Pest resistant plants

Abstract

The disclosure provides an isolated nucleic acid molecule encoding a 7-epizingiberene synthase, a chimeric gene comprising said nucleic acid molecule, vectors comprising the same, as well as isolated 7-epizingiberene synthase proteins themselves. In addition, transgenic plants and plant cells comprising a gene encoding a 7-epizingiberene synthase, optionally integrated in its genome, and methods for making such plants and cells, are provided. Especially Solanaceae plants and plant parts (seeds, fruit, leaves, etc.) with enhanced insect pest resistance are provided.

Claims

1. A Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit, comprising a nucleotide sequence encoding a 7-epizingiberene synthase comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence comprising at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1.

2. The Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit according to claim 1, which has enhanced 7-epizingiberene production compared to a wild-type Solanum lycopersicum plant.

3. The Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit according to claim 1, which has enhanced insect pest resistance compared to a wild-type Solanum lycopersicum plant.

4. The Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit according to claim 1, which has enhanced resistance to whiteflies compared to a wild-type Solanum lycopersicum plant.

5. The Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit according to claim 1, further comprising a nucleic acid sequence encoding a Z,Z-farnesyl diphosphate synthase comprising the amino acid sequence of SEQ ID NO:6 or an amino acid sequence comprising at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO:6.

6. The Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit according to claim 5, which has enhanced 7-epizingiberene production compared to a wild-type Solanum lycopersicum plant.

7. The Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit according to claim 5, which has enhanced insect pest resistance compared to a wild-type Solanum lycopersicum plant.

8. The Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit according to claim 5, which has enhanced resistance to whiteflies compared to a wild-type Solanum lycopersicum plant.

9. The Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit according to claim 1, wherein the Solanum lycopersicum plant, plant cell or tissue culture, seed, or fruit is non-transgenic.

Description

FIGURES

(1) FIG. 1 shows Gas Chromatography Mass Spectrometry (GCMS) results of production of 7-epizingiberene using zFPP as precursor by E. coli transformed with the nucleotide sequence encoding 7-epizingiberene synthase. 7-epizingiberene was identified by its MS ion mass fingerprint, retention time and Kovats index.

(2) FIGS. 2A and 2B show a mass spectra of 7-epizingiberene produced by expression of the nucleotide sequence encoding 7-epizingiberene synthase (FIG. 2A) in E. coli and 7-epizingiberene produced by S. habrochaites PI127826 trichomes (FIG. 2B).

(3) FIG. 3A shows the determination of the enantiomer of ShZIS

(4) Enantioselective gas chromatography on the cyclodextrin coated column allowed identification of the different zingiberene stereoisomers (Astec CHIRALDEX™ B-DM column, Supelco). From top to bottom: S. habrochaites=positive control for 7-epizingiberene; F2 ShxSI=the F2 produces 7-epizingiberene with zFPP as precursor gingeroil=positive control for alpha-zingiberene. ShZIS+ginger oil=ShZIS with zFPP produces 7-epizingiberene and ginger oil contains alpha-zingiberene

(5) FIG. 3A indicates that ShZIS synthesizes 7-epizingiberene when provided with zFPP. Moreover, it shows evidence that F2 plants used in the bioassays also produce 7-epizingiberene.

(6) FIG. 3B shows the determination of the enantiomer of the zingiberene produced by ShZIS. Enantioselective gas chromatography on the cyclodextrin coated column allowed identification of the different zingiberene stereoisomers (Astec CHIRALDEX™ B-DM column, Supelco). From top to bottom: S. habrochaites=positive control for 7-epizingiberene; αZIS+FPP=alpha-zingiberene synthase provided with FPP yields α-zingiberene; αZIS+FPP & ShZIS+zFPP=alpha-zingiberene synthase provided with FPP yields alpha-zingiberene and ShZIS synthesizes 7-epizingiberene when provided with zFPP. Ginger oil=positive control for alpha-zingiberene. ShZIS+zFPP=the protein ShZIS, when supplied with zFPP as a precursor produces 7-epizingiberene.

