Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the invention relates to a method for producing plants in which expression of an insecticidal protein is regulated by the wound-inducible TR2′ promoter, to the chimeric genes used in this method and the plants obtained thereby.
• the currently favored strategy is the combination of 100% toxicity of the transgenic plants to the target pest(s), obtained by high dose expression of a specific toxin and this during the full life-cycle of the pest, with the use of refuges of non-transgenic plants, which allow the maintenance of the target pest population (De Maagd et al. 1999).
• the use of strong constitutive promoters in the engineering of insect-resistant plants has further been encouraged.
• transgenic cabbage leaves transformed with the cry1Ab3 gene placed under the control of the inducible vspB promoter from soybean were as toxic to diamondback moth as those transformed with the same gene under control of the 35S promoter, but wound-inducibility was not demonstrated (Jin et al., 2000).
• TR2′ promoter of the mannopine synthase gene of Agrobacterium tumefaciens originally considered to direct constitutive expression (Velten et al. 1984; Vaeck et al. 1987), has been used to direct wound-inducible expression of a native Cry1Ab gene in tomato, which led to relatively low expression and only moderate insect control (Reynaerts & Jansens, 1994). Though a possible broad application for expression of Bt proteins has been suggested (Peferoen et al. 1997), there appear to be discrepancies between the reports on the expression pattern of the TR2′ promoter in tobacco and other dicots (Ni et al. 1995).
• the present invention describes how the TR2′ promoter can be used to direct wound-induced expression of an insecticidal protein in monocot plants to obtain insect resistance.
• Such wound-inducible expression of the TR2′ promoter leads to a strong but localized increase of expression of the insecticidal protein.
• the putative effect on plant vigor and growth, observed with high level expression of some Bt proteins particularly upon repeated inbreeding is likely to be reduced as the limited expression of the protein should minimize any burden on functions important for maintaining the agronomic qualities of the engineered crop. This is also an important factor when stacking of different traits (or different Bt proteins) is envisaged.
• the present invention relates to a method for obtaining wound-induced expression of an insecticidal protein in a monocot plant, which method comprises introducing into the genome of the plant a foreign DNA which is a chimeric gene comprising a DNA sequence encoding the insecticidal protein under control of a promoter region comprising the TR2′ promoter.
• the insecticidal protein is a Bacillus thuringiensis toxin.
• the insecticidal protein is an insecticidal protein active against pests of monocot plants; most preferably, the insecticidal protein can be a Cry1Ab, Cry1F or Cry2Ab protein.
• a preferred embodiment of the present invention relates to a method for obtaining insect resistance in plants, more particularly in monocotyledonous plants, especially in graminae, most particularly in corn, by providing the plants with a foreign DNA comprising a DNA sequence encoding an insecticidal protein under control of a promoter region comprising the TR2′ promoter.
• the TR2′ promoter is used to increase expression of the insecticidal protein upon wounding, e.g. by insect feeding.
• expression of the insecticidal protein in monocotyledonous plants preferably measured in the greenhouse, is low (i.e. below 0.005% total soluble protein) in leaves and most other plant tissues, in the absence of wounding or infestation and is increased, preferably at least doubled, most preferably increased 5-100 fold, in the wounded or infested tissues, within 24 hours.
• a chimeric gene comprising a DNA sequence encoding an insecticidal protein under control of the wound-inducible TR2′ promoter is used to confer insect resistance to monocotyledonous plants, especially to gramineae, most particularly to corn, by directing local expression of the insecticidal protein at the site of insect feeding.
• the invention relates to chimeric genes for obtaining wound-inducible expression in monocot plants.
• expression of an insecticidal protein in the plants is such that, in the absence of wounding of the plant (e.g., when grown in the greenhouse) the insecticidal protein is expressed at low or undetectable levels (i.e. below 0.005% protein) in leaves, stalk, seed and pollen and, upon feeding by insects, is increased in the wounded tissue to a level which is sufficient to kill the feeding pest, preferably to a concentration of at least 0.01% soluble protein.
• the TR2′ promoter is used in monocotyledonous plants to confer insect resistance by directing wound-inducible expression of an insecticidal protein which is a Bt toxin.
