072] According to another aspect, there is provided a composition comprising a compound represented by Formula II:

[0073] In some embodiments, a compound represented by Formula II is also termed herein "Dehydroxyuttroside B".

[0074] In some embodiments, the active ingredient(s) of the composition comprise or consist of the compound represented by Formula II. In some embodiments, the activeingredient(s) of the composition comprise or consists essentially of the compound represented by Formula II.

[0075] In some embodiments, the composition of the invention further comprises an acceptable carrier. In some embodiments, the carrier is an agriculturally acceptable carrier.

[0076] In some embodiments, the composition is an agricultural composition.

[0077] In some embodiments, the composition is an anti-insect composition. In some embodiments, the composition is insecticide composition.

[0078] In some embodiments, the composition is characterized by having an insecticide activity.

[0079] In some embodiments, insecticide activity comprises killing insects, inhibiting insect reproduction, growth, or both, or any combination thereof.

[0080] As used herein, the phrase “agriculturally acceptable” excipient or carrier is suitable for use in agriculture without undue adverse side effects to the plants, the environment, or to humans or animals who consume the resulting agricultural products derived therefrom commensurate with a reasonable benefit / risk ratio.

[0081] In some embodiments, the composition of the invention further comprises at least one additional agent or compound having an insecticide activity.Compounds

[0082] According to another aspect, there is provided a compound represented by Formula III:

[0083] In some embodiments, a compound represented by Formula III is also termed herein "25(S)-Uttroside B".

[0084] According to another aspect, there is provided a compound represented by Formula IV:

[0085] In some embodiments, a compound represented by Formula IV is also termed herein "25(S)-Dehydroxyuttroside B".

[0086] In some embodiments, there is provided a composition comprising a compound represented by Formula III, Formula IV, or a combination thereof. In some embodiments, the composition further comprises an acceptable carrier. In some embodiments, the carrier is an agriculturally acceptable carrier.Polynucleotides and protein products

[0087] According to another aspect, there is provided an artificial DNA molecule comprising at least one nucleic acid sequence selected from: (a) a nucleic acid sequence comprising the sequence:ATGGCTATTGCAGTTTTAATTGCTTTAGCAGTATTCTTACCATTCTCCTTTTGGT GCCTAAAATTGCTCTACTTTGTATGGTGGCGTCCCAAAACAGTAGAAAATGAG TTGCGGCAGCAAGGAATATATGGCCGTCCATATAGATTTCTTTTTGGAAATCTA AAAGAGATGATAGAGATGAATAAAATAGCCAAATCTAAACCCATGCCTTTGCA CCACGATTTCACACCTCGACTTAATCCATTGTTCTATGAACTCGCCACCACTTA CAAGAAACTTTACTTGTTTTGGCTAGGACCGATACCTCGATTGACCATTTTGGA TCCCAAGTTAATAAAGGAAGTACTATCGAACAAATCGGGTGAATTCAGTAAAC CGAAAATCAGTGCTTTCCTCAAGCTATTTGTAACGGGTCTAGGGACTTACGATG GTGAAAAATGGGCCAAACACAGAAAAATTCTTAATCCGGCTTTCCATATGGAA AAATTGAAGTTGATGTTGGGAGCATTTGCTCACTGTACAGAAGATATGAAGTG CAGATGGGATAAGCTAACTGGATCAACAGGTTCTTGTGAATTGGATATCGCTC AAGAATTTCATAGTTTAACTGGAGATATGCTATCGAAAGCAGCTTTTGGAAGC AATTTTGAAGAAGGGAAATTGGTATTTTCACTTCTGAGGGAACAATGTGAACT AATTTTCACTGCAAAGCTTGCTATTAATGTCTTCCCATGGTTAAGATTTGTGCC AACGAAAACTAATAGGAGAAGATTGTACATCTATAACACAGTTCGTAGTTCTC TAAAAGGAATAATAGAGAAGCGAGAGAAAGAGGTACTATCAGGAAAGTCACACAATGAAGATCTGTTGGGATTGTTAATGAAATCTAATCAAGAGGAACAGCAAG GGAATAAGAACTCGAATAAAGGAATGAGTACAGAGGATGTGATAGAGGAGTG CAACTCTTTCTACTTTGCTGGCCAAGAGACTACTGCTACTTTGTTAACATGGAC TGCAATTGTCTTGACCATGCATCCAGATTGGCAAGAGAAAGCCAGGAAAGAAG TTCTTGAAATCATTGGAAAAGATGAACCTAAGTTTGATCAACTCAACCAGCTA AAGATTGTAACTATGATCTTGCACGAGGTTCTGAGGTTATATCCATCAGGTTCT CTTGTTAGAGAAACAAACAAAAAGACAAAGCTCGGAGAGTATACAATTCCAG CAGGTGCACAACTTTTAGTGCCACTACAAACAATCCATCGCGATACAGAGGCG TGGGGAGAAGACGCACTAATTTTCCGCCCAGAAAGGTTTGCAGAAGGGGTATC AAAAGCATCAAAGGATTTGATGTACTTTCCCTTTGGTTGGGGTTCTAGGATATG CCTTGGAATGAATTTTGCCATGATTCAAGTTAAGCTTGTTTTGGCTAAAATCTT ACAGAACTACTCATTTGAGCTTTCCCCATCCTATGCTCATGGTCCAACCATGCC AGCACTTGTTCTACAACCACAATATGGTGCTCCTATGATCGTTCGAAAGCTATA A (SEQ ID NO: 1); (b) a nucleic acid sequence comprising the sequence: ATGGCAGACCTCCTCTCAAACTGGTCAAGCACACTACAAGCAGTTCCTCCAAG TCATCGCATACCAGCGCATGAAAGACCAACGGATCCAGTTGAAATTGTGAACA CTATTCCAGTCATTGATTTGGGAAAAGCTAACGGAGAAGAAAGATCTGTCGTT GTTCAAGATCTTTTGAAAGCTTTTGAAGAATATGGGTTTTTTCAGATAATCAAC CACGGAGTACCGGAAAATCTTATGGAGGAGGCAATGAAAGTATACAAAGAAT TTTTCAGTCTTCCAGCTGAGGAGAAAGAAAATTATGCAAAGGATGCTGCTAAT AATACAAATAGAGGTGCTGCTACACTGTACAGTAGCAGTGCTAAGCATTATGA TTCAGAGGAGCATCGATACTGGAGAGATGTCTTGGAACATAGCTGCAATCCTG ATGGGGAAGACAAAAAAACTTGGCCTGATAACCCTCCAAGATATCGGGAGGTT ATTGGTGAATATTCAAATGAATTGAGAAGGGTGAGCAAAGTCATCTTGGGTAT GTTAGCTGAAGGGCTAGGATTGGAGCCAGGTTACTTTGACAACGAACTTGGGC AGAGAATGCTTGTGAATCACTATCCAGCTTGTCCAGATCCAAGTTTAACATTGG GAGTTGGCGGACATTGTGATCCTAATCTCATAACCATTATCCAACAAGAAGTG TATGGTCTTCAAATATTGAAGGATGACAAATGGATTGGTGTGGAGCCTATTCCT AAGGCATTTGTGGTCAATTCTGGTTTACCAATACAGGTAATTAGTAATGGGAA GCTCACTAGTGTTGCACATCGTGTGGTGACGAACACAACTCATCCTCGAACTTC CATTGGTACTTTTATTTGTCCCCATGAAATCGTTGAACCTGCAAAATCACTTGT TGGTCCAGAGAATCCTCCACAGTTCAAATCCTTCCATTGGGGCATTGATTTTAT GCCACATTACCTCAGCAAGAAATCTGTTTACCACGCATCATTGGAGCCATTCA AAATCGATGCTTAA (SEQ ID NO: 2); (c) a nucleic acid sequence comprising the sequence:ATGGCAGACCTCCTCTCAAACTGGTCAAGCACACTACAAGCAGTTCCTCCAAG TCATCGCATACCAGCGCATGAAAGACCAACGGATCCAGTTGAAATTGTGAACACTATTCCAGTCATTGATTTGGGAAAAGCTAACGGAGAAGAAAGATCTGTCGTTGTTCAAGATCTTTTGAAAGCTTTTGAAGAATATGGGTTTTTTCAGATAATCAACCACGGAGTACCGGAAAATCTTATGGAGGAGGCAATGAAAGTATACAAAGAATTTTTCAGTCTTCCAGCTGAGGAGAAAGAAAATTATGCAAAGGATGCTGCTAATAATACAAATAGAGGTGCTGCTACACTGTACAGTAGCAGTGCTAAGCATTATGATTCAGAGGAGCATCGATACTGGAGAGATGTCTTGGAACATAGCTGCAATCCTGATGGGGAAGACAAAAAAACTTGGCCTGATAACCCTCCAAGATATCGGGAGGTTATTGGTGAATATTCAAATGAATTGAGAAGGGTGAGCAAAGTCATCTTGGGTATGTTAGCTGAAGGGCTAGGATTGGAGCCAGGTTACTTTGACAACGAACTTGGGCAGAGAATGCTTGTGAATCACTATCCAGCTTGTCCAGATCCAAGTTTAACATTGGGAGTTGGCGGACATTGTGATCCTAATCTCATAACCATTATCCAACAAGAAGTGTATGGTCTTCAAATATTGAAGGATGACAAATGGATTGGTGTGGAGCCTATTCCTAAGGCATTTGTGGTCAATTCTGGTTTACCAATACAGGTAATTAGTAATGGGAAGCTCACTAGTGTTGCACATCGTGTGGTGACGAACACAACTCATCCTCGAACTTCCATTGGTACTTTTATTTGTCCCCATGAAATCGTTGAACCTGCAAAATCACTTGTTGGTCCAGAGAATCCTCCACAGTTCAAATCCTTCCATTGGGGCATTGATTTTAT GCCACATTACCTCAGCAAGAAATCTGTTTACCACGCATCATTGGAGCCATTCAAAATCGATGCTTAA (SEQ ID NO: 3); (d) a nucleic acid sequence comprising the sequence:ATGAAAAAAACCATGGAGCTCAACAAAAGTACTGTTCCACAACCTATCACCACCATTTACCGACTCCACATGTTTCTCCACTCTCTAATAATGCTTGCATTAGTATACTACCGTCTATCTAATTTGCTCAACTTCGAAAACATCCTTAGTTCACAAGCACTTGCTTGGCTACTTATCACTTTAGGTGAACTTAGTTTCATTCTCAAGTGGTTCTTTGGACAAGGAACTCGTTGGCGCCCTGTTGAAAGGGAAGTGTTCCCTGAAAACATTACTTGCAAAGATTCTGAGTTACCACCAATTGATGTCATGGTATTTACAGCTAATCCTAAGAAAGAGCCAATTGTGGATGTCATGAATACTGTGATATCCGCAATGGCTCTTGATTACCCTACCGATAAATTAGCTGTGTATCTGGCTGATGATGGTGGATGTCCTTTGTCGTTGTATGCCATGGAGGAAGCATGTGTTTTTGCCAAGATGTGGCTACCTTTCTGTAGGAAGTATGGAGTTAAAACAAGGTGTCCTAAAGCGTTTTTTTCTCCGTTAGGAGAAGATGACCGTGTTTTGAAGAATGATGATTTTGTTGATGAAATGAAAGAAATTAAATCTAAATATGAAGAGTTCCAGCAGAATGTGGAACGTGCT GGTGAATCCGGAAAAATCAATGGTAACGTAGTGCCTGATAGAGCCTCGTTTGTTAAGGTAATAAATGACAGGAAAACGGAGAGCGAAAAGAGTGACGACGATTTAACAAAAATGCCTTTGGTAGTATACGTATCCCGTGAAAGAAGATATCACCGTCT TCATCACTTCAAGGGCGGATCTGCAAATGCTCTTTTTCGAGTTTCTGCGCTAAT GAGTAATGCCCCCTTTTTACTGGTTTGGGATTGTGATTTCTTTTGTCATGATCCA ATATCAGCTAGGAAGGCTATGTGTTTCCATCTTGATCCAAAGCTATCATCTGAC TTAGCTTATGTTCAGTTCCCTCAAGTCTTTTACAATGTCAGCAAGTCTGAAATT TACGATGTCAAAATTAGACAGGCTTACAAGACAATATGGCACGGTATGGATGG TATCCAAGGCCCAGTGTTATCAGGAACTGGTTATTTTCTCAAGAGGAAGGCGT TATACACGAGTCCAGGCATCAAGGAGGAGTATCCTAGTTCACCAGAAAAACAT TTCGGAACAAGTAAAAAGTTCCTTGCTTCACTAGAGGAAAATAATTATGTTAAGCAAGAGAGAGTTATATCAGAAGATATTATAGAGGAAGCTCAGAACCTAGCT ACTTGTGCATACGAAGATGGCACACATTGGGGTCAAGAGATCGGTTATTCATA CGATTGTCATTTGGAGAGCACTTTTACTGGTTATCTTTTGCACTGCAAAGGGTG GAGATCGACCTATTTGTATCCAGACAGGCCGTCTTTCTTAGGTTGTGCCCCAGT TGATATGCAAGGTTTCTCCTCACAGCTCATTAAATGGGTTGCTGCACTTACACAAGCTGGTCTATCACATCTCAATCCCATCACTTATGGCTTCAATAGCAGAATGAA AACTCTCCAATGCATGTGTTATGCCTATTTAATGTATTTCTCTCTTTACTCTTGG GGAATGGTTCTACATGCTAGTGTTCCTTCACTTGGCCTTTTATTTGGCTTCCAAG TCTATCCCGAGGTGTATGATCCATGGTTTGCAGTGTATGCGATCGCTTTTGTGT CAACAATTTTGGAGAATATGTCGGAGTCAATTCCAGAAGGAGGATCGGTTAAA ACATGGTGGATGGAATACAGGGCGTTGATGATGATGGGAGTTAGTGCAATTTG CTTAGGAGGAGTGAAAGCTATAATAGACAAAATCATCGGAACGCAAGGAGAG AAATTATATTTGTCAGACAAAGCAATTGATAAGGAAAAGCTGAAGAAATACG AGAAGGGGAAATTTGATTTTCAAGGGATTGGGATACTTGCTGTGCCATTGATTGCATTTTCTGTGTTGAATTTGGTAGGCTTTTTGGTTGGAGCTAATCAAGTGTTT ATTACTATGAAGTTTGCAGATGTGCTTGGACAATTGCTCGTATCATCCTTCTTC GTGTTTGTGGTGGTTACTGTTGTTATTGATGTTGTATCTTTCTTAAAGGATTCTTAA (SEQ ID NO: 4); (e) a nucleic acid sequence comprising the sequence: ATGGCAATTGTTACAGTAATTGGTGCAACGATTGGCATTTTGATAGCCCTATTT TTTGTAAAATCGTTTTATACATTATGGTGGTGGCCAAAGATGATCGAAAAGAA GCTGAAGAAGGAAGGTATTCATGGTCAGCCGTACCAATTTCTGTTTGGAAATC TGAAAGAGATGACGAGAATGTCTAGAGAAGCAAAGAAAAAACCATTAGTAAA TCACGATATTGTTCCTTGGGTGAATCCTTTTATTCTTCATCTTTCTAAAACTTAC GAGAGATTATTTGTGATGTGGGCTGGACCAACCCCTCGGATTACAGTAACAGA TCCAAAGCTAATAAAAGAAGTGGTGAACAGACATAATGAATTTCAAAAGCCTC AAGCCAATGCCTTCATTGATATGTTTGTTACTGGACTTGCTAGTTACAATGGTCAAAAATGGGATCACCATAGAAAGATACTAAACCCGGCTTTTCATATAGAGAAGATTAAGAGGTTGTACCCAGCATTTTGCGAGTGTTGTGATGAAATGATAAATAGATGGGAGGACTTGGTTAGCAAAACTGGAAGTTGTGAATTGGATGTAGCAGATGAATTTCTAAATGTAGGTGGAGATGTTATATCGAGAGCTGCTTTTGGTAGCAATATTGAAGAAGGAAGGACTATTTTCATACTTCAGAAAGAGCAGTGCGATCTTATTTTGGCTTCTCCATTTACTCTCTTCTTTCCCTTACTAAGATTCTTTCCAACAGAATCAAACAGAAGAGCAAGATACATCTACAAAAAAGTGTTATCATTGATCAAAGGAATCATAGAGAAGAAAGAAGACGCTATGCGAAGAGGAGTCTCAGAAAGTGATGATATATTAGGATTACTCTTAAAAGGAGGACTGTCAACCACTGAAATAATTGAAGAATGTAAGGAATTCTATCTTGCAGGACAAGATACAACCACAGCTTTGCTCTCCTGGACATTGGTAGCCTTGAGTATGCATCCTGAGTGGCAAGACAAAGCTAGAAATGAAGTATTTCAAGTACTTGGAAAAAACAAACCAAAGTTTGAGGACTTGAATCAATTAAAAATAATGAACATGATCTTCCAAGAGGTGTTGAGATTATACCCAGCACTCACCCTTATGCGAAGCACCTCAAAGGACACTAAATTGGGAGAAATGACAATTCCTGCAGGAGTACAAATTTTTGTGCCTATATACATAGCACATCGCGACCCCCAAGTATGGGGAGACGATGCACTGATTTTCAATCCAAATAGGTTCTCAGAAGGGGTATCCAAAGCTGCAAAAGAGCCATTGTATTTCCCCTTCGGTTGGGGTCCTCGAATGTGCATTGGTAATAACTTTGGAATGGCAGAAGCAAAGCTCGTTTTATCTC AAATTCTGCAGAGGTTTTGGTTCAAGCTCTCTCCTTCCTATGTTCATGCCCCTCAGGCAATACTCGTTATGAAGCCTCAGTATGGTGCTCAGATAATCCTCAACAAGC TCTGA (SEQ ID NO: 14); (f) any combination of (a) to (e).