(7) The figure indicates that ShZIS synthesizes 7-epizingiberene when provided with zFPP. Lemon basil zingiberene synthase (ObZIS; Iijima et al., 2004) is a bona-fide alpha-zingiberene synthase when provided with FPP.

(8) FIGS. 4A and 4B show production of 7-epizingiberene in transgenic S. lycopersicum plants, when both zFPS and ShZIS are expressed under trichome specific promoters. FIG. 4A. The production of 7-epizingiberene as measure by GCMS. Depicted are the terpenoid profiles of untransformed control (S. lyc) plants, S. lycopersicum plants transformed only with zFPS, and S. lycopersicum plants transformed with both zFPS and ShZIS under trichome-specific promoters. 7-epizingiberene was only produced in plants transformed with zFPS and ShZIS (both under trichome-specific promoters). FIG. 4B. Enantioselective gas chromatography on the cyclodextrin coated column proved that zingiberene production in S. lycopersicum plants transformed with zFPS and ShZIS (S. lycopersicum zFPS-ZIS in the figure), like in wild S. habrochaites, is 7-epizingiberene.

(9) FIG. 5 shows the concentration of zingiberene (ng terpenes per mg leaf FW) in three different genotypes. An interspecies cross between S. lycopersicum and S. habrochaites was performed and F2 lines were tested for the production of zingiberene. Cuttings were made of zingiberene producing F2 lines, S. lycopersicum C32 (Moneymaker) and of S. habrochaites (PI127826). F2 and S. habrochaites (PI127826) plants showed similar amounts of 7-epizingiberene, no 7-epizingiberene was detectable in S. lycopersicum C32.

(10) FIG. 6A shows the percentage of dead B. tabaci adults (mortality) in three different genotypes. Clip cage experiments were performed on cuttings of F2 lines, S. lycopersicum C32 (Moneymaker) and of S. habrochaites (PI127826). Compared to S. habrochaites and F2 plants, the percentage dead adults after 5 days was significantly lower on S. lycopersicum (FIG. 4A; One-way ANOVA, LSD; p<0.05 for both comparisons).

(11) FIG. 6B shows the total number of eggs deposited by whitefly adults in five days. Clip cage experiments were performed on cuttings of F2 lines, S. lycopersicum C32 and of S. habrochaites (PI127826). The number of eggs deposited by female B. tabaci adults was significantly higher on S. lycopersicum C32 compared to either F2 or S. habrochaites plants (FIG. 4B; One-way ANOVA, LSD; p<0.05 for both comparisons).

(12) FIG. 7A shows 7-epizingiberene levels F2 plants expressed as [zingiberene] (ng mg-1 FW leaf).

(13) FIG. 7B shows Colorado Potato Beetle (CPB) neonate larvae survival in a bio-assay (24 hrs feeding).

(14) FIG. 7C shows feeding damage by CPB—24 hrs of feeding.

(15) FIG. 7D shows feeding damage by CPB—24 hrs of feeding—damage is classified as arbitrary units (pixels).

(16) FIG. 8 demonstrates the preference of the Greenhouse whitefly (Trialeurodes vaporariorum) preference in a choice-assay for low 7-epizingiberene producing plants (line F2-45) over high 7-epizingiberene (line F2-40) plants.

(17) FIG. 9 shows performance of the potato/tomato aphid (Macrosiphum euphorbiae) in a no-choice-assay between low 7-epizingiberene producing plants (line F2-45) and high 7-epizingiberene producing (line F2-40) plants.

(18) FIG. 10A demonstrates oviposition of Tuta absoluta. Tuta absoluta moths were released in a cage on F2 plants that produced a range of 7-epizingiberene. These plants have arisen from an interspecies cross between S. lycopersicum (C32) and S. habrochaites (PI127826). The number of eggs per tomato genotype was determined after 5 days.

(19) FIG. 10B shows production of 7-epizingiberene in these F2 plants arisen from an interspecies cross between S. lycopersicum (C32) and S. habrochaites (PI127826).