• an insecticidal protein which is a Bt toxin. Examples of such DNA sequences encoding Bt toxins are well-known in the art and are described herein.
• the present invention further relates to monocotyledonous plants that are resistant to insects while expressing very low basal levels of insecticidal protein in non-wounded leaves of the plant.
• monocotyledonous plants, more particularly corn are obtained which combine efficient insect resistance with optimal agronomic characteristics, without penalty on agronomic performances due to expression of the insecticidal protein, as can be ascertained by assessing plant phenotype, segregation, emergence, vigor and agronomic ratings.
• monocotyledonous plants particularly corn plants are provided, which are resistant to (a) target pest(s), but for which production of the insecticidal protein is low to undetectable in the absence of wounding, limiting exposure of non-target organisms to the insecticidal protein.
• the plants with the characteristics described above are obtained by introduction into the genome of the plant of a DNA sequence encoding an insecticidal protein under control of the TR2′ promoter which is demonstrated to function as wound-inducible promoter in monocotyledonous plants, more particularly in corn plants.
• the term “gene” as used herein refers to any DNA sequence comprising several operably linked DNA fragments such as a promoter region, a 5′ untranslated region (the 5′UTR), a coding region (which may or may not code for a protein), and an untranslated 3′ region (3′UTR) comprising a polyadenylation site.
• the 5′UTR, the coding region and the 3′UTR are transcribed into an RNA of which, in the case of a protein encoding gene, the coding region is translated into a protein.
• a gene may include additional DNA fragments such as, for example, introns.
• chimeric when referring to a gene or DNA sequence refers to a gene or DNA sequence which comprises at least two functionally relevant DNA fragments (such as promoter, 5′UTR, coding region, 3′UTR, intron) that are not naturally associated with each other and/or originate, for example, from different sources.
• “Foreign” referring to a gene or DNA sequence with respect to a plant species is used to indicate that the gene or DNA sequence is not naturally found in that plant species, or is not naturally found in that genetic locus in that plant species.
• foreign DNA will be used herein to refer to a DNA sequence as it has incorporated into the genome of a plant as a result of transformation in that plant or in a plant from which it is a progeny.
• a “fragment” or “truncation” of a DNA molecule or protein sequence as used herein refers to a portion of the original DNA or protein sequence (nucleic acid or amino acid) referred to or a synthetic version thereof (such as a sequence which is adapted for optimal expression in plants), which can vary in length but of which the minimum size is sufficient to ensure the (encoded) protein to be biologically active, the maximum size not being critical.
• a “variant” of a sequence is used herein to indicate a DNA molecule or protein of which the sequence (nucleic or amino acid) is essentially identical to the sequence to which the term refers.
• Sequences which are “essentially identical” means that when two sequences are aligned, the percent sequence identity, i.e. the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, is higher than 70%-80%, preferably 81-85%, more preferably 86-90%, especially preferably 91-95%, most preferably 96-100%.
• the alignment of two nucleotide sequences is performed by the algorithm as described by Wilbur and Lipmann (1983) using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4.
• target insects are the pests of monocotyledonous plants, most particularly of corn, such as, but not limited to major lepidopteran pests, such as Ostrinia nubialis (European corn borer or ECB), Sesamia nonagroides (Mediterranean Stalk borer) and Helicoverpa zea (corn earworm), and major coleopteran pests, such as Diabrotica virgifera (Corn rootworm).
• major lepidopteran pests such as Ostrinia nubialis (European corn borer or ECB), Sesamia nonagroides (Mediterranean Stalk borer) and Helicoverpa zea (corn earworm)
• major coleopteran pests such as Diabrotica virgifera (Corn rootworm).
• insecticidal protein or ‘toxin’ as used herein should be understood as a protein which is toxic to insects.
• examples of such an insecticidal protein are the Bt Cry toxins (such as those reviewed by Höfte et al., 1989 and described in WO 00/26378, WO 97/40162, and U.S. Pat. No. 6,023,013), more particularly the Cry2Ab, Cry1F and Cry1Ab proteins.
