Wound-inducible expression in plants

FIELD OF THE INVENTION

[0001] 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.

BACKGROUND ART

[0002] Most successful attempts to make plants that are resistant to insect attack have been obtained by, and are believed to be dependent on, high level expression of insecticidal toxins (Estruch et al., 1997; Witowsky & Siegfried, 1997). Most particularly for the Bt toxins, which as full-length proteins were found to be expressed poorly in plants, efforts have concentrated on increasing expression of these toxins in plants by use of strong constitutive promoters and by modification of the genes encoding them (Vaeck et al., 1987; Barton et al., 1987). More recently spatial regulation of expression of the insecticidal protein in those tissues susceptible to attack or triggered by feeding of the insect has been suggested as possibly providing advantages for resistance management (Peferoen & Van Rie, 1997). One of the major conditions for obtaining regulatory approval for transgenic insect resistant plants is the availability of an insect resistance management strategy. 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). In order to be able to comply with such a strategy, the use of strong constitutive promoters in the engineering of insect-resistant plants has further been encouraged.

[0003] Inducible expression of insect resistance has primarily been examined for the potato proteinase-inhibitor genes (pin1 and pin2), which are part of the natural defense system in plants and upon wounding, ensure the generation of a systemic signal throughout the plants (Green and Ryan, 1972; Hilder et al. 1987). Introduction of the pin2 gene in rice resulted in high-level accumulation of the protein in rice plants, which showed increased resistance to major pests (Duan et al., 1996). Breitler et al. (2001) describes the use of the C1 region of the maize proteinase inhibitor (MPI) gene to drive wound-inducible expression of the cry1B coding sequence in rice and the first transformants were found to effectively protect rice against stemborer attack. In a small-scale laboratory experiment, 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).

[0004] The 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). In general, the use of monocot promoters is preferred for optimal expression of genes in monocots (Shimamoto, 1994) and certain promoters have been found to have different cis-acting elements in monocots and dicots (Luan et al. 1992). The contribution of different elements of the TR2′ promoter on its activity was investigated in maize protoplast (Fox et al., 1992), but there have been no reports on the expression pattern of the TR2′ promoter in a monocotyledonous plant. Furthermore, the strongest deletion mutant was found to be 20 times less active than the CaMV 35S promoter (Fox et al., 1992).

[0005] 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 (such as reported in WO 00/26378 for Cry2Ab) 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. As high-dose expression levels are attained upon contact with the target pest, such plants should comply with current IRM strategies. Additionally, the combination of the specificity of the insect toxin produced with a spatially and temporally limited expression pattern (i.e. in tissues susceptible to wounding, upon wounding) is likely to reduce exposure of non-target organisms, which can be considered advantageous over constitutive production of the toxin by the plants. Thus, an effective system of regulated expression ensuring effective insect resistance for major crops such as corn and rice is of interest from both a regulatory and agricultural point of view.

SUMMARY OF THE INVENTION

[0006] 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. According to a particular embodiment of the invention, the insecticidal protein is a Bacillus thuringiensis toxin. In a preferred embodiment of the present invention, 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.

[0007] Thus, 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. According to the present invention the TR2′ promoter is used to increase expression of the insecticidal protein upon wounding, e.g. by insect feeding. Thus, according to a particular embodiment of the invention, 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.

[0008] According to a preferred embodiment of the present invention, 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. Thus, the invention relates to chimeric genes for obtaining wound-inducible expression in monocot plants. In a particular embodiment of the invention, expression (as measured in the greenhouse) is low or not detectable (<0.005% protein) in leaves of non-wounded, non-infected plants, and, upon infection, increased levels of insecticidal protein (>0.02% protein) are induced locally in the infected tissues, within 18 hours. According to this aspect of the invention, plants, more particularly monocotyledonous plants are provided that are insect resistant due to the presence in their genome of a foreign DNA comprising a DNA sequence encoding an insecticidal protein, under the control of the TR2′ promoter which ensures expression in wounded tissues. According to one embodiment of the invention, 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.