[0088] In some embodiments, the artificial DNA molecule comprises the nucleic acid sequences set forth in SEQ ID Nos: 1-4, and 14, or any combination thereof.

[0089] In some embodiments, the nucleic acid sequence encoding the enzyme GAME 6 comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the nucleic acid sequence encoding the enzyme GAME 8 comprises the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 15, or both. In some embodiments, the nucleic acid sequence encoding the enzyme GAME 11 comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the nucleic acid sequence encoding the enzyme GAME 15 comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0090] According to another aspect, there is provided an artificial DNA molecule comprising at least one nucleic acid sequence selected from: (a) a first nucleic acid sequence encoding the enzyme GLYCOALKALOID METABOLISM 6 (GAME6) comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) a second nucleic acid sequenceencoding the enzyme GAME8 comprising the amino acid sequence set forth in SEQ ID NO: 11, SEQ ID NO: 15, or both; (c) a third nucleic acid sequence encoding the enzyme GAME 11 comprising the amino acid sequence set forth in SEQ ID NO: 12; and (d) a fourth nucleic acid sequence encoding the enzyme GAME 15 comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0091] In some embodiments, the artificial DNA molecule further comprises a fifth nucleic acid sequence encoding a glucose transferring (GT) enzyme. In some embodiments, artificial DNA molecule comprises at least one fifth DNA molecule encoding a glucose transferring (GT) enzyme. In some embodiments, the artificial DNA molecule further comprises a fifth nucleic acid sequence encoding a sugar transferring. In some embodiments, the artificial DNA molecule further comprises a fifth nucleic acid sequence encoding a glucuronosyltransferase. In some embodiments, the artificial DNA molecule further comprises a fifth nucleic acid sequence encoding a glucuronosyltransferase. In some embodiments, the artificial DNA molecule further comprises a fifth nucleic acid sequence encoding a uridine 5'-diphospho-glucuronosyltransferase (UDP- glucuronosyltransferase; (UGT)). In some embodiments, the at least one fifth nucleic acid encodes at least one polypeptide selected from: Solatium lycopersicum (Sl)GAMEl, S1GAME17, S1GAME18, S1GAME2, S1GAME25, Sct5R2, or any combination thereof. In some embodiments, the at least one fifth nucleic acid encodes at least one polypeptide selected from: S1GAME1, S1GAME17, S1GAME18, S1GAME2, or any combination thereof.

[0092] The nucleic acid and amino acid sequences of any one of: S1GAME1, S1GAME17, S1GAME18, S1GAME2, S1GAME25, 5-alpha-reductase (Sa5R2), are readily available in the Genbank, as would be apparent to one of ordinary skill in the art.

[0093] In some embodiments, the artificial DNA molecule is a polynucleotide. In some embodiments, the artificial DNA molecule is an isolated DNA molecule or an isolated polynucleotide. In some embodiments, the artificial DNA molecule is a complementary DNA (cDNA) molecule.

[0094] As used herein, the terms "isolated polynucleotide" and "isolated DNA molecule" refers to a nucleic acid molecule that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the nucleic acid in nature. Typically, a preparation of isolated DNA or RNA contains the nucleic acid in a highly purified form, e.g., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. In some embodiments, the isolated polynucleotide is any one of DNA, RNA, and cDNA. In someembodiments, the isolated polynucleotide is a synthesized polynucleotide. Synthesis of polynucleotides is well known in the art and may be performed, for example, by ligating or covalently linking multiple nucleic acid molecules together by primer linkers.

[0095] The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to any molecule (e.g., a strand) of DNA, RNA or a derivative or analog thereof, comprising nucleotides. Nucleotides are comprised of nucleosides and phosphate groups. The nitrogenous bases of nucleosides include, for example, naturally occurring purine or pyrimidine nucleosides as found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C).

[0096] The term "nucleic acid molecule" includes but is not limited to single- stranded RNA (ssRNA), double-stranded RNA (dsRNA), single- stranded DNA (ssDNA), double- stranded DNA (dsDNA), small RNAs, circular nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, amplification products, modified nucleic acids, plasmid or organellar nucleic acids, and artificial nucleic acids such as oligonucleotides.

[0097] In some embodiments, the artificial DNA molecule comprises a nucleic acid sequence with at least 90%, at least 98%, at least 99%, or 100% homology or identity to any one of SEQ ID Nos: 1-4, and 14, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the artificial DNA molecule comprises a nucleic acid sequence with 90% to 100%, 95% to 100%, or 99% to 100% homology or identity to any one of SEQ ID Nos: 1-4, and 14. Each possibility represents a separate embodiment of the invention.

[0098] In some embodiments, any one of the nucleic acid sequences disclosed herein, e.g., the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, etc., or any combination thereof, is codon optimized for expression in the cell.

[0099] In some embodiments, the artificial DNA molecule of the invention is codon optimized for expression in the cell. In some embodiments, the cell is a target cell. In some embodiments, a target cell is a competent cell. In some embodiments, any cell as disclosed herein, e.g., a target cell, a competent cell, etc., are suitable for recombinant expression of at least one protein encoded the artificial DNA molecule, or by any of the first to fourth nucleic acid sequences (SEQ ID Nos: 1-4, and 14).

[0100] In some embodiments, the artificial DNA molecule comprises a plurality of polynucleotides. In some embodiments, the artificial DNA molecule comprises a pluralityof types of polynucleotides. As used herein, the term “plurality” comprises any integer equal to or greater than 2. In some embodiments, the polynucleotide comprises at least 2, at least 3, at least 4, at least 5, or at least 6 different nucleic acid sequences, or any value and range therebetween, wherein each of the different nucleic acid sequences is selected from SEQ ID Nos: 1-4, and 14. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises 2-3, 2-4, or 3-4 different nucleic acid sequences, wherein each of the different nucleic acid sequences is selected from SEQ ID Nos.: 1-4, and 14.

[0101] According to another aspect, there is provided a plasmid or an expression vector comprising the artificial DNA molecule of the invention. In some embodiments, the artificial DNA molecule comprises or is an artificial vector.

[0102] In some embodiments, the artificial vector comprises a plasmid. In some embodiments, the artificial vector comprises or is an agrobacterium comprising the artificial nucleic acid molecule. In some embodiments, the artificial vector is an expression vector. In some embodiments, the artificial vector is a plant expression vector.

[0103] As used herein, the terms "artificial" and "synthetic" are used herein interchangeably.

[0104] Expressing polynucleotide within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell's genome. In some embodiments, the polynucleotide is in an expression vector such as plasmid or viral vector. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly- Adenine sequence.