• insecticidal proteins are for instance the VIPs (Estruch et al., 1996, WO 96/10083), or the proteins encoded by the mis, war and sup sequences (WO98/18932, WO99/57282), the toxins isolated from Xhenorabdus and Photorabdus ssp. such as those produced by Photorabdus luminescens (Forst et al., 1997).
• insecticidal proteins include, but are not limited to, the potato proteinase inhibitor I and II, the cowpea proteinase inhibitor, the cystein proteinase inhibitor of soybean (Zhao et al., 1996) or the cystatins such as those isolated from rice and corn (Irie et al., 1996), cholesterol oxidases, chitinases, and lectins.
• An insecticidal protein can be a protoxin (i.e. the primary translation product of a full-length gene encoding an insecticidal protein). Also included are equivalents and variants, derivatives, truncations or hybrids of any of the above proteins that have insecticidal activity.
• a Bt toxin as used herein refers to an insecticidal protein as previously defined which is directly or indirectly derived (e.g., modified so as to improve expression in plants or toxicity to insects) from a protein naturally produced by Bacillus thuringiensis and comprises a sequence that is essentially identical to the toxic fragment of a naturally produced Bt toxin.
• a DNA encoding an insecticidal protein as used herein includes a truncated, modified, synthetic or naturally occurring DNA sequence, encoding an insecticidal protein.
• the DNA encoding an insecticidal protein is a DNA sequence encoding a Bt toxin, more preferably a DNA sequence modified to increase expression of the insecticidal protein in plants.
• the DNA sequence encoding an insecticidal protein is a modified cry1Ab DNA sequence which encodes at least part of the Cry1Ab5 protein described by Höfte et al. (1986), preferably a DNA sequence encoding a protein comprising the amino acids 1-28 to 607-725 thereof, most preferably comprising amino acids 1-616.
• the encoded modified Cry1Ab protein has an insertion of an alanine codon (GCT) behind the ATG start codon (A1aAsp2 . . . Asp616).
• the expression level of an insecticidal protein in plant material can be determined in a number of ways described in the art, such as by quantification of the mRNA encoding the insecticidal protein produced in the tissue using specific primers (such as described by Cornelissen & Vandewiele, 1989) or direct specific detection of the amount of insecticidal protein produced, e.g., by immunological detection methods. More particularly, according to the present invention the expression level of insecticidal protein is expressed as the percentage of soluble insecticidal protein as determined by immunospecific ELISA as described herein related to the total amount of soluble protein (as determined, e.g., by Bradford analysis).
• a ‘wound-inducible’ promoter or a promoter directing an expression pattern which is wound-inducible as used herein means that, at least in the leaves, upon wounding expression of the coding sequence under control of the promoter is significantly increased, i.e. is at least doubled, preferably 5 times increased, most preferably 20 to 100 times increased.
• wounding as used herein is intend to mean either mechanical damage or perforation of at least the plant epidermis or outer cell layer by any kind of insect feeding.
• wound-inducible expression of an insecticidal protein in a plant means that basal expression (i.e.
• the protein in the leaves of the plant at V4 stage is low, most preferably below 0.005% total soluble protein content (average value of multiple measurements taken from one plant), and, upon wounding rises to a level of 0.04% to 0.5% total soluble protein content or higher.
• expression of the insecticidal protein at least in the wounded leaves rises to 0.1% total soluble protein content.
• ‘High dose’ expression refers to a concentration of the insecticidal protein (as measured in percentage of total protein) which kills a developmental stage of the target insect which is significantly less susceptible, preferably between 25 to 100 times less susceptible to the toxin than the first larval stage of the insect and can thus can be expected to ensure full control of the target insect.
• High dose when referring to ECB control as used herein refers to the production of insecticidal protein by the plant in mid-whorl stage in an amount that is toxic to ECB larvae of the L4 stage (European Corn Borer. Ecology and Management. 1996. North Central Regional Extension Publication No. 327. Iowa State University, Ames, Iowa) as can be determined by toxicity assays with artificial infestation described herein, wherein mortality of at least to 90%, more preferably 97-100%, most preferably 99-100% of the L4 ECB larvae is obtained.