[0009] According to a preferred embodiment of the invention 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. Examples of such DNA sequences encoding Bt toxins are well-known in the art and are described herein.

[0010] According to one embodiment of the present invention use of the TR2′ promoter in corn to direct wound-inducible expression of a Bt toxin is particularly suited to engineer resistance of corn against the European Corn Borer (ECB), based on the local high-dose expression of the toxin in the plant upon feeding by target insects. The plants or plant parts (cells or tissues) of the present invention, comprising in their genome a foreign DNA comprising a DNA sequence encoding an insecticidal protein under the control of the TR2′ promoter upon wounding produce levels of insecticidal protein that are toxic ECB, more particularly to ECB larvae of the fourth stage as can be determined by insect efficacy assays described herein. Preferably, mortality rates of ECB fourth instar larvae of 90 to 96%, more preferably 97-100%, most preferably 99-100% are obtained.

[0011] 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. According to a particular aspect of this invention, 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.

[0012] According to another aspect of this invention, monocotyledonous plants, particularly corn plants are obtained that are insect resistant and particularly suited for stacking with other traits (e.g. other types of insect resistance, herbicide resistance or agronomic traits).

[0013] According to another aspect of the invention, 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.

[0014] According to the present invention, 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.

DETAILED DESCRIPTION

[0015] 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. Typically in plant cells, 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.

[0016] The term “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. The term “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.

[0017] A genome of a plant, plant tissue or plant cell, as used herein, refers to any genetic material in the plant, plant tissue or plant cell, and includes both the nuclear and the plastid and mitochondrial genome.

[0018] 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.

[0019] 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.

[0020] ‘Insecticidal’ is used herein to mean toxic to insects that are crop pests. More particularly, in the context of the present invention 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).

[0021] An ‘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. Other 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). Other 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.

[0022] In a particular embodiment of the present invention, 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. According to a particular embodiment of the present invention 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. Most preferably, the encoded modified Cry1Ab protein has an insertion of an alanine codon (GCT) behind the ATG start codon (A1aAsp2 . . . Asp616).

[0023] 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).

[0024] 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. Preferably, according to the present invention, wound-inducible expression of an insecticidal protein in a plant means that basal expression (i.e. in the absence of wounding, preferably as measured in the greenhouse) of 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. According to a particular embodiment of the invention, expression of the insecticidal protein at least in the wounded leaves rises to 0.1% total soluble protein content.

[0025] ‘High dose’ expression as used herein 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.

[0026] According to the present invention ‘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. This as opposed to a systemic effect, which directly or indirectly (through a cascade of reactions) ensures a widespread effect, more particularly wide-spread expression of proteins involved in the natural defence mechanisms of the plant. Preferably, 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).

[0027] The ‘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. Most particularly, the TR2′ promoter as used herein 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. Alternatively such 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.

[0028] The plants of the present invention are protected against insect pests, by the wound-inducible expression of a controlling amount of insecticidal protein. By controlling is meant a toxic (lethal) or combative (sublethal) amount. Preferably, upon induction, a high dose (as hereinbefore defined) is produced. At the same time, the plants should be morphologically normal and may be cultivated in a usual manner for consumption and/or production of products. Furthermore, said plants should substantially obviate the need for chemical or biological insecticides (to insects targetted by the insecticidal protein).

[0029] 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). In the field, 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.

[0030] 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 Liberty™. Tolerance to Liberty™ can be tested in different ways. For instance, tolerance can be tested by Liberty™ 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 Liberty™+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. Examples of other herbicide resistance genes are 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. 4,810,648) 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), and genes encoding resistance to cyanamide (such as described in WO 98/48023 and WO 98/56238) and 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).

[0031] According to a preferred embodiment of the invention, 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’). Similarly, 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.