[0105] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno- associated viral vector, a virgaviridae viral vector, or a poxviral vector. The barley stripe mosaic virus (BSMV), the tobacco rattle virus and the cabbage leaf curl geminivirus (CbLCV) may also be used. The promoters may be active in plant cells. The promoters may be a viral promoter.

[0106] In some embodiments, the artificial DNA molecule as disclosed herein is operably linked to a promoter. The term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation systemor in a host cell when the vector is introduced into the host cell). In some embodiments, the promoter is operably linked to the polynucleotide of the invention. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is the endogenous promoter.

[0107] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), such as biolistic use of coated particles, and needle-like particles, Agrobacterium Ti plasmids and / or the like.

[0096] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. The promoter may extend upstream or downstream of the transcriptional start site and may be any size ranging from a few base pairs to several kilobases.

[0108] In some embodiments, the artificial DNA molecule or a nucleic acid sequence(s) thereof is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells, known to catalyze the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.

[0109] In some embodiments, a plant expression vector is used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO I. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3: 1671-1680 (1984); and Brogli et al., Science 224:838- 843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0110] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDS VE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0111] In some embodiments, recombinant viral vectors, which offer advantages such as systemic infection and targeting specificity, are used for in vivo expression. In one embodiment, systemic infection is inherent in the life cycle of, for example, the retrovirus and is the process by which a single infected cell produces many progeny virions that infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread systemically. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0112] In some embodiments, plant viral vectors are used. In some embodiments, a wildtype virus is used. In some embodiments, a deconstructed virus such as are known in the art is used. In some embodiments, Agrobacterium is used to introduce the vector of the invention into a virus.

[0113] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, lohn Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, agrobacterium Ti plasmids and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0114] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield, or activity of the expressed polypeptide.

[0115] In some embodiments, the artificial vector comprises a polynucleotide encoding a protein comprising an amino acid sequence as described herein.

[0116] According to some embodiments, there is provided a protein encoded by: (a) the artificial DNA molecule disclosed herein; (b) the artificial vector disclosed herein; or the plasmid or agrobacterium disclosed herein.

[0117] In some embodiments, the protein is encoded by a nucleic acid sequence (or polynucleotide) comprising or consisting of SEQ ID Nos: 1-4.

[0118] In some embodiments, the protein is an isolated protein.

[0119] As used herein, the terms "peptide", "polypeptide" and "protein" are interchangeable and refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides, polypeptides and proteins described have modifications rendering them more stable while in the organism or more capable of penetrating into cells. In one embodiment, the terms "peptide", "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms "peptide", "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0120] As used herein, the terms "isolated protein" refers to a protein that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the nucleic acid in nature. Typically, a preparation of an isolated protein contains the protein in a highly purified form, e.g., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. In some embodiments, the isolated protein is a synthesized protein. Synthesis of protein is well known in the art and may be performed, for example, by heterologous expression in a transformed cell, such as exemplified herein.

[0121] In some embodiments, the protein comprises or consists of the amino acid sequence: MAIAVLIALAVFLPFSFWCLKLLYFVWWRPKTVENELRQQGIYGRPYRFLFGNLK EMIEMNKIAKSKPMPLHHDFTPRLNPLFYELATTYKKLYLFWLGPIPRLTILDPKLI KEVLSNKSGEFSKPKISAFLKLFVTGLGTYDGEKWAKHRKILNPAFHMEKLKLMLGAFAHCTEDMKCRWDKLTGSTGSCELDIAQEFHSLTGDMLSKAAFGSNFEEGKLV FSLLREQCELIFTAKLAINVFPWLRFVPTKTNRRRLYIYNTVRSSLKGIIEKREKEVL SGKSHNEDLLGLLMKSNQEEQQGNKNSNKGMSTEDVIEECNSFYFAGQETTATLL TWTAIVLTMHPDWQEKARKEVLEIIGKDEPKFDQLNQLKIVTMILHEVLRLYPSGS LVRETNKKTKLGEYTIPAGAQLLVPLQTIHRDTEAWGEDALIFRPERFAEGVSKAS KDLMYFPFGWGSRICLGMNFAMIQVKLVLAKILQNYSFELSPSYAHGPTMPALVL QPQYGAPMIVRKL (SEQ ID NO: 10).

[0122] In some embodiments, the protein comprises an amino acid sequence with at least 90%, at least 93%, at least 95%, or at least 97% homology or identity to SEQ ID NO: 10, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 92% to 100%, 93% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 10. Each possibility represents a separate embodiment of the invention.

[0123] In some embodiments, the protein comprises or consists of the amino acid sequence: MAAATIIAAIFGILIAYFCGKVLYTIWWWPKMIEKKLKKEGIHGEPYKLLFGNLKE MMKMSRES KKKPLLDHDIIPW VNPFLLHLS KTYKKIFVLWAGPTPRVTVTDPKLIR EVLNRYNEFHKPEANAFIHLFVTGLASYDGEKWDTHRKILNPAFHVEKLKRMFPA ISVCCDEMINRWEEMVSKSGSCELDVADEFLNLGGDVISRAAFGSNIEEGRSIFLLQ KEQCELILASPFTLFFPSLRFLPTASNRKAKYIHKKVISLIRGIIEKREEAVRRGMSEN DDILGLLLKARNQENKSKAGKGSLSTDDVIEECKEFYFAGQDTTTALLSWTLVILSMHPEWQDKARNEVFQVIGKNKPKFDDLNQLKIINMIFQEVLRLYPAIFLIRSTSKDT KLGDMTIPKGVQVCVPTHLVHRDPEVWGDDALVFNPERFSEGVTKAAKEQLYFP FGWGARMCIGMNFGMLEAKLIFAQIVQHFWFELSPSYTHAPLLTLIMRPQYGAQII VHKL (SEQ ID NO: 11).

[0124] In some embodiments, the protein comprises an amino acid sequence with at least 90%, at least 93%, at least 95%, or at least 97% homology or identity to SEQ ID NO: 11, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 92% to 100%, 93% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 11. Each possibility represents a separate embodiment of the invention.

[0125] In some embodiments, the protein comprises or consists of the amino acid sequence: MADLLSNWSSTLQAVPPSHRIPAHERPTDPVEIVNTIPVIDLGKANGEERSVVVQD LLKAFEEYGFFQIINHGVPENLMEEAMKVYKEFFSLPAEEKENYAKDAANNTNRG AATLYSSSAKHYDSEEHRYWRDVLEHSCNPDGEDKKTWPDNPPRYREVIGEYSNE LRRVSKVILGMLAEGLGLEPGYFDNELGQRMLVNHYPACPDPSLTLGVGGHCDPN LITIIQQEVYGLQILKDDKWIGVEPIPKAFVVNSGLPIQVISNGKLTSVAHRVVTNTT HPRTSIGTFICPHEIVEPAKSLVGPENPPQFKSFHWGIDFMPHYLSKKSVYHASLEPF KIDA (SEQ ID NO: 12).

[0126] In some embodiments, the protein comprises an amino acid sequence with at least 90%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 12, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 90% to 100%, 80% to 100%, 88% to 100%, or 90% to 100% homology to SEQ ID NO: 12. Each possibility represents a separate embodiment of the invention.

[0127] In some embodiments, the protein comprises or consists of the amino acid sequence: MKKTMELNKSTVPQPITTIYRLHMFLHSLIMLALVYYRLSNLLNFENILSSQALAW LLITLGELSFILKWFFGQGTRWRPVEREVFPENITCKDSELPPIDVMVFTANPKKEPI VDVMNTVISAMALDYPTDKLAVYLADDGGCPLSLYAMEEACVFAKMWLPFCRK YGVKTRCPKAFFSPLGEDDRVLKNDDFVDEMKEIKS KYEEFQQNVERAGES GKIN GNVVPDRASFVKVINDRKTESEKSDDDLTKMPLVVYVSRERRYHRLHHFKGGSA NALFRVSALMSNAPFLLVWDCDFFCHDPISARKAMCFHLDPKLSSDLAYVQFPQV FYNVSKSEIYDVKIRQAYKTIWHGMDGIQGPVLSGTGYFLKRKALYTSPGIKEEYP SSPEKHFGTSKKFLASLEENNYVKQERVISEDIIEEAQNLATCAYEDGTHWGQEIG YSYDCHLESTFTGYLLHCKGWRSTYLYPDRPSFLGCAPVDMQGFSSQLIKWVAAL TQAGLSHLNPITYGFNSRMKTLQCMCYAYLMYFSLYSWGMVLHASVPSLGLLFG FQVYPEVYDPWFAVYAIAFVSTILENMSESIPEGGSVKTWWMEYRALMMMGVSA ICLGGVKAIIDKIIGTQGEKLYLSDKAIDKEKLKKYEKGKFDFQGIGILAVPLIAFSV LNLVGFLVGANQVFITMKFADVLGQLLVSSFFVFVVVTVVIDVVSFLKDS (SEQ ID NO: 13).

[0128] In some embodiments, the protein comprises an amino acid sequence with at least 90%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 13, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 90% to 100%,80% to 100%, 88% to 100%, or 90% to 100% homology to SEQ ID NO: 13. Each possibility represents a separate embodiment of the invention.

[0129] In some embodiments, the protein comprises or consists of the amino acid sequence: MAIVTVIGATIGILIALFFVKSFYTLWWWPKMIEKKLKKEGIHGQPYQFLFGNLKE MTRMSREAKKKPLVNHDIVPWVNPFILHLSKTYERLFVMWAGPTPRITVTDPKLIK EVVNRHNEFQKPQANAFIDMFVTGLASYNGQKWDHHRKILNPAFHIEKIKRLYPA FCECCDEMINRWEDLVSKTGSCELDVADEFLNVGGDVISRAAFGSNIEEGRTIFILQ KEQCDLILASPFTLFFPLLRFFPTESNRRARYIYKKVLSLIKGIIEKKEDAMRRGVSE SDDILGLLLKGGLSTTEIIEECKEFYLAGQDTTTALLSWTLVALSMHPEWQDKARN EVFQVLGKNKPKFEDLNQLKIMNMIFQEVLRLYPALTLMRSTSKDTKLGEMTIPA GVQIFVPIYIAHRDPQVWGDDALIFNPNRFSEGVSKAAKEPLYFPFGWGPRMCIGN NFGMAEAKLVLSQILQRFWFKLSPSYVHAPQAILVMKPQYGAQIILNKL (SEQ ID NO: 15).

[0130] In some embodiments, the protein comprises an amino acid sequence with at least 90%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 15, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 90% to 100%, 80% to 100%, 88% to 100%, or 90% to 100% homology to SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention.

[0131] The terms “homology” or “identity”, as used interchangeably herein, refer to sequence identity between two amino acid sequences or two nucleic acid sequences, with identity being a stricter comparison. The phrases “percent identity or homology” and “% identity or homology” refer to the percentage of sequence identity found in a comparison of two or more amino acid sequences or nucleic acid sequences. Two or more sequences can be anywhere from 0-100% identical, or any value there between. Identity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison to a reference sequence. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of homology of amino acid sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

[0132] The following is a non-limiting example for calculating homology or sequence identity between two sequences (the terms are used interchangeably herein). The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non- homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences.

[0133] In some embodiments, % homology or identity as described herein are calculated or determined using the basic local alignment search tool (BLAST). In some embodiments, % homology or identity as described herein are calculated or determined using Blossum 62 scoring matrix.

[0134] In some embodiments, the protein comprises or is characterized by having oxidation enzymatic activity.Transgenic cell, tissue, or plant

[0135] According to another aspect, there is provided a transgenic cell comprising: (a) the artificial DNA molecule of the invention; (b) the plasmid or expression vector of the invention; or (c) both (a) and (b).

[0136] As used herein, the term "transgenic cell" refers to any cell that has undergone human manipulation on the genomic or gene level. In some embodiments, the transgenic cell has had exogenous polynucleotide, such as an isolated DNA molecule as disclosed herein, introduced into it. In some embodiments, a transgenic cell comprises a cell that has an artificial vector introduced into it. In some embodiments, a transgenic cell is a cell which has undergone genome mutation or modification. In some embodiments, a transgenic cell is a cell that has undergone CRISPR genome editing. In some embodiments, a transgenic cell is a cell that has undergone targeted mutation of at least one base pair of its genome. In some embodiments, the exogenous polynucleotide (e.g., the isolated DNA molecule disclosed herein) or vector is stably integrated into the cell. In some embodiments, the transgenic cell expresses a polynucleotide of the invention. In some embodiments, the transgenic cellexpresses a vector of the invention. In some embodiments, the transgenic cell expresses a protein of the invention. In some embodiments, the transgenic cell, is a cell that is devoid of a polynucleotide of the invention that has been transformed or genetically modified to include the polynucleotide of the invention. In some embodiments, CRISPR technology is used to modify the genome of the cell, as described herein.