(20) FIG. 11A demonstrates Spider mite (T. urticae) fecundity on S. lycopersicum (C32), 7-epi-zingiberene producing transgenic S. lycopersicum (line 2) and S. habrochaites (PI127826).

(21) FIG. 11B shows Spider mite (T. urticae) survival on S. lycopersicum (C32), 7-epi-zingiberene producing transgenic S. lycopersicum (line 2) and S. habrochaites (PI127826).

(22) FIG. 11C displays Spider mite (T. evansi) fecundity on S. lycopersicum (C32), 7-epi-zingiberene producing transgenic S. lycopersicum (line 2) and S. habrochaites (PI127826).

(23) FIG. 11D demonstrates Spider mite (T. evansi) survival on S. lycopersicum (C32), 7-epi-zingiberene producing transgenic S. lycopersicum (line 2) and S. habrochaites (PI127826).

(24) The following non-limiting Examples illustrate the different embodiments of the invention. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

(25) All references recited in the present disclosure are herein incorporated by reference.

EXAMPLES

Example 1

E. coli Expression Assay to Determine Production of 7-epizingiberene

(26) The full length gene (comprising SEQ ID NO:4 5′ of SEQ ID NO:2 (SEQ ID NO:4-SEQ ID NO:2)) was cloned into the pGEX-KG expression vector (Guan and Dixon 1991). Constructs were transformed to C41 (DE3) E. coli cells (Dumon-Seignovert et al., 2004). As a control, empty pGEX-KG vector was transformed. A culture was grown to an OD.sub.600 of 0.5-0.6 at 37° C. and placed at 4° C. for 30 min. Protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After 16 hours incubation at 16° C., cells were harvested by centrifugation. The supernatant was removed and the pellet was resuspended in assay buffer (25 mM HEPES, pH 7.2, 10 mM MgCl.sub.2, 10% (v/v) glycerol) with added lysozyme (1 mg mL.sup.−1) and proteinase inhibitors and incubated on ice for 30 minutes and subsequently sonicated. The lysate was centrifuged and the supernatant was stored at −80° C. Activity assays were performed in 500 μL 50 mM HEPES, pH7.2, 100 mM KCl, 7.5 mM MgCl.sub.2, 20 μM MgCl.sub.2, 5% (v/v) glycerol, 5 mM DTT with 50 μL protein and 2 mM cis-FPP(2Z-6Z-farnesyl diphosphate) as substrate. Enzyme products were analyzed by GC-MS with Solid Phase Micro Extraction fiber (SPME). Terpene products were identified using standards and comparing ion spectra, retention time and Kovats Index (see FIGS. 1 and 2).

Example 2

Determining the Enantiomer of Zingiberene Produced by the Various Genes/Proteins

(27) Activity assays were performed in 20 mL glass vials in a total volume of 500 μL 50 mM HEPES, pH7.2, 100 mM KCl, 7.5 mM MgCl.sub.2, 20 μM MgCl.sub.2, 5% (v/v) glycerol, 5 mM DTT with 50 μL protein and either 2 mM cis-FPP (2Z-6Z-farnesyl diphosphate), trans-FPP (E-E-farnesyl diphosphate), GPP (geranyl diphosphate), NDP (neryl diphosphate), or GGPP (geranylgeranyl diphosphate) as a substrate (Echelon Biosciences Incorporated, Salt Lake City, USA). Vials were closed with a teflon lined crimp cap immediately and incubated under moderate shaking for 1 hour at 30° C.

(28) Enzyme products were sampled with a Solid Phase Micro Extraction fiber (SPME) for 10 minutes after the vial had been agitated and heated to 50° C. The fiber was desorbed for 1 minute in an Optic injector port (ATAS GL Int. Zoeterwoude, NL) which was kept at 220° C. For liquid injection 1-3 μL of sample in hexane was injected.