[0032] Introduction of a foreign DNA or a chimeric gene into the genome of a plant, cell or tissue can be achieved in different ways and is not critical to the present invention. Successful genetic transformation of monocots has been obtained by a number of methods including Agrobacterium infection (as described, for example for corn in U.S. Pat. No. 6,074,877 or U.S. Pat. No. 6,140,553), microprojectile bombardment (as described, for example by Chen et al., 1994), direct DNA uptake into protoplasts (as described, for example by Data et al. 1999; Poulsen, 1996) and electroporation (D'Halluin et al., 1992,).

[0033] The following non-limiting examples describe the construction of chimeric genes comprising a DNA sequence encoding an insecticidal protein under control of the TR2′ promoter, for expression in plants and insect resistant plants obtained therewith. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). 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. Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR—Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

[0034] Throughout the description and Examples, reference is made to the following sequences represented in the sequence listing:

1SEQ ID NO: 1:nucleotide sequence of a preferred embodiment of theTR2′ promoterSEQ ID NO: 2:sequence of pTSVH0212SEQ ID NO: 3:sequence of a modified cry1Ab coding sequence

EXAMPLES

Example 1

[0035] Generation of Events With a Foreign Gene Under Control of the TR2′ Promoter

[0036] a) Development of Events

[0037] For the evaluation of wound-induced expression of a DNA sequence encoding an insecticidal gene, 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).

2PTSVH02123′nos-bar-p35S3><Tr2-modcry1Ab-(SEQ ID NO: 2)3′ocs

[0038] The structure of the PTSVH0212 construct is provided in Table 1. For control plants with constitutive expression of the insecticidal protein, 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. 1988)(also referred to as ‘Gos/cab-cry1Ab’) or the 5′ leader sequence of the GOS2 gene from rice, containing the second exon, the first intron and the first exon of the GOS transcript (de Pater et al., 1992)(referred to as ‘Gos/gos-cry1Ab’). All constructs included the 35S-bar gene.

3TABLE 1Nucleotide positions of the genetic elements in pTSVH0212 (TR2′-cry1Ab)Nt positionsDirectionDescription and references25-1 CounterLeft Border sequence of TL-DNA of pTiB6S3 (Gielen et al.,clockwise1984).Synthetic polylinker sequence316-56 CounterFragment containing polyadenylation signals from theclockwise3′untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982).Synthetic polylinker sequence887-336Counterbar: the coding sequence of phosphinothricin acetyl transferase ofclockwiseStreptomyces hygroscopicus (Thompson et al., 1987)1720-888 CounterP35S3: promoter region from the Cauliflower Mosaic Virus 35Sclockwisetranscript (Odell et al., 1985).Synthetic polylinker sequence2252-1170CounterTR2′ Promoter fragment derived from the right T-DNA ofclockwiseoctopine type Agrobacterium strain ACH5 (Velten et al., 1984)Synthetic polylinker sequence2261-4114Modified cry1Ab: The modified cry1Ab coding sequence encodespart of the Cry1Ab5 protein described by Höfte et al. (1986) andhas an insertion of an alanine codon (GCT) 3′ of the ATG startcodon (SEQ ID NO: 3).Synthetic polylinker sequence4128-4422Fragment containing polyadenylation signals from the3′ untranslated region of the octopine synthase gene from the TL-DNA of pTiAch5 (De Greve et al., 1982).Synthetic polylinker sequence4470-4446CounterRight Border sequence of TL-DNA of pTiB6S3 (Gielen et al.,clockwise1984).

[0039] Regenerated plantlets were selected based on tolerance to Liberty.

[0040] b) Evaluation of Events

[0041] 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).

Example 2

[0042] Wound-Induced Expression of an Insecticidal Protein

[0043] i) Basal Expression of the Insecticidal Protein

[0044] 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. As a comparison, 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.