[0137] In some embodiments, the transgenic tissue, plant, or both, comprises at least one transgenic cell, as disclosed herein.

[0138] In some embodiments, the cell comprises a unicellular organism, a cell of a multicellular organism, a cell in a culture, or any combination thereof.

[0139] In some embodiments, a unicellular organism comprises a fungus or a bacterium.

[0140] In some embodiments, the fungus comprises a yeast cell.

[0141] In some embodiments, the cell comprises an insect cell. In some embodiments, the cell comprises an insect cell line. In some embodiments, the cell comprises a plant cell. In some embodiments, the cell comprises a plant cell line.

[0142] Types of insect cell lines suitable for transformation and / or heterologous expression are common and would be apparent to one of ordinary skill in the art. Non-limiting examples of such insect cell lines include, but are not limited to, Sf-9 cells, SR+ Schneider cells, S2 cells, and others.

[0143] According to some embodiments, there is provided an extract derived from a transgenic cell disclosed herein, or any fraction thereof.

[0144] In some embodiments, the extract comprises a compound represented by Formula I.

[0145] In some embodiments, the extract comprises the artificial DNA molecule of the invention, a protein as disclosed herein, or any combination thereof.

[0146] According to some embodiments, there is provided a homogenate, lysate, extract, derived from a transgenic cell disclosed herein, any combination thereof, or any fraction thereof.

[0147] Methods and / or means for extracting, lysing, homogenizing, fractionating, or any combination thereof, a cell or a culture of same, are common and would be apparent to one of ordinary skill in the art of cell biology and biochemistry. Non-limiting examples include, but are not limited to, pressure lysis (e.g., such as using a French press), enzymatic lysis, soluble-insoluble phase separation (such for obtaining a supernatant and a pellet), detergent-based lysis, solvent (e.g., polar or nonpolar solvent), liquid chromatography mass spectrometry, or others.

[0148] According to some embodiments, there is provided a transgenic plant, a transgenic plant tissue or a plant part. In some embodiments, there is provided a transgenic plant, or any portion, seed, tissue or organ thereof, comprising at least one transgenic plant cell of the invention. In some embodiments, the transgenic plant, transgenic plant tissue or plant part, comprises: (a) the DNA molecule disclosed herein; (b) the artificial disclosed herein; (c) the plasmid or agrobacterium disclosed herein; (d) the protein of the invention; (e) the transgenic cell disclosed herein; or any combination thereof.

[0149] In some embodiments, the transgenic plant, transgenic plant tissue, or plant part consists of transgenic plant cells of the invention. In some embodiments, the transgenic plant, transgenic plant tissue, or plant part comprises at least: 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% transgenic cells of the invention, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the transgenic plant, transgenic plant tissue, or plant part comprises 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, or 20%-100% transgenic cells of the invention. Each possibility represents a separate embodiment of the invention.

[0150] In some embodiments, the transgenic plant, transgenic plant tissue, or plant part is or derived from a Solanum plant. In some embodiments, the transgenic plant is a Solatium plant.

[0151] In some embodiments, the transgenic plant is characterized by an increased amount or abundance of a compound represented by Formula I compared to a control plant. In some embodiments, a control plant comprises a genetic reference of the transgenic plant. In some embodiments, a transgenic plant comprises a gene-edited plant. In some embodiments, the transgenic plant is characterized by increased expression (e.g., over-expression) of at least one nucleic acid sequence selected from SEQ ID Nos: 1-4, and 14, or any combination thereof, in at least one cell thereof. In some embodiments, the transgenic plant comprises an exogenous DNA molecule comprising at least one nucleic acid sequence encoding at least one protein comprising an amino acid sequence selected from SEQ ID Nos: 10-13, and 15, or any combination thereof.

[0152] According to some embodiments, there is provided a composition comprising any one of the herein disclosed: (a) artificial DNA molecule of the invention (for example, an isolated DNA molecule or an isolated polynucleotide); (b) artificial vector; (c) plasmid oragrobacterium; (d) protein as disclosed herein; (e) transgenic cell; (f) extract; (g) transgenic plant tissue or plant part; and (h) any combination of (a) to (g), and an acceptable carrier.

[0153] As used herein, the term “carrier”, “excipient”, or “adjuvant” refers to any component of a composition, e.g., pharmaceutical or nutraceutical, that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some nonlimiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate) as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non- toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful in the present compositions include distilled water, physiological saline, Ringer'ssolution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8thEd., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18thEd., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21stEd., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the presently described peptides are formed from standard vesicle -forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0154] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.Methods of synthesis

[0155] According to another aspect, there is provided a method for synthesizing a compound represented by Formula I. According to another aspect, there is provided a method for synthesizing a compound represented by Formula II. According to another aspect, there is provided a method for synthesizing a compound represented by Formula III. According to another aspect, there is provided a method for synthesizing a compound represented by Formula IV.

[0156] In some embodiments, the method comprising the steps: (a) providing a cell comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 2, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and culturing the cell from step (a) such that at least one protein being encoded by any one of the first polynucleotide, the secondpolynucleotide, the third polynucleotide, and the fourth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula I, Formula II, or both.

[0157] In some embodiments, the method comprising the steps: (a) providing a cell comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 14, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and culturing the cell from step (a) such that at least one protein being encoded by any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula III, Formula IV, or both.

[0158] In some embodiments, the artificial DNA molecule further comprises a fifth nucleic acid sequence encoding a GT enzyme.

[0159] In some embodiments, the method comprising the steps: (a) providing a cell comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 2, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and further comprising at least one fifth polynucleotide comprising a nucleic acid sequence encoding at least one enzyme selected from: S1GAME1, S1GAME17, S1GAME18, S1GAME2, S1GAME25, Sa5R2, and any combination thereof, and culturing the cell from step (a) such that at least one protein being encoded by any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula I.

[0160] In some embodiments, the method comprising the steps: (a) providing a cell comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 2, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and further comprising at least one fifth polynucleotide comprising a nucleic acid sequence encoding at least one enzyme selected from: S1GAME1, S1GAME17, S1GAME18, S1GAME2, and any combination thereof, and culturing the cell from step (a) such that at least one protein being encoded by any one of thefirst polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula II.

[0161] In some embodiments, the method comprising the steps: (a) providing a cell comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 14, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and further comprising at least one fifth polynucleotide comprising a nucleic acid sequence encoding at least one enzyme selected from: S1GAME1, S1GAME17, S1GAME18, S1GAME2, S1GAME25, Sa5R2, and any combination thereof, and culturing the cell from step (a) such that at least one protein being encoded by any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula III.

[0162] In some embodiments, the method comprising the steps: (a) providing a cell comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 14, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and further comprising at least one fifth polynucleotide comprising a nucleic acid sequence encoding at least one enzyme selected from: S1GAME1, S1GAME17, S1GAME18, S1GAME2, and any combination thereof, and culturing the cell from step (a) such that at least one protein being encoded by any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula IV.

[0163] In some embodiments, the culturing comprises supplementing the cell with an effective amount of cholesterol or a precursor thereof.

[0164] In some embodiments, the method further comprises a step before step (a), comprising introducing or transfecting the cell with the artificial DNA molecule.

[0165] In some embodiments, the method further comprises a step comprising isolating, extracting, purifying, or any combination thereof, the compound represented by Formula I.

[0166] According to some embodiments, the method comprises contacting cholesterol or a precursor thereof with at least one protein comprising an amino acid sequence set forth in SEQ ID Nos: 10-13, and 15, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0167] According to some embodiments, the method comprises contacting cholesterol or a precursor thereof with a plurality of proteins comprising the amino acid sequences set forth in SEQ ID Nos: 10-13, and 15.

[0168] According to some embodiments, there is provided a method for obtaining an extract from a transgenic cell or a transfected cell.

[0169] In some embodiments, the method comprises culturing a transgenic cell or a transfected cell in a medium and extracting the transgenic cell or the transfected cell.

[0170] In some embodiments, the method comprises the steps: (a) culturing a transgenic cell or a transfected cell in a medium; and (b) extracting the transgenic cell or the transfected cell, thereby obtaining an extract from the transgenic cell or the transfected cell.

[0171] In some embodiments, the transgenic cell or the transfected cell comprises an artificial vector comprising at least one nucleic acid sequence set forth in SEQ ID Nos: 1-4, and 14, or any combination thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the transgenic cell or the transfected cell comprises an artificial vector comprising a plurality of nucleic acid sequences set forth in SEQ ID Nos: 1- 4, and 14.

[0172] In some embodiments, the transgenic cell or the transfected cell comprises the DNA molecule of the invention or a plurality thereof, as disclosed herein. In some embodiments, the transgenic cell or the transfected cell comprises the artificial nucleic acid molecule or vector as disclosed herein. In some embodiments, the cell is a transgenic cell, or a cell transfected with a DNA molecule as disclosed herein.

[0173] In some embodiments, the transgenic cell or the transfected cell comprises an artificial vector comprising a nucleic acid sequence of SEQ ID Nos: 1-4, and 14, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0174] According to some embodiments, the method comprises the steps: (a) providing a cell comprising an artificial vector comprising SEQ ID Nos: 1-4, and 14, or any combination thereof; and (b) culturing the cell from step (a) such that a protein encoded by the artificial vector is expressed. Each possibility represents a separate embodiment of the invention.

[0175] According to some embodiments, the method comprises contacting cholesterol, with a first protein comprising an amino acid sequence set forth in SEQ ID NO: 10, a second protein comprising an amino acid sequence set forth in SEQ ID NO: 11, a third protein comprising an amino acid sequence set forth in SEQ ID NO: 12, and a forth protein comprising an amino acid sequence set forth in SEQ ID NO: 13.

[0176] According to some embodiments, the method comprises contacting cholesterol, with a first protein comprising an amino acid sequence set forth in SEQ ID NO: 10, a second protein comprising an amino acid sequence set forth in SEQ ID NO: 15, a third protein comprising an amino acid sequence set forth in SEQ ID NO: 12, and a forth protein comprising an amino acid sequence set forth in SEQ ID NO: 13.

[0177] In some embodiments, the method further comprises contacting the cholesterol or a metabolite thereof with at least one enzyme selected from: S1GAME1, S1GAME17, S1GAME18, S1GAME2, S1GAME25, Sa5R2.

[0178] Method for introducing or transfecting a cell with an artificial nucleic acid molecule or vector are common and would be apparent to one of ordinary skill in the art.

[0179] In some embodiments, introducing or transfecting comprises transferring an artificial nucleic acid molecule or vector comprising the polynucleotide / DNA molecule disclosed herein into a cell; or modifying the genome of a cell to include the polynucleotide / DNA molecule disclosed herein. In some embodiments, the transferring comprises transfection. In some embodiments, the transferring comprises transformation. In some embodiments, the transferring comprises lipofection. In some embodiments, the transferring comprises nucleofection. In some embodiments, the transferring comprises viral infection.

[0180] As used herein, the terms “transfecting” and “introducing” are interchangeable.

[0181] In some embodiments, the contacting is in a cell-free system.

[0182] Types of suitable cell-free systems utilizing any one of: the polynucleotide / DNA molecule of the invention or a plurality thereof, as disclosed herein, and the protein of the invention, or a plurality thereof, would be apparent to one of ordinary skill in the art.

[0183] In some embodiments, the method further comprises a step preceding step (b), comprising separating the cultured transgenic cell or the cultured transfected cell from the medium.

[0184] Method for separating cell from a medium are common and may include, but not limited to, centrifugation, ultracentrifugation, or other, as would be apparent to one of ordinary skill in the art.

[0185] According to some embodiments, there is provided an extract of a transgenic cell or a transfected cell obtained according to the herein disclosed method.