(29) In order to separate alpha-zingiberene from 7-epizingiberene the Astec CHIRALDEX™ B-DM column (30m×0.25 mm×0.12 μm film thickness; Supelco) was selected. The column was placed in an 6890 N gas chromatograph (Agilent, Amstelveen, NI). The programme was initially set to 115° C. for 3 minutes, increased to 140° C. by 4° C. min.sup.−1 where it was kept for an additional minute after which the temperature slowly increased (2° C. min.sup.−1) to 166° C. by where it was kept for 5 minutes prior to a rapid increase with 40° C. min.sup.−1 to 220° C. Helium was used as a carrier gas. Mass spectra were generated with the ion source set to −70V at 200° C. and collected with a Time-of-Flight MS (Leco, Pegasus III, St. Joseph, Mich., USA) at 1850 V, with an acquisition rate of 20 scans per second.

(30) Run time 1800 sec; Initial injector temperature: 220° C.; Final injector temperature: 220° C.; Transfer column flow: 1.5 mL min.sup.−1; Transfer time: 120 sec; Initial column flow: 1 mL min.sup.−1; Final column flow: 1 mL min.sup.−1; Split flow: 25 mL min.sup.−1

(31) More columns specifics can be obtained via the supplier or via: http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N5=SEARCH_CONCAT_PNO|BRAND_KEY&N4=66023AST|SUPELCO&N25=0&QS=ON&F=SPEC

(32) Results

(33) Enantioselective gas chromatography on the cyclodextrin coated column allowed identification of the different zingiberene stereoisomers in S. habrochaites and ginger, previously inseparable by our GC-MS analysis. By NMR it was determined that S. habrochaites PI127826 produces 7-epizingiberene, whereas gingeroil contains alpha-zingiberene (Bleeker et al., 2011). Extracts from S. habrochaites and gingeroil were used as positive controls to study the enantiomer status of zingiberene synthesized by ObZIS (sweet lemon basil zingiberene synthase; Iijima et al., 2004) and ShZIS. Analysis (both liquid and SPME) indicated that the enzymes synthesize different stereoisomers. ShZIS is responsible for the production of 7-epizingiberene (similar to the enantiomer found in S. habrochaites), whereas ObZIS is a bona-fide alpha-zingiberene synthase (FIG. 3A,B).

(34) FIG. 3A: Liquid injection of samples in hexane. S. habrochaites leaf wash: standard for 7-epizingiberene (RT:844). Ginger oil: standard for alpha-zingiberene (RT: 851) and S-curcumene (RT:829). Mix leafwash and gingeroil: S-curcumene (RT:829), 7 epizingiberene (RT:844) and alpha-zingiberene (RT:851). Hexane overlay of E. coli C41 (DE3) transformed with pGEX:ZIS2 incubated with zFPP: 7-epizingiberene (RT:844). This experiment shows separation of 7-epizingiberene and alpha-zingiberene (previously identified with NMR in Bleeker et al., 2011) on the chiral column and proves that heterologously expressed 7-epizingiberene synthase (ShZIS) is responsible for 7-epizingiberene in PI127826. It also shows that the F2 plant produces 7-epizingiberene.

(35) FIG. 3B: SPME: S. habrochaites leaf material as standard for 7-epizingiberene (RT: 850) and R-curcumene (RT:841). Gingeroil as standard for alpha-zingiberene (RT:856) and S-curcumene (RT:835). Sweet lemon basil ObZIS (lijima, R. Davidovich-Rikanati, E. Fridman, D. R. Gang, E. Bar, E. Lewinsohn, and E. Pichersky (2004). The Biochemical and Molecular Basis for the Divergent Patterns in the Biosynthesis of Terpenes and Phenylpropenes in the Peltate Glands of Three Cultivars of Basil. Plant Physiology 136; 3724-3736.) heterologously expressed and provided with E-E-FPP made alpha-zingiberene (RT:856). PI127826 ShZIS heterologously expressed and provided with Z-Z-FPP made 7-epizingiberene (RT:850). Mixed ObZIS and ShZIS showed both peaks. This experiments shows that ShZIS makes a different zingiberene stereoisomer than known plant zingiberene synthase ObZIS.