• ‘wound-inducible’ expression is furthermore preferably characterized in that the effect of the promoter is local, i.e. is confined essentially to those tissues directly affected by wounding or immediately surrounding the wounded tissue.
• the effect of the promoter is local, i.e. is confined essentially to those tissues directly affected by wounding or immediately surrounding the wounded tissue.
• expression of the insecticidal protein in undamaged tissues of the plant is on average not more than 0.01% of the total soluble protein concentration, more preferably not more than 0.005% total soluble protein, as measured by ELISA (see above).
• TR2′ promoter as used herein relates to any promoter comprising the TR2′ (or mas) functional part of the TR1-TR2 dual promoter element from Agrobacterium (Velten et al. 1984; Langridge et al. 1989). Thus this can comprise the TR2′ element either alone or in combination with the divergent TR1 element (Guevara-Garcia et al., 1998) or other (regulatory) elements.
• the TR2′ promoter refers to a promoter region comprising a fragment of SEQ ID NO: 1 spanning from nucleotide 1-336 to nucleotide 483, preferably comprising the sequence of nucleotides 96 to 483 of SEQ ID NO: 1, most preferably comprising SEQ ID NO: 1 or a functional equivalent thereof, ie a modification thereof capable of directing wound-induced expression in plants, more particularly in monocotyledonous plants.
• Such functional equivalents include sequences which are essentially identical to a nucleotide sequence comprising at least nucleotides 328 to 483 (comprising the TR2′ promoter element, Velten et al., 1984) of SEQ ID NO: 1.
• Such sequences can be isolated from different Agrobacterium strains.
• functional equivalents correspond to sequences which can be amplified using oligonucleotide primers comprising at least about 25, preferably at least about 50 or up to 100 consecutive nucleotides of nucleotides 328 to 483 of SEQ ID NO: 1 in a polymerase chain reaction.
• Functional equivalents of the TR2′ promoter can also be obtained by substitution, addition or deletion of nucleotides of the sequence of SEQ ID NO: 1 and includes hybrid promoters comprising the functional TR2′ part of SEQ ID NO: 1.
• Such promoter sequence can be partly or completely synthesized.
• the plants of the present invention are protected against insect pests, by the wound-inducible expression of a controlling amount of insecticidal protein.
• a controlling amount of insecticidal protein By controlling is meant a toxic (lethal) or combative (sublethal) amount.
• a high dose (as hereinbefore defined) is produced.
• the plants should be morphologically normal and may be cultivated in a usual manner for consumption and/or production of products.
• said plants should substantially obviate the need for chemical or biological insecticides (to insects targetted by the insecticidal protein).
• Different assays can be used to measure the effect of the insecticidal protein expression in the plant. More particularly, the toxicity of the insecticidal protein produced in a corn plant to Ostrinia nubilalis or ECB (also referred to herein as ECB efficacy) can be assayed in vitro by testing of protein extracted from the plant in feeding bioassays with ECB larvae or by scoring mortality of larvae distributed on leaf material of transformed plants in a petri dish (both assays described by Jansens et al., 1997).
• first brood ECB larvae (ECB1) infestation is evaluated based on leaf damage ratings (Guthrie, 1989) while evaluation of the total number of stalk tunnels per plant and stalk tunnel length are indicative of second brood ECB (ECB2) stalk feeding damage.
• the plants of the present invention optionally also comprise in their genome a gene encoding herbicide resistance. More particularly, the herbicide resistance gene is the bar or the pat gene, which confers glufosinate tolerance to the plant, i.e. the plants are tolerant to the herbicide LibertyTM. Tolerance to LibertyTM can be tested in different ways. For instance, tolerance can be tested by LibertyTM spray application. Spray treatments should be made between the plant stages V2 and V6 for best results.
• Tolerant plants are characterized by the fact that spraying of the plants with at least 200 grams active ingredient/hectare (g.a.i./ha), preferably 400 g.a.i./ha, and possibly up to 1600 g.a.i./ha (4 ⁇ the normal field rate), does not kill the plants.
• a broadcast application should be applied at a rate of 28-34 oz LibertyTM+3# Ammonium Sulfate per acre. It is best to apply at a volume of 20 gallons of water per acre using a flat fan type nozzle while being careful not to direct spray applications directly into the whorl of the plants to avoid surfactant bum on the leaves.