[0186] According to some embodiments, there is provided a medium or a portion thereof separated from a cultured transgenic cell or a cultured transfected cell, obtained according to the herein disclosed method.

[0187] According to some embodiments, there is provided a composition comprising: (a) the extract disclosed herein; (b) the medium disclosed herein or a portion thereof; or (c) any combination of (a) and (b), and an acceptable carrier, as described herein.

[0188] In some embodiments, a portion comprises a fraction or a plurality thereof.

[0189] In some embodiments, the method comprises culturing a transgenic plant comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 2, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4.

[0190] In some embodiments, the method comprises providing a transgenic plant comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 2, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4.

[0191] In some embodiments, the method comprises: (a) providing a transgenic plant comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 2, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and culturing the transgenic plant from step (a) such that at least one protein being encoded by any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula I, Formula II, or both.31

[0192] In some embodiments, the method comprises culturing a transgenic plant comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 14, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4.

[0193] In some embodiments, the method comprises providing a transgenic plant comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 14, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4.

[0194] In some embodiments, the method comprises: (a) providing a transgenic plant comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 14, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and culturing the transgenic plant from step (a) such that at least one protein being encoded by any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula III, Formula IV, or both.

[0195] In some embodiments, the method further comprises a step before or preceding step (a), comprising introducing or transfecting at least one plant cell or a plant of a cell with the artificial DNA molecule, thereby obtaining or producing at least one transgenic plant cell. In some embodiments, transgenic plant cell is further cultured, propagated, or both, thereby obtaining or producing a transgenic plant tissue or a transgenic plant.

[0196] Methods for producing plants from transgenic cells are common and would be apparent to a person of ordinary skill in the art. Non-limiting examples for such methods include, but are not limited to, agrobacterium-mediated transformation, biolistic particle delivery (gene gun), electroporation, microinjection, protoplast transformation, PEG- mediated transformation, floral dip method, leaf disc transformation, callus culture and regeneration, and somatic embryogenesis, to name a few.

[0197] In some embodiments, the method further comprises a step comprising isolating, extracting, purifying, or any combination thereof, the compound represented by Formulae I-IV, or any combination thereof, from a cell, a tissue, a plant part, or the transgenic plant, or any combination thereof.Methods of use

[0198] According to another aspect, there is provided a method for controlling an insect infection in a plant.

[0199] In some embodiments, the method comprises contacting a plant or a plant part with an effective amount of a compound represented by Formula I.

[0200] In some embodiments, controlling comprises treating and / or preventing.

[0201] In some embodiments, controlling comprises increasing resistance of a plant to an insect.

[0202] In some embodiments, controlling comprises: repelling an insect from a plant, reducing feeding rate of an insect on a plant, reducing number of eggs laid by an insect on a plant, or any combination thereof.

[0203] In some embodiments, a plant comprises any plant part. In some embodiments, a plant part comprises a plant cell, a plant tissue, a plant organ, a plant organelle, or any combination thereof. In some embodiments, a plant part comprises: root, stem, trunk, seed, fruit, leaf, flower, or any combination thereof.

[0204] Types of pathogenic insect or insects that induce infections or infectious disease or disorders in plants are common and would be apparent to one of ordinary skill in the art.

[0205] In some embodiments, an insect is: Empoasca decipiens, Leptinotarsa decemlineata, or both. In some embodiments, an infection in the plant is induced by an insect selected from: E. decipiens, L. decemlineata, or both.

[0206] In some embodiments, an insect includes any insect pest afflicting wellbeing, appearance, survival, quality, or any combination thereof, of a plant or a part thereof.

[0207] Types of insect plant pests would be apparent to one of ordinary skill in the art, including methods of identifying same.

[0208] In some embodiments, the method comprises comprising contacting a plant or a plant part with an effective amount of: (a) the insecticide of the invention; (b) the composition of the invention; (c) a compound represented by Formula I and / or being synthesized according to the method disclosed herein; (d) an extract as disclosed herein; or (e) any combination of (a) to (d).

[0209] In some embodiments, contacting comprises pre-harvest contacting, post-harvest contacting, or both. In some embodiments, contacting comprises pre-harvest contacting and post-harvest contacting.

[0210] In some embodiments, contacting comprises: spraying, dipping, drenching, irrigating, soaking, injecting, or any combination thereof.

[0211] In some embodiments, the plant belongs to the order Solanales.

[0212] In some embodiments, inhibiting or reducing comprises at least 5%, at least 15%, at least 25%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, at least 97%, at least 99%, or 100% inhibiting or reducing, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, inhibiting or reducing comprises 5-50%, 10-90%, 20-99%, or5-100% inhibiting or reducing. Each possibility represents a separate embodiment of the invention.

[0213] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0214] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ± 100 nm.

[0215] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0216] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the artwould understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".

[0217] 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 sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0218] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0219] 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.EXAMPLES

[0220] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland(1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8thEdition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Materials and MethodsPlant sources and growth conditions

[0221] Solanum nigrum CV Linn inbred line Sn30 (originally collected at a field site in Jena, Germany) was used as the wild type (WT) genetic background to generate knockout mutants of game! 5, as well as WT control plant in all experiments. To induce germination, 5. nigrum seeds were first incubated in 1 M KNO3 solution (101 g in 1,000 mL distilled water) for 48 h at 4 °C and then transferred in a peat-based substrate with clay additions in 50 mL Teku pots (JP3050 / 72P; Poeppelmann). After 10 to 14 days, germinated young seedlings were transferred to 1 L pots and continued to grow under a day / night cycle of 16 h (23-26 °C) / 8 h (16-22 °C) in a glasshouse at the Max Planck Institute for Chemical Ecology (MPI-CE), Jena, Germany. Light was supplemented when required. Nicotiana benthamiana seeds were germinated directly in 9x9 square containers (Goettinger) and grown in the greenhouse at 23-26 °C during the day and 16-22 °C during the night, with a 16 / 8 h photoperiod for four to six weeks. For transient expression experiments, plants were transferred to walk-in growth chamber (York) with a 16 / 8 day / night cycle and 21 °C all time at 55% humidity.Chemicals and solvents

[0222] MS grade solvents for the UHPLC-MS analysis were purchased from Fisher Scientific. Methanol and ethanol used for the extraction of steroidal metabolites from 5. nigrum tissues were HPLC grade and purchased from Fisher Scientific. Analytical standards a-solasonine and a-solamargine were purchased from Sigma-Aldrich. Kanamycin sulfate, gentamicin sulfate, rifampicin, spectinomycin were purchased from Sigma- Andrich. D-luciferin was purchased from Promega. Uttroside B and malonyl- solamargine standardswere purified from S. nigrum leaves and unripe berries respectively, as reported by Grzech et al., in revision (uttroside B) and Lucier et al., in review (malonyl- solamargine).Molecular biology and cloning

[0223] All the genes reported in this study were amplified by PCR using Phusion High- Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer’s instructions. Restriction enzymes and T4 ligase (New England Biolabs) and In-Fusion kit (Takara Bio) were used to clone the corresponding PCR amplicons into in-house generated plant binary vectors (301-for overexpression, and 3Ql:Cas9 for CRISPR knockout). Primers were purchased from Sigma-Aldrich. E. coli TOPIO cells (Invitrogen) and Agrobacterium tumefaciens (strain GV 3101) were used for plasmid isolation and for transient expression / stable transformation in N. benthamiana / S. nigrum plants, respectively. For plasmid isolation, Wizard Plus sv-miniprep DNA purification system (Promega) was used, while for DNA recovery from PCR gels, Zymoclean gel DNA recovery kit (ZymoResearch) was used.RNA-seq analysis

[0224] Total RNA was extracted from different tissues (young and old leaves, roots, stems) of S. nigrum using the RNeasy Mini Kit (Qiagen). Subsequently, the extracted samples were submitted for mRNA library preparation and RNA-seq analysis at BGI (bgi.com / ) following the company's standard protocols, employing paired-end sequencing (PE 2x150) with an approximate yield of 40 million reads per sample. De Novo transcriptome assemblies were generated from cleaned, trimmed reads using Trinity. Transdecoder (github.com / TransDecoder / TransDecoder) was used to identify candidate-coding regions within transcript sequences. Functional annotation was then performed with seven functional databases (NR, NT, GO, KOG, KEGG, SwissProt and InterPro). Gene expression measured by FPKM (fragments per kilobase of transcript per million mapped reads) was calculated using RS EM.Co-expression network analysis

[0225] The co-expression analysis was performed using 5. nigrum RNA-Seq transcriptome data generated in this study from different tissues (young and old leaves, stem and roots). Briefly, the Pearson correlation of the logarithm, base 2, of the gene expression in FPKM of all transcripts against GAME6, GAME8 and GAME11 was calculated using the R base package v3.6.1 (r-project.org / ). An adjacency matrix was obtained by keeping the highest10% correlation values as edges using the igraph library. The resulting graphs were exported to Cytoscape v3.8.2 using the RCy3 package.Knockout of game 15 in S. nigrum

[0226] For the knockout of game15, three RNA guides were designed (Fig. 2A) using CRISPR-P v2.0 (crispr.hzau.edu. cn / CRISPR2 / ) and CRISPR RGEN tools (rgenome.net / ). The sgRNAs (sgRNAl: TGTCATGATCCAATATCAAGCT (SEQ ID NO: 5), sgRNA2: ATCCAAGGCCCAGTGTTATC (SEQ ID NO: 6), sgRNA3:TGGCACACATTGGGGTCAAG (SEQ ID NO: 7)) were introduced into an in-house developed 3Q1 plasmid, containing the Cas9 and kanamycin-resistance-expression (nptll) cassettes by Goldenbraid cloning. The sgRNAs were inserted in the 301 :Cas9 plasmid under the control of the Arabidopsis U6-26 promoter.S. nigrum stable transformation and genotyping

[0227] For S. nigrum stable transformation, seeds were incubated in the sterilizing solution for 5 min, washed 3 times with deionized sterile water and then incubated in 5 rnL sterile 1 M KN03 solution at 4 °C in darkness, overnight. Seeds were germinated on Gamborg B5 medium for 7 days at the following day / night cycle: 16 / 8 day / night, at 26 °C. The game15- Cas9 construct was transformed into A. tumefaciens (strain GV 3101) by electroporation. One-single colony was used to inoculate 8 mL LB media culture supplemented with 200 pg mL1spectinomycin, 100 pg mL-1rifampicin and 50 pg mL1gentamicin and grown overnight (28 °C, 200 rpm). The cells were pelleted by centrifugation at 4000 x g, room temperature for 5 min. The pellet was re-suspended in 7 mL agrobacterium washing media (Basal media: 4.41 g L1Murashige and Skoog containing vitamins, 30 g L1Sucrose; supplemented with 0.02 mg L’1Indole-3-acetic Acid (IAA), 1 mg L16-Benzylaminopurine (BAP); pH 5.80). The seedling hypocotyls were cut in 3 mm long pieces using a sterile scalpel dipped in the A. tumefaciens suspension. The explants were transferred onto callus induction media (Basal media supplemented with 3 g L’1Phytagel, 0.02 mg L’1Indole-3- acetic Acid (IAA), 1 mg L16-Benzylaminopurine (BAP); pH 5.80) and incubated at 26 °C in darkness for 3 days. The explants were then sub-cultured onto fresh callus induction media plates containing antibiotics (25 mg L1kanamycin and 125 mg L timentin) and incubated at 30 °C for 16 / 8 h day / night at 30 °C for a total of 14 days. The resulting callus was transferred onto shoot induction media (Basal media supplemented with 0.5 mg L1BAP, 25 mg L1kanamycin, 125 mg L1timentin) and incubated at 30 °C, 16 / 8h photoperiod for a total of 7 days. For shoot maturation the calli with primodia shoots were subcultured on maturation media (Basal media supplemented with 25 mg L1kanamycin, 125 mg L1timentin) at 28 °C for 7 days. The resulting plantlets were separated and transferred onto rooting media (292 mg L1of Peter’s hydrosol salts, 2 mL of 500x MS vitamins solution, 6 g L1plant agar supplemented with 125 mg L1timentin; pH 6.0). The plantlets were placed in a culture room with a constant 22 °C temperature and 16 h of light and 8 h of darkness photoperiod and were sub-cultured onto fresh rooting media plates every 14 days. After rooting, the plants were transferred to soil in Magenta boxes (100 mm x 77 mm x 77 mm) and finally planted in 2 L pots in the green house for acclimatization and hardening under a long-day photoperiod (16 h light, 8 h dark) at 25 °C and 65 % relative humidity. CRISPR / Cas9 induced mutations were genotyped in Ti plants using DNA sequencing. Briefly, the genomic DNA from leaf tissue of S. nigrum plants (Ti) was isolated using CTAB method. Three independent game15-ko lines (game! 5ko#l , game15ko#2 and game15ko#3) were selected based on the mutations confirmed by Sanger sequencing of clones amplified using GAME15 target site-specific oligonucleotide pairs (F: AAATGACAGGAAAACGGAGAG (SEQ ID NO: 8); R:TAGACCAGCTTGTGTAAGTGCA (SEQ ID NO: 9)).LC-MS-based SGA and steroidal saponins analysis

[0228] SGAs and saponins analysis was performed as previously described (Itkin et al., 2011; Sonawane et al., 2018, and Cardenas et al., 2016). Unless stated otherwise, at least three biological replicates (n=3) were used for metabolite analysis. Briefly, 100 mg of frozen powdered leaf tissues were extracted with 300 pL of 80% methanol and 0.1% formic acid. The samples were vortexed and then sonicated for 20 min in a water bath at room temperature. Extracts were centrifuged for 20 min at 20,000 x g and the supernatant was filtered through PTFE (0.2 pm). Metabolites were analyzed by UHPLC- MS / MS using a Thermo Scientific UltiMate 3000 RS ultra-high performance liquid chromatography (UHPLC) system (Thermo Scientific, Germering, Germany) coupled to a triple quadrupole mass spectrometer (EVOQ Elite™, Bruker Daltonics, Bremen, Germany).