Example 3

Development of Transgenic S. lycopersicum Plants

(36) Tomato Cotyledon Explant Transformation Experiments

(37) Tomato (S. lycopersicum) line C32 was used for transformations with Agrobacterium tumefaciens (GV3101). The tomato transformation protocol has been described in Koornneef et al (1986) (Koornneef, Maarten, Jongsma, Maarten, Weide, Rob, Zabel, Pim, and Hille, Jacques. (1986); Transformation of tomato. In: Tomato Biotechnology, Donald Nevins and Richard Jones, eds. Alan Liss Inc., New York, USA, pg. 169-178.) and in Koornneef et al (1987) (Koornneef, M., Hanhart, C. J., and Martinelli, L. (1987); A genetic analysis of cell culture traits in tomato. Theor. Appl. Genet. 74: 633-641). Trichome-specific targeting was ensured using MKS1 (methylketone synthase 1; Fridman et al., 2005 (Fridman E, Wang J, Iijima Y, Froehlich J E, Gang D R, Ohlrogge J, Pichersky E (2005). Metabolic, genomic, and biochemical analyses of glandular trichomes from the wild tomato species Lycopersicon hirsutum identify a key enzyme in the biosynthesis of methylketones. Plant Cell 17: 1252-1267)) and MTS1 (monoterpene synthase 1; WO2009082208) from S. habrochaites and S. lycopersicum, respectively. For co-transformation, Agrobacterium carrying a binary vector with pMKS1:zFPS and pMTS1:ShZIS were diluted cultures are mixed in a ratio of 1:1. The remainder of the described protocol has been unchanged. When tomato shoots appeared, they were harvested and rooted on solid MS20 medium containing 1 mg L.sup.−1 IBA, 200 mg L.sup.−1 cefotaxime, 200 mg L.sup.−1 vancomycin, and 100 mg L.sup.−1 kanamycin.

(38) Genomic DNA was isolated from transgenic plants and PCR was performed on TO plants to confirm successful insertion of the plasmids. Leaf material of TO plants was harvested and analyzed by GC-MS as described above.

(39) Results:

(40) Bleeker et al. (2009) previously have shown that 7-epizingiberene is produced by S. habrochaites PI127826. The gene responsible for the production of 7-epizingiberene, called ShZIS, was isolated from S. habrochaites PI127826. Transgenic plants were produced by Agrobacterium-mediated transformation of S. lycopersicum C32. Whereas no 7-epizingiberene was formed in S. lycopersicum (C32) control plants or plants transformed with MKS1:zFPS only, 7-epizingiberene was present in transgenic S. lycopersicum plants with zFPS and ShZIS inserted into their genome (FIG. 4).

Example 5

Effect of the Expression of the 7-epizingiberene Synthase-encoding Nucleotide Sequence on Insect Pest Resistance

(41) Methodology Bioassays:

(42) An interspecies cross between S. lycopersicum and S. habrochaites was performed and F2 lines were transferred to the greenhouse at the University of Amsterdam. The F2 plants were tested for their production of 7-epizingiberene. Cuttings were made of 7-epizingiberene producing F2 lines, S. lycopersicum C32 (moneymaker) and of S. habrochaites (PI127826). Both a parental line of the initial cross.

(43) Bioassay B. tabaci (Whitefly)

(44) Two cuttings of genotypes PI127826 and C32 and the respective F2s received 4 clip cages, each of which contained 20 adult B. tabaci (biotype Q) initially collected in Almeria (Spain) and reared continuously on cucumber under laboratory conditions (see Bleeker et al., 2009—Plant Physiol.). After 5 days, the total number and percentage of dead flies and total nr of eggs (combined abaxial and adaxial side of leaves) was determined.

(45) In addition, leaf material of same leaflet was used to determine the terpene concentrations.

(46) F2 plants have 7-epizingiberene levels comparable to S. habrochaites (PI127826). 7-epizingiberene was not detected in S. lycopersicum (C32).