• the herbicide effect should appear within 48 hours and be clearly visible within 5-7 days.
• herbicide resistance genes examples include the genes encoding resistance to phenmedipham (such as the pmph gene, U.S. Pat. No. 5,347,047; U.S. Pat. No. 5,543,306), the genes encoding resistance to glyphosate (such as the EPSPS genes, U.S. Pat. No. 5,510,471), genes encoding bromoxynil resistance (such as described in U.S. Pat. No.
• genes encoding resistance to sulfonylurea such as described in EPA 0 360 750
• genes encoding resistance to the herbicide dalapon such as described in WO 99/27116
• genes encoding resistance to cyanamide such as described in WO 98/48023 and WO 98/56238
• genes encoding resistance to glutamine synthetase inhibitors such as PPT (such as described in EP-A-0 242 236, EP-A-0 242 246, EP-A-0 257 542).
• the chimeric gene comprising a DNA encoding an insecticidal protein under control of the TR2′ promoter can be introduced (simultaneously or sequentially) in combination with other chimeric genes into a plant, to obtain different traits in the plant (also referred to as ‘stacking’).
• the plant of the invention comprising a foreign DNA comprising a DNA encoding an insecticidal protein under control of the TR2′ promoter is particularly suited for combination with other traits.
• Such other traits include, but are not limited to traits such as those encoded by chimeric genes which confer insect resistance, herbicide resistance, stress or drought tolerance, or which modify other agronomic characteristics of the plant. Such a trait can also encompass the synthesis of a product to be recovered from the plant.
• SEQ ID NO: 1 nucleotide sequence of a preferred embodiment of the TR2′ promoter
• SEQ ID NO: 2 sequence of pTSVH0212
• SEQ ID NO: 3 sequence of a modified cry1Ab coding sequence
• a construct was made comprising a promoter region comprising the TR2′ promoter (Velten et al. 1984) directing the expression of a modified cry1Ab protein.
• the plasmid pTSVH0212 containing the genes of interest placed between the T-DNA borders (also referred to as ‘TR2′-Cry1Ab’) was used for Agrobacterium-mediated transformation (WO 98/37212).
• the structure of the PTSVH0212 construct is provided in Table 1.
• transformations were performed with constructs comprising a DNA sequence encoding the modified Cry1Ab protein under control of either the 35S promoter from Cauliflower Mosaic Virus (Franck et al. 1980)(referred to as 35S-cry1Ab), or the promoter of the GOS2 gene from rice (de Pater et al., 1992) with the cab22 leader from Petunia (Harpster et al.
• Synthetic polylinker sequence 316-56 Counter Fragment containing polyadenylation signals from the clockwise 3′untranslated region of the nopaline synthase gene from the T- DNA of pTiT37 (Depicker et al., 1982).
• Synthetic polylinker sequence 887-336 Counter bar: the coding sequence of phosphinothricin acetyl transferase of clockwise Streptomyces hygroscopicus (Thompson et al., 1987) 1720-888
• Counter P35S3 promoter region from the Cauliflower Mosaic Virus 35S clockwise transcript (Odell et al., 1985).
• Synthetic polylinker sequence 4470-4446 Counter Right Border sequence of TL-DNA of pTiB6S3 (Gielen et al., clockwise 1984).
• the Agrobacterium transformants were checked for presence of vector sequence at the left border of the T-DNA. Southern blot analyses were performed with leaf material of the primary transformants (T0).
• the basal level of expression of the modified Cry1Ab insecticidal protein was determined by a Cry1Ab sandwich ELISA with a polycondensated IgG fraction of a polyclonal rabbit antiserum against Cry1Ab as first antibody and a monoclonal antibody against Cry1Ab as second antibody.
• Samples of leaves at V3 stage plants, pollen and leaf of R1 stage plants and leaves, stalk and pollen at harvest were taken of plants in the greenhouse.
• samples were taken from plants transformed with the Gos/gos-cry1Ab constructs. Results are provided in Table 2. The mean value represents the average of 5 different samples taken from one plant.

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