[0229] For the steroidal saponin uttroside B, the metabolites were separated by reversed - phase liquid chromatography using an Acquity UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 pm particle size, 130 A, Massachusetts, United States) kept at 35 °C. The following gradient was used for analysis of uttroside B: 5% B for 1 min; 5 % to 28% B in 10.5 min; 28% to 100% B in 7 min, 100 % B for 1.5 min, returning to the initial conditions (5% phase B) within 0.5 min and a final re-equilibration to 5% B for another 2.5 min before next injection.

[0230] For the SGAs, a-solamargine, a-solasonine and malonyl-solamargine, the metabolites were separated by reserved-phase liquid chromatography using an Acquity Premier BEH VanGuard FIT C18 column (100 mm x 2.1 mm, 1.7 pm particles, 130 A) by Waters (Milford, Massachusetts, United States) kept at 35 °C. The following gradient was used for analysis of SGA’s: 5% B for 1 min; 5% to 28% B in 22 min; 28% to 100% B in 14 min, 100% B for 3 min, returning to the initial conditions (5% phase B) within 0.5 min and a final re-equilibration to 5% B for another 2.5 min before next injection.

[0231] For both LC methods, the mobile phases consisted of 0.1% formic acid in water (phase A) and 100% acetonitrile (phase B), with a flow rate of 0.3 mL min1. Both analysis were carried in positive mode (ESI) and the samples were kept at 10 °C in the autosampler. The injection volume of both the standard solutions and the samples was 2 pL. The EVOQ source parameters were as follows: capillary voltage was 4,000 V; the source was kept at 400 °C; cone temperature was 350 °C; cone gas flow 20 arbitrary units; the heated probe gas flow 45 arbitrary units, nebulizer gas flow, 55 arbitrary units and exhaust gas on. The following precursor ions were recorded: m / z 1197.6 for uttroside B (11.7 min), m / z 868.5 for a-solamargine (22.2 min), m / z 884.5 for a-solasonine (21.3 min) and m / z 954.5 for malonyl- solamargine (23.1 min). Data analysis was performed using Bruker Daltonics MS Workstation software (Data Review version 8.2.1).GC-MS sterols analysis

[0232] Leaf and green unripe berries frozen tissues (100 mg) were powdered and saponified at 65 °C for 2 h in 600 pL of 20% KOH (w / v) in 50% ethanol in a thermoshaker (400 rpm, Eppendorf ThermoMixer). Samples were extracted three times with 500 pL hexane, and the combined phases were evaporated to dryness using a gentle stream of nitrogen. The samples were resuspended in 50 pl of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and incubated for 10 min at room temperature and then for 10 min at 65 °C. Samples were transferred to a glass inserts and or not, depending on the run conditions and analyte concentration if necessary, a sample dependent dilution was performed to be within the linear range of the detector. One (1) pL of final volume for each sample was injected onto GC-MS system comprised a GC PAL auto sampler (CTC Analytics), a trace 1310 GC ultra-gas chromatograph equipped with a split-splitless injector and ISQ LT quadrupole mass spectrometer (Thermo Scientific). GC was performed on a 30 m x 0.25 mm * 0.25 pm Zebron ZB-5 column with 10 m guard column (Phenomenex). Samples were analyzed in the split mode (split 1:20) and the inlet temperature was set at 250 °C. Separation of analytes was performed using the following chromatographic conditions: helium was used as carriergas at a flow rate of 1.1 ml min1. The thermal gradient used started at 160 °C (hold 1.5 min), ramped up to 270 °C at 30 °C min1, ramped up to 290 °C at 1 °C min1(hold 10 min) and then finally up to 300 °C at 30 °C min-1(hold 2.5 min). Eluents were fragmented in the electron impact mode with an ionization voltage of 70 eV, and the mass spectrometry transfer line temperature was set at 290 °C and the ion source at 250 °C. The chromatograms and mass spectra were evaluated using Xcalibur software (v.4.2.47; Thermo Scientific). Sterol compounds were identified by comparing their retention time and mass spectrum with those generated for authentic standards (trimethylsilylated) analyzed on the same instrument and those reported in the literature. Data analysis was performed with Xcalibur software (Thermo Scientific).Split luciferase assay

[0233] The plasmids pCAMBIA-nLUC and pCAMBIA-cLUC were digested by KpnI / Sall and KpnI / PstI restriction enzymes, respectively. GAME15 was cloned into pCAMBIA- nLUC and potential interacting partners (GAME6, GAME8, GAME11, GAME4, GAME12, and AtCYP90B l) were cloned separately into pCAMBIA-cLUC vectors by In-Fusion (TakaraBio) cloning. Each GAME gene used in the assay was amplified from cDNA of 5. nigrum (young leaves and unripe berries) while CYP90B 1 was amplified from cDNA of A. thaliana. The nLUC and cLUC constructs harboring respective gene of interest were transformed into A. tumefaciens (strain GV 3101) by electroporation. One- single colony of each construct was used to inoculate 10 mL LB media culture supplemented with 50 pg mL’1kanamycin, 100 pg mL1rifampicin and 50 pg mL-1gentamycin. The bacterial cultures were incubated overnight at 28 °C with shaking (200 rpm). The cultures were centrifuged at 2,000 x g for 20 min and cell pellets were washed once with 5 ml of infiltration buffer [50 mM MES buffer (pH 5.6), 10 mM MgC12, 150 pM acetosyringone]. Finally, each pellet was resuspended in 10 mL of infiltration buffer and incubated at room temperature for 1-2 h. For combinatorial infiltrations, optical density (ODeoo) for each strain was set at 0.4. Agrobacterium suspensions were infiltrated into 4-6- week-old N. benthamiana leaves. Leaves overexpressing the nLUC / cLUC constructs were imaged 48 hours post-infiltration in a Nightshade LB 985 (Barthold Technologies). The leaf material was sprayed with 0.5 mM solution of d- luciferin and incubated in the dark for 15 minutes, before being imaged on the adaxial side. Images were exposed for 0.1 seconds and luminescence emission was exposed for 20 seconds with 8 x 8 pixel binning.Reconstitution of steroidal scaffolds in N. benthamiana

[0234] Genes of interest (GAME6, GAME8, GAME11 and GAME 15) were amplified by PCR using cDNA prepared from RNA isolated from either green berries or young leaves. The PCR amplified genes were further cloned into binary 3Q1 destination vector using Goldenbraid cloning or In-Fusion cloning and transformed into A. tumefaciens (GV 3101) by electroporation. Single colonies with each target construct were inoculated and N. benthamiana infiltration (ODeoo=0.2 for each construct) was performed as mentioned above. After 5 days, infiltrated leaves were harvested for further LC-MS based analysis. Biological replicates consisted of several leaves collected from different infiltrated plants. Various GAME gene combinations with or without GAME 15 were used to examine the formation of furostanol / diosgenin (steroidal saponin) scaffold. Targeted profiling of steroidal scaffolds was performed on LC-Triple Quadrupole (TQ)-MS as described here. Due to the non-polar nature of the steroidal scaffolds (e.g., diosgenin), their recovery from N. benthamiana leaf samples using methanolic extraction method was rather poor. Therefore, the inventors adopted a non-polar extraction method for these samples as described above in the section ‘GC-MS sterols analysis’. Briefly, powdered leaf tissues (100 mg) was first saponified, extracted with hexane, dried and resuspended in 150 pl of ethanol. Extracts were filtered through 0.22 pm PTFE filter and 2 pL were injected on a Thermo Scientific UltiMate 3000 RS ultra-high performance liquid chromatography on a (UHPLC) system (Thermo Scientific, Germering, Germany) and analysed by a triple quadrupole mass spectrometer (EVOQ Elite™, Bruker Daltonics, Bremen, Germany) as described below. The metabolites were separated by reversed-phase liquid chromatography using an Acquity UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 pm particle size, 130 A, Waters, Milford, Massachusetts, United States) kept at 35 °C. The mobile phases consisted of 0.1% formic acid in water (phase A) and 100% acetonitrile (phase B), with a flow rate of 0.3 mL min1. The following gradient was used for analysis of diosgenin: 5% B for 1 min; 5% to 50% B in 9 min; 50% to 100% B in 7 min, 100% B for 1 min, returning to the initial conditions (5% phase B) within 0.1 min and a final re-equilibration to 5% B for another 2.9 min before next injection. The samples were kept at 10 °C in the autosampler during analysis. The mass spectrometer was operated in positive ionization mode. The EVOQ source parameters were as follows: heated ESI spray voltage (+) 4000 V; cone gas flow 20 arbitrary units at 350 °C; probe gas flow 45 arbitrary units at 400°C; nebulizer gas flow 55 arbitrary units; and exhaust gas on. The analysis was performed in MRM mode operating the QI mass analyzer under unit resolution (0.7 Da FWHM) and Q3 mass analyzer at 2.0 Da FWHM. MRM transitions were determined from the analytical standards and used to record during sample analysis: diosgenin (quantifier: m / z 415.1-^271.1, qualifier 1: m / z 415.1-^253.1, qualifier 2:m / z 415.1-^157.1). The EVOQ chromatograms were analysed using Data Review version 8.2.1 of the MS workstation software (Bruker Daltonics, Bremen, Germany).Sub-cellular localization studiesSub-cellular localization of GAME15

[0235] For the subcellular localization of GAME 15 protein, the corresponding gene sequence was fused in frame to an eGFP marker, both under the control of UbilO promoter and terminator in 3Ql-overexpression vector as described previously. Three different cellular compartment markers, an ER targeted mCHERRY, plasma membrane mCHERRY and nucleus mCHERRY markers (Addgene: #61170, #61180, #61168), were used for localization studies. Constructs were introduced in A. tumefaciens GV 3101 by electroporation and infiltrated in N. benthamiana leaves as described previously. Two days post-infiltration, micrographs were acquired from freshly punched and water embedded 5 mm leaf discs on a cLSM 880 (Zeiss, Oberkochen, Germany) with a C-Apochromat 40x / 1.20 W or Plan- Apochromat 63x / 1.4 Oil objective. Excitation wavelengths were generated with an Argon 488 and Helium-Neon 543 laser for GFP (8-10 % transmission, 525 PMT gain) and mCHERRY (40-100% transmission, 750 PMT gain) respectively. The spectral detector range was set to 490-550 nm for GFP and 550-650 nm for mCHERRY, both combined with MBS 488 / 543. Pinhole was set to 1 Airy Unit and the pixel dwell time was adjusted to 1 us with an 8-fold unidirectional line averaging, controlled with ZEN black (Zeiss, Oberkochen, Germany). Pixel dimensions were optimized depending on the used objective, excitation wavelength and zoom factor. Transmitted light signal was acquired with a T-PMT in the mCHERRY -track (T-PMT gain 250). Channels were scanned sequentially to reduce crosstalk. Contrast and brightness improvement, cropping and scale bar insertion was performed using Image J and Photoshop.Sub-cellular localization ofGAME6 and GAME8