(47) Results Bioassay B. tabaci:

(48) Cuttings of an F2 plant and S. habrochaites (PI127826) showed similar amounts of 7-epizingiberene. No 7-epizingiberene could be detected in S. lycopersicum C32 (FIG. 5). Moreover, increased resistance to whiteflies was observed in cuttings of the F2 plant and S. habrochaites (PI127826) (FIG. 6A,B). Compared to S. habrochaites and F2 plants, the percentage dead adults after 5 days was significantly lower on S. lycopersicum (FIG. 6A; One-way ANOVA, LSD; p<0.05 for both comparisons). Moreover, the number of eggs deposited by female B. tabaci adults was significantly higher on S. lycopersicum C32 compared to either F2 or S. habrochaites plants (FIG. 6B; One-way ANOVA, LSD; p<0.05 for both comparisons). Both the mortality and oviposition characteristics show that 7-epizingiberene produced by plants enhances resistance to whiteflies.

(49) Bio-assay Colorado Potato Beetle (CPB)

(50) Larvae of the CPB, Leptinotarsa decemlineata (order: Coleoptera) were reared on potato (cultivar Bintje). A no-choice assay with performed for 24 hours. CPB larvae (neonates) were allowed to feed on leaf discs (1.2 cm diameter) of F2 plants arisen from an interspecies cross between S. lycopersicum (C32) and S. habrochaites (PI127826).

(51) F2 plant 40 shows high levels of zingiberene (similar to S. habrochaites), whereas F2 plant 45 only produced minute levels of zingiberene. Leaf discs from both genotypes were placed on wetted filter paper in a Petri dish and one larvae was allowed to feed for 24 hours (10 biological replicates per plant genotype). Subsequently, larvae survival and feeding damage were assessed.

(52) Result Bio-assay CPB

(53) 7-epizingiberene levels were measured using the method described above. The F2 plant 40 shows high concentration of 7-epizingiberene, whereas F2 plant 45 produces only minute levels of 7-epizingiberene (concentration at the detection limit; FIG. 7A).

(54) Larvael survival after 24 hours of feeding was significantly different on the two genotypes. Only 20% of the larvae survived on the high 7-epizingiberene producing F2-40 plant. In contrast, most larvae survived (70%) and were feeding from the low-producing plant (F2-45; FIG. 7B). Feeding damage was assessed by scanning the leaf discs. Significantly more damage was observed on plants with low 7-epizingiberene (plant F2-45; FIG. 7C). Moreover, damage due to CPB feeding was quantified by using ImageJ. The analysis determines the number of pixels (arbitrary units) of scanned leaf discs. Damage was determined as the number of pixels for undamaged leaf discs compared to that of CPB damaged leaf discs. FIG. 7D indicates that significantly more damage was observed on leaf discs of plant F2-45, compared to F2-40 (high 7-epizingiberene production).

(55) Bio-assay Trialeurodes vaporariorum (Greenhouse Whitefly)

(56) Trialeurodes vaporariorum (order: Hemiptera) were reared on tomato (S. lycopersicum). A choice assay with performed for 24 hours. Adults were released in a cage with two F2 plants arisen from an interspecies cross between S. lycopersicum (C32) and S. habrochaites (PI127826). Subsequently, adult settling preference was determined on leaves of the following F2 plants (10 leaves per plant). F2 plant 40 showed high levels of 7-epizingiberene (similar to S. habrochaites), whereas F2 plant 45 only produced minute levels of 7-epi-zingiberene (FIG. 7A).

(57) Results Bio-assay Trialeurodes vaporariorum

(58) Greenhouse whitefly preference was different on the two genotypes (FIG. 8). Compared to the high 7-epizingiberene producing plants (F2-40), twice as many greenhouse whitefly adults settled on the low 7-epizingiberene producing plants (F2-45).