[0236] For the subcellular localization of GAME6 and GAME8 proteins, the corresponding gene sequence was fused in frame to a mCERULEAN marker (Addgene #54730), both under the control of UbilO promoter and terminator by Goldenbraid (for GAME6) and In-Fusion (for GAME8) cloning. An ER targeted mCHERRY and GAME15 fused to eGFP were used for co-localization study purposes. Constructs were introduced in A. tumefaciens GV 3101 by electroporation and infiltrated in N. benthamiana leaves. The micrographs were acquired in a similar way as mentioned above. mCERULEAN was additionally excited using an Argon 458 laser (20-50% transmission, 700 PMT gain) simultaneous with the mCHERRYdetection, here combined with MBS 458 / 543. The spectral detector range was set to 460- 485 nm for mCERULEAN to minimize bleed through of the eGFP signal.Ecological studies and analysis

[0237] When cultivating Solanum nigrum wild type (WT) and game15ko plants in the greenhouse, the inventors noticed an increased infestation of pest insects. Usually insect pests are not a problem in 5. nigrum cultures in the glasshouse, as this Solanum species is well defended. To test whether this observation corresponds to reality, the inventors started an experiment in another greenhouse in Isserstedt close to Jena, which allows the inventors to let insects behave freely without influencing other experiments. The inventors first grew wild type and edited plants under pest free conditions in a glasshouse in Jena and transferred plants six weeks after germination to the greenhouse in Isserstedt. Ten plants of each line (WT, game 15ko#l, game 15ko#2~) were randomly placed on the table in a distance of at least 60 cm from each other. The experimental plants were distributed on two neighboring tables and other greenhouse plants with different pest have been placed on the third one. In addition to these pests, the inventors released 25 freshly caught Empoasca decipiens leafhoppers (see below). After two weeks, all plants were examined for the presence of insects. The inventors noticed the presence of E. decipiens on two WT, nine game15ko#l, and eight game15ko#2 plants. While no thrips {Echinothrips americanus~), aphids (Aulacorthum solani) or white flies (Trialeurodes vaporariorum) were observed on WT, the inventors found thrips on three game15ko#l and four game! 5ko#2 plants, aphids on game! 5ko#l plants and white flies on one of both game15ko# lines. These results encouraged the inventors to carry out feeding experiments with individual insect species to prove the effect of steroidal saponins on the choice and feeding behavior of insects.

[0238] E. decipiens was collected on potato plants (July to September 2023) and Buddleja (butterfly lilac) plants (September to December 2023) grown in the surroundings of the MPI- CE, Jena, Germany. E. decipiens can be found on the underside of the leaves. After harvesting the potato fields, they move to overwintering plants such as Buddleja, where they spend the entire cold season and are easy to catch in cold temperatures. The insects were used in the experiments within 4 hours of collection. None of the insects were used twice, and newly caught insects were used for each experiment. For the choice assay, WT leaves have been paired either with same sized leaves of game15ko#l or game15ko#2 mutant plants. Each leaf was taken from a different plant. Leaves were put individually in 2 mL labeled Eppendorf vials with a hole on top and filled with fresh tap water. The paired leaves were put together with a single leafhopper in a transparent 370 ml glass jar (WeekRundrandglas 100). The position of the leafhopper has been evaluated after 1 h, 3 h, 24 h and one week. Damage was recorded after one week using a ruler.

[0239] Colorado potato beetle (Leptinotarsa decemlineata say) larva and eggs have been collected in Auengrund (50.45420710.88961°) and Burgwenden (51.22527711.30243°), Germany, to start a colony under lab conditions. The colony was established in rearing cages (shop.bugdorm.com / product_mfo.php?cPath=l_l l&sort=la&products_id=34) in the greenhouse at a 16 / 8 h day-night cycle at 23-25 °C day and 16-22 °C night temperature. Beetles of the second generation have been used for all experiments. Each individual has been used only once. For the “forced-feeding” bioassay, one single Colorado potato beetle was placed in a Petri dish together with single detached leaves of either WT, game15ko#l or game15ko#2 S. nigrum plants. Each leaf was taken from a different plant. Leaves were put individually in 2 mL labeled Eppendorf vials with a hole on top and filled with fresh tap water. Damage caused by the feeding was recorded after 6 h using a ruler. For the choice assay, the same set up as for E. decipiens was employed. The position and the damage of the beetle have been evaluated after 3 h and 18 h.EXAMPLE 1GAME15 controls the biosynthesis of steroidal specialized metabolites in .S', nigrum

[0240] In tomato and potato steroidal glycoalkaloid (SGA) biosynthesis, the starting precursor cholesterol undergoes three hydroxylation / oxidation reactions catalyzed by two cytochrome P450, family 72, subfamily A (CYP45072A) enzymes, GLYCOALKALOID METABOLISM6 (GAME6, CYP72A188), and GAME8 (CYP72A208) at position C22 and C26 respectively, and at C16 by a 2-oxoglutarate-dependent dioxygenase, GAME 11 to generate the furostanol intermediate. This proposed furostanol is supposedly further oxidized (GAME4, CYP88B 1) and transaminated (GAME12, aminotransferase) to form steroidal alkaloid aglycones (e.g., dehydrotomatidine), which generate SGAs (e.g., dehydrotomatine) through the action of different UDP-glycosyltransferases (UGTs). In S', nigrum berries, solasodine is the core steroidal alkaloid aglycone, which is further glycosylated and / or acylated to produce diverse SGA structures such as a-solasonine, a-solamargine and malonyl-solamargine (Fig. 1A). In contrast, uttroside B, a steroidal saponin, is the major product that accumulates in the leaves of 5. nigrum. (Figs. 1A and 4A). Notably, uttroside B shares a common furostanol scaffold with SGAs, strongly suggesting that the furostanol scaffold is the branching point for the production of the steroidal saponins in the leaves and SGAs in the berries of S. nigrum. Therefore, GAME6, GAME8 and GAME11, which convert cholesterol to furostanol, likely act not only in SGA biosynthesis as has beenpreviously reported, but also in steroidal saponin biosynthesis (Fig. 1A). In fact, the identified orthologues of GAME6, GAME8 and GAME11 genes were found to be expressed in young leaves of S. nigrum plants (5-7 weeks old) (Fig. 4B), the tissue with the highest accumulation of uttroside B (Fig. 4A).

[0241] Co-expression analysis using transcriptomic data from 5. nigrum revealed a cellulose synthase like protein of the class G herein named GAME15 that exhibited strong coexpression with GAME6, GAME8 and GAME11 genes (Fig. IB). Together with GAME15, four UGTs that could also potentially be involved in uttroside B biosynthesis were also coexpressed in the analysis. Earlier studies proposed the involvement of GAME15 in SGA metabolism in the genus Solanum (e.g., tomato, potato) based on the strong co-expression pattern of GAME15 with SGA associated genes as well as the fact that GAME15 is a part of SGA biosynthetic metabolic gene cluster (chromosome 7) in tomato, potato and eggplant. The inventors hypothesized that GAME 15 could be involved in both steroidal saponin and SGA pathways in S. nigrum. However, the role of GAME15 in Solanum steroidal specialized metabolism remained unproven, and the mechanism by which a cellulose-like synthase protein would facilitate SGA or saponin biosynthesis was unknown.

[0242] To unequivocally determine the function of GAME 15, the inventors deleted the gene in S. nigrum using CRISPR / Cas9 (Figs. 2A and 5). Editing of game15 (game! 5ko) had no observable effect on the growth and developmental phenotype of the mutant lines (Fig. 6). However, metabolite profile analysis by liquid chromatography-triple-quadrupole mass spectrometry (LC-QqQ-MS) showed that uttroside B, the major steroidal saponin in the leaves of wild type (WT) plants, was lost in game15ko edited plants (Figs. 2B and 7A). The leaves of wild type 5. nigrum do not produce any SGAs (Fig. 2C). However, the SGAs a- solasonine, a-solamargine and malonyl-solamargine that are found in green unripe berries of wild type plants were also lacking in the unripe berries of edited plants (Figs. 2C and 7B). Conversely, there was a substantial increase in cholesterol, the starting biosynthesis precursor for SGAs and saponins (Figs. 2D-2E). These findings clearly demonstrate that the cellulose synthase like gene GAME 15 is crucial for the biosynthesis of both steroidal saponins and steroidal glycoalkaloids in S. nigrum plants.EXAMPLE 2GAME 15 localizes to the endoplasmic reticulum

[0243] Since it was not clear what role a cellulose synthase like protein could play in these natural product biosynthetic pathways, the inventors first investigated the sub-cellularlocalization of this protein by transient expression in Nicotiana benthamiana leaves. Confocal microscopy analysis of the infiltrated N. benthamiana leaves co-expressing GAME 15 fused to a fluorescent reporter (GAME15:eGFP; enhanced green fluorescent protein) and endoplasmic reticulum marker fused to mCHERRY (ER:mCHERRY) established that GAME 15 is localized to the membrane of the endoplasmic reticulum (Figs. 2F and 8). Accumulation of starting cholesterol precursor in game!5 mutant lines led the inventors to believe that earlier pathway steps in biosynthesis are somehow disrupted by the elimination of GAME15. The first two enzymes involved in saponin biosynthesis, GAME6 and GAME8, are both CYP450s, which typically operate in the ER membrane. The inventors next tested the sub-cellular localization of GAME6 and GAME8 (fused separately to a fluorescent reporter; GAME6:mCERULEAN and GAME8:mCERULEAN) in N. benthamiana leaves along with ER:mCHERRY marker and GAME15:eGFP. Confocal microscopy analysis showed that both CYPs are co-localized with GAME15 to the ER membrane (Figs. 9-10). The inventors hypothesized that GAME 15, which has no obvious catalytic function, might interact with these other ER-localized enzymes of the pathway, and that this interaction would facilitate the production of furostanol, the key central intermediate of steroidal saponin biosynthesis. To test whether pairwise protein -protein interactions occur between GAME15 and the earlier GAME enzymes involved in furostanol biosynthesis, the inventors carried out firefly luciferase complementation imaging assays by expressing the appropriately labeled proteins in N. benthamiana leaves. GAME 15 was fused to the N- terminus of the luciferase fragment, while the potential interacting partners were fused to the C-terminus of luciferase. The assay suggested that interaction occurs between GAME 15 and GAME6 (CYP72A188), GAME15 and GAME8 (CYP72A208) and GAME15 and GAME11 (dioxygenase) (Figs. 11-12). No luciferase activity was observed between GAME15 and GAME4 or GAME12, downstream enzymes that are specifically involved in steroidal glycoalkaloid biosynthesis (Figs. 11-12). The inventors also noted that there was no pairwise interaction between GAME 15 and the Arabidopsis thaliana CYP90B1, which catalyzes C22 hydroxylation of cholesterol in bras sino steroid biosynthesis, suggesting that the interactions observed with GAME 15 are specific for upstream Solanum steroidal pathway enzymes (Figs. 11-12). Although these experiments suggest that GAME15 acts as a structural protein in these pathways, the inventors cannot definitively rule out the possibility that GAME 15 has some cryptic catalytic activity.EXAMPLE 3Steroidal saponin scaffold generation in planta

[0244] Having established GAME15 as an essential component of steroidal metabolite biosynthesis, the inventors attempted to reconstitute steroidal saponin scaffold biosynthesis by Agrobacterium-mediated transient expression in the leaves of N. benthamiana. The inventors transiently expressed the respective GAME genes together with or without GAME15 in N. benthamiana leaves. No formation of furostanol, the common central intermediate for SGA and steroidal saponins, was observed when GAME6, GAME8 and GAME11 were co-expressed. However, when GAME15 was added to this combination of GAME genes, the cyclized form of furostanol (diosgenin) was clearly observed (Fig. 13). The inventors suspect that an endogenous enzyme of N. benthamiana oxidizes furostanol, which in turn triggers spontaneous cyclization to yield diosgenin.EXAMPLE 4GAME15 knockout reveals that steroidal saponins play a key role in defense

[0245] While loss of GAME15 did not visibly affect the phenotype of the mutant lines (Fig. 6), the inventors noticed that these plants were more susceptible to pest insects compared to the wild type 5. nigrum plants (Figs. 3A and 14). While SGAs have been reported to play a role in defense against pests, the inventors’ understanding of the ecological functions of steroidal saponins is much more limited. To investigate the role that steroidal saponins play in plant defense, the inventors then tested how the game15ko S. nigrum plants, deprived of steroidal saponins (Fig. 3B), would fare against Empoasca decipiens (family: Cicadellidae'), a native pest which feeds on leaves of various Solanum species, including tomato and potato. Steroidal glycoalkaloids, which are produced exclusively in berries, are not present in any detectable levels in leaves, suggesting that any defense response for leaves must be mediated by steroidal saponins, not alkaloids. The inventors performed a choice and feeding experiment in which detached wild type leaves were paired with leaves from game! 5 knock out lines (game15ko#1 or game15ko#2), and one individual E. decipiens. After one week, the bioassay illustrated the dramatic preference of E. decipiens for game15ko leaves (Figs. 3C-3D). Next, the inventors tested whether this class of compounds would affect an unrelated pest that has not co-evolved with S. nigrum, the Colorado potato beetles (Leptinotarsa decemlineata say - family: Chrysomelidae) (Fig. 3E), an international ‘superpest’ that is one of the most destructive insect herbivore of cultivated potato, tomato and eggplant leaves. Notably, Colorado potato beetle does not survive on leaves of wild type 5. nigrum. The inventors performed a “forced-feeding” bioassay in which single detached leaves of either wild type, game15ko#l or game 15ko#2 S. nigrum plants were placed individually together with one beetle. As with E. decipiens, game15ko plants lackingsteroidal saponins were highly susceptible to Colorado potato beetle, as evidenced by the consumed leaf biomass (Figs. 3F and 15). Notably, almost no visible consumption of leaf biomass occurred on wild type .S', nigrum detached leaves. The deterrent ability of steroidal saponins was further confirmed through a choice assay, wherein adult Colorado potato beetles were placed in a glass jar with detached leaves of wild type paired with either game15ko#l or game15ko#2 leaves. In this choice bioassay, adult beetles displayed a strong preference for game!5ko leaves over those of saponin-containing wild type plants (Figs. 3G and 16).

[0246] The current study shows that leaves deficient in steroidal saponins are highly susceptible to a variety of insect herbivores, providing the first definitive evidence for the role of steroidal saponins in plant defense, including uttroside B.EXAMPLE 5

[0247] Explored to a lesser extent, uttroside B is a major steroidal saponin accumulated in 5. nigrum leaf. Recently, a 25(R)-configuration in uttroside B was revealed using NMR and showed the role of S. melongena GAME8 (SmGAME8, C26 hydroxylase) in the formation of 25(1?) configuration in the steroidal scaffold. In contrast, .S', lycopersicum GAME8 (S1GAME8, C26 hydroxylase) was reported in the formation of 25(5) configuration steroidal scaffold (Itkin et al. 2013). With this GAME8 stereochemistry, the inventors hypothesized that a reconstitution-based approach could be used to evaluate the activity of known SGA biosynthesis enzymes for production of steroidal saponins (e.g., uttroside B) in N. benthamiana.

[0248] First, the inventors infiltrated N. benthamiana leaves with Agrobacterium tumefaciens harboring S1GAME15 (glucuronosyltransferase), S1GAME6 (cholesterol C22 hydroxylase), eggplant SmGAME8 (C26 hydroxylase), S1GAME11 (C16 hydroxylase), and four UGTs (S1GAME1, 2, 17, and 18) (Fig. 17). Analysis of N. benthamiana leaves extract using a LC-MS method (40 min run time) revealed an efficient production of dehydrouttroside B (m / z 1195.5750; C56H91O27) (Fig. 17C). Substituting SmGAME8 in the above combination with tomato S1GAME8 facilitated the conversion of cholesterol to 25(5)- dehydrouttroside B (m / z 1195.5753; C56H91O27), possibly ‘new to nature’, steroidal saponin in N. benthamiana (Fig. 17C). Next, infiltration of N. benthamiana leaves with same set of the above genes with S1GAME25 and 5-alpha-reductase (C5 double bond reduction) resulting in the efficient production of uttroside B (m / z 1197.5903; C56H93O27) and new 25(5)-uttroside B (m / z 1197.5912; C56H93O27) from SmGAME8 and S1GAME8, respectively (Figs. 17A and 17D). Notably, addition of glucose on furostanol C26 may be performed byan endogenous N. benthamiana UGT (Fig. 17A), because other UGTs (S1GAME1,2,17,18) reported to act on C3 (Itkin et al. 2011; Itkin et al. 2013). Production of C25 stereospecific saponins (e.g., uttroside B) in N. benthamiana from S1GAME8 and SmGAME8 suggesting their activity is important in formation of diverse steroidal scaffolds and further validated Jozwiak et al (unpublished) work on GAME8. Uttroside B and dehydrouttroside B are not commercially available, but compounds with corresponding masses (m / z 1197.5903, m / z 1195.5751) were observed in the leaves of S. nigrum (Figs. 17C-17D). All metabolites (A. benthamiana infiltration products) were putatively identified by comparing their retention times, accurate mass-derived elemental composition, and mass fragmentation pattern with those described for the same by Itkin et al (2013) (Fig. 18). An additional MS / MS analysis was performed to analyze the structures of identified saponin products (Fig. 18).

[0249] Altogether, these results showed that GAME8 activity not only is capable of producing natural steroidal saponins (uttroside B and dehydrouttroside B) but also allows the production of new to nature, 25(S)-uttroside B and 25(5)-dehydrouttroside B, steroidal saponins in N. benthamiana. In summary, characterization of the crucial enzymes (like GAME8 in this study) allows the engineering of structurally diverse products in plants. Therefore, this study sets the base for metabolic engineering efforts to improve production of commercial valuable and diverse class of metabolites.EXAMPLE 6

[0250] Solanum nigrum, like other species in the Solanum genus, produces steroidal alkaloid aglycones with varying chirality at the C25 position, influenced by the activity of GAME8 enzymes, which are cytochrome P450 enzymes responsible for modifying cholesterol derivatives in the plant. This species predominantly accumulates the 25R isomer in its leaves and stems, while its roots contain both 25R and 255 isomers, indicating tissue-specific expression of GAME8 genes (Fig. 19A). Solanum nigrum has two GAME8 genes; the tomato-like GAME8 gene is mainly expressed in the roots, forming the 25S isomer, while the eggplant-like GAME8 gene is expressed across all tissues, leading to the predominant formation of the 25R isomer (Figs. 19B-19C). This dual expression and functionality suggest an evolutionary adaptation that has allowed Solanum nigrum to develop a complex system of chemical defense through the production of diverse glycoalkaloids. The stereochemistry of these alkaloids, which is a result of gene duplication and neofunctionalization events, likely plays a crucial role in the plant's defense mechanisms and adaptability to various environmental pressures.

[0251] Further to the above, to investigate the role of the tomato GAME8 gene (SIGAMES) in altering SGA stereochemistry, the inventors have overexpressed this gene in Solatium nigrum. By introducing and overexpressing SIGAME8 in 5. nigrum, the inventors observed a significant increase in the 25 S isomer, demonstrating that the tomato GAME8 enzyme can successfully alter the stereochemical makeup of SGAs in this plant (Figs. 19D-19E). This shift highlights the flexibility of SGA biosynthesis pathways in Solanum species and underscores the potential of genetic manipulation for enhancing or modifying plant secondary metabolites for agricultural or pharmaceutical purposes.

[0252] Accordingly, it is submitted that a method including the expression and / or overexpression of a tomato GAME8, or a functional analog thereof, may be applicable so as to increase production of Uttroside B 255 isomer(s) in a Solanum plant.

[0253] 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.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising an effective amount of a compound represented by FormulaI:, and an agriculturally acceptable carrier, wherein said composition being an insecticide.

2. The composition of claim 1, wherein said compound represented by said Formula I is biosynthetically produced.

3. The composition of claim 2, wherein said biosynthetically produced is by a recombinant or transgenic cell.

4. The composition of claim 3, wherein said recombinant or transgenic cell is any one of: a unicellular organism, a cell of a multicellular organism, and a cell in a culture.

5. The composition of claim 4, wherein unicellular organism comprises a fungus or a bacterium.

6. The composition of claim 5, wherein said fungus is a yeast cell.

7. The composition of any one of claims 1 to 6, wherein said compound represented by said Formula I is present in said composition as a purified extract of a Solatium plant.

8. The composition of claim 7, wherein said Solatium plant is Solarium nigrum.

9. A method for controlling an insect infection in a plant, the method comprising contacting said plant with an effective amount of a compound represented by Formula I.

10. The method of claim 9, wherein said controlling comprises treating and / or preventing.

11. The method of claim 9 or 10, wherein said controlling comprises increasing resistance of said plant to said insect.

12. The method of claim 9 or 10, wherein said controlling comprises: repelling said insect from said plant, reducing feeding rate of said insect on said plant, reducing number of eggs laid by said insect on said plant, or any combination thereof.

13. The method of any one of claims 9 to 12, wherein said insect is any one of: Empoasca decipiens, Leptinotarsa decemlineata, and both.

14. A method for synthesizing a compound represented by Formula I, the method comprising the steps: a. providing a cell comprising an artificial DNA molecule comprising a first polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 1, a second polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 2, a third polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 3, and a fourth polynucleotide comprising the nucleic acid sequence as forth in SEQ ID NO: 4; and b. culturing said cell from step (a) such that at least one protein being encoded by any one of said first polynucleotide, said second polynucleotide, said third polynucleotide, and said fourth polynucleotide, is expressed, thereby synthesizing the compound represented by Formula I.

15. The method of claim 14, wherein said artificial DNA molecule further comprises a fifth polynucleotide encoding a protein being a glucose transferring (GT) enzyme.

16. The method of claim 14 or 15, wherein said at least one protein comprises an amino acid sequence set forth in any one of SEQ ID Nos: 10-13.

17. The method of any one of claims 14 to 16, wherein said culturing comprises supplementing said cell with an effective amount of cholesterol.

18. The method of any one of claims 14 to 17, wherein said artificial DNA molecule is an expression vector.

19. The method of any one of claims 14 to 18, wherein said cell is a prokaryote cell or a eukaryote cell.

20. The method of any one of claims 14 to 19, wherein said cell is a transgenic cell or a cell transfected with said artificial DNA molecule.

21. The method of any one of claims 14 to 20, further comprising a step before step (a), comprising introducing or transfecting said cell with said artificial DNA molecule.

22. The method of any one of claims 14 to 21, further comprising a step comprising isolating, extracting, purifying, or any combination thereof, said compound represented by Formula I.

23. An extract obtained according to the method of claim 22.

24. An artificial DNA molecule comprising: a. a first nucleic acid sequence encoding the enzyme GLYCOALKALOID METABOLISM 6 (GAME6) comprising the amino acid sequence set forth in SEQ ID NO: 10; b. a second nucleic acid sequence encoding the enzyme GAME8 comprising the amino acid sequence set forth in SEQ ID NO: 11; c. a third nucleic acid sequence encoding the enzyme GAME 11 comprising the amino acid sequence set forth in SEQ ID NO: 12; and d. a fourth nucleic acid sequence encoding the enzyme GAME15 comprising the amino acid sequence set forth in SEQ ID NO: 13.

25. The artificial DNA molecule of claim 24, further comprising a fifth nucleic acid sequence encoding a protein being a GT enzyme.

26. A plasmid or an expression vector comprising the artificial DNA molecule of claim 24 or 25.

27. A transgenic cell comprising any one of: a. the artificial DNA molecule of claim 24 or 25; b. the plasmid or expression vector of claim 26; or c. both (a) and (b).

28. The transgenic cell of claim 27, being any one of: a unicellular organism, a cell of a multicellular organism, and a cell in a culture.

29. The transgenic cell of claim 28, wherein said unicellular organism comprises a fungus or a bacterium.

30. The transgenic cell of claim 29, wherein said fungus is a yeast cell.

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