(59) Bio-assay Macrosiphum euphorbiae (Potato/Tomato Aphid)

(60) Macrosiphum euphorbiae (order: Hemiptera) were reared on tomato (S. lycopersicum). A no-choice assay was performed for 48 hours. One adult aphid was placed in a clip-cage on either of two F2 plants arisen from an interspecies cross between S. lycopersicum (C32) and S. habrochaites (PI127826). Subsequently, aphid performance (survival and number of offspring) was determined on the following F2 plants (3 clip-cages per plant; 6 plants per genotype). F2 plant 40 shows high levels of 7-epizingiberene (similar to S. habrochaites), whereas F2 plant 45 only produced minute levels of 7-epizingiberene (FIG. 7A).

(61) Results Bio-assay Macrosiphum euphorbiae

(62) Aphid performance was different on the two genotypes (FIG. 9). Compared to the high 7-epizingiberene producing plants (F2-40), aphids performed better in terms of survival and number of offspring produced on low 7-epizingiberene producing plants (F2-45).

(63) Bio-assay Tuta absoluta

(64) Tuta absoluta (order: Lepidoptera) were reared on tomato (S. lycopersicum). A no-choice assay was performed for 7 days. 5 adults were allowed to oviposit their eggs on S. lycopersicum (C32) plants and on F2 plants arisen from an interspecies cross between S. lycopersicum (C32) and S. habrochaites (PI127826). After 7 days, Tuta abosoluta oviposition (number of eggs deposited) was determined on the abaxial and adaxial side of six leaves per plant genotype. F2 plants were characterized for 7-epizingiberene content after the assay and Tuta abosoluta oviposition (number of eggs deposited) was correlated to the content of 7-epizingiberene.

(65) Results Bio-assay Tuta absoluta

(66) Oviposition by Tuta absoluta females was significantly reduced on F2 plants producing 7-epizingiberene (FIG. 10a). FIG. 10b indicates the 7-epizingiberene concentration in the F2 plants tested for Tuta absoluta ovipostition. Oviposition was negatively correlated with 7-epizingiberene content (combination of FIGS. 10A and 10B).

(67) Bio-assay Spider Mites

(68) Spider mites, like insects, belong to the arthropods but are a different class of organisms. The effect of 7-epizingiberene was tested on two spider mite species, Tetranychus urticae and T. evansi. Both arthropod species were reared on common garden bean. A 4-day no-choice assay was performed with synchronized populations of T. urticae and T. evansi. Mites were place on leaf discs of susceptible control plants (S. lycopersicum), resistant S. habrochaites PI127826 plants and on 7-epizingiberene producing transgenic S. lycopersicum plants (line 2). Subsequently, mite survival and fecundity (number of eggs/mite) was assessed. Transgenic plants were made as described above. In short, plants were co-transformed with two constructs to produce 7-epizingiberene in glandular trichomes of S. lycopersicum (pMKS1:zFPS and pMTS1:ShZIS). In this experiment one transgenic line was used (line 2).

(69) Results Bio-assay Spider Mites

(70) Mite fecundity was reduced by the production of 7-epi-zingiberene in transgenic S. lycopersicum plants. Compared to S. lycopersicum, transgenic plants that produced 7-epi-zingiberene showed reduced mite survival (both species). Moreover, FIGS. 11A and 11C indicate a strong reduction of mite fecundity (eggs/mite) for both T. urticae and T. evansi, 81% and 54% reduction, respectively.

(71) Overall survival was also impacted for both spider mite species. FIGS. 11B and 11D indicate that the percentage of dead spider mites was higher on transgenic plants producing 7-epizingiberene compared to non-7-epizingiberene-producing S. lycopersicum plants (S. lyc 32).

Example 5

7-epi-zingiberene Production in Various Plants

(72) Arabidopsis thaliana, Nicotiana tabacum, Cucumis melo, Lactuca sativa, Glycine max, and Gossypium hirsutum are co-transformed with ShzFPS (additional zFPP precursor) and ShZIS (encoding 7-epizingiberene synthase). 7-epi-zingiberene production in the leaves of co-transformed plants is compared to 7-epi-zingiberene production in mock-transformed plants of the same species. Arabidopsis thaliana, Nicotiana tabacum, Cucumis melo, Lactuca sativa, Glycine max, and Gossypium hirsutum are capable of producing 7-epi-zingiberene.