4TABLE 2Expression of Cry1Ab protein in different tissues of plants transformed with the TR2′-cry1Ab and Gos/gos-cry1Ab constructsCry1Ab in % soluble protein / total proteinEventV3 stageR1 stageHarvest(construct)LeafLeafPollenLeafStalkPollenWI602-0402mean00.0000.0000.0100.000(TR2′-cry1Ab)st. dev.000.0000.0000.0100.010WI604-1602Mean000.0000.0000.0100.000(TR2′-cry1Ab)st. dev.000.0000.0000.0100.000WI606-0802Mean000.0000.0000.0200.000(TR2′-cry1Ab)St. dev.000.0000.0000.0400.000WI606-1206Mean000.0000.0000.0100.000(TR2′-cry1Ab)St. dev.000.0000.0000.0100.000WI600-0218Mean000.000(TR2′-cry1Ab)St. dev.000.000WI602-0802Mean000.0000.0000.0100.010(TR2′-cry1Ab)St. dev.000.0000.0000.0100.010CE2168-0202Mean0.310.260.0400.2300.8000.030(Gos/gos-cry1Ab)st. dev.0.040.060.0100.0600.3600.010CE2168-1602Mean0.340.230.0500.2700.8700.030(Gos/gos-cry1Ab)st. dev.0.040.050.0200.1501.4500.010

[0045] For the plants obtained from transformation with the TR2′-cry1Ab construct, average values for the basal expression of the modified Cry1Ab protein was found to be below detection limit (0.005% soluble protein) in all leaf samples taken from plants at V3 stage, R1 stage or at harvest. Average basal expression of the insecticidal protein in the stalk was found to be around 0.01% total soluble protein content for most plants at harvest. Plants obtained with the Gos/gos-cry1Ab construct, expression of the Cry1Ab protein was above 0.2% in all leaf samples and up to more than 1% in some of the stalk samples taken at harvest.

[0046] ii) Wound-Induced Expression of the Insecticidal Protein

[0047] Studies were performed in the greenhouse to determine the expression of the insecticidal Cry1Ab protein upon mechanical damage in untransformed plants, plants obtained with the TR2′-cry1Ab construct and plants obtained with the Gos/cab-cry1Ab construct. Leaves and roots were damaged by cutting. Leaf samples were taken before and 18h after mechanical damage and Cry1Ab protein levels were measured by ELISA (Table 2). Means represent averages of 5 samples from one plant.

5TABLE 2expression of insecticidal protein in different plant parts before and after mechanicalwoundingCry1Ab in % soluble protein / total proteinV4 stageFloweringEventLeafLeafRootHarvest(construct)LeafinducedLeafinducedrootinducedPollenLeafStalkKernelWI600-0218Mean0.0000.0200.0000.0200.0230.0360.0000.0000.0000.000(TR2′-Cry1Ab)st. dev.0.0000.0080.0000.0070.0080.0110.0000.0000.0000.000WI606-0406Mean0.0050.0460.0000.0070.0120.0080.000\0.0020.002(TR2′-Cry1Ab)st. dev.0.0070.0050.0000.0060.0090.0090.0000.004WI604-1602Mean0.0020.1040.0010.0200.0200.0240.0000.0040.0040.001(TR2′-Cry1Ab)st. dev.0.0030.0120.0000.0090.0080.0080.0000.0020.0030.000WI606-0802Mean0.0030.0640.0010.0100.0270.0370.0000.0020.0040.003(TR2′-Cry1Ab)st. dev.0.0040.0000.0090.0120.0200.0000.0010.0030.001WI606-1206Mean0.0010.0810.0000.0220.0320.0240.0000.0040.0020.001(TR2′-Cry1Ab)st. dev.0.0160.0000.0100.0050.0110.0000.0030.0010.001CE048-2402Mean0.830.6140.2800.3970.1960.2530.0000.3761.1200.047(35S-Cry1Ab)st. dev.0.1080.1110.0270.1330.0830.0000.0980.1750.014CE048-2602Mean0.6360.5270.0070.0060.0870.0450.0000.1060.0400.015(35S-Cry1Ab)st. dev.0.160.0460.0020.0020.0440.0070.0000.0110.0130.004Control 1Mean0.0000.0000000000.0010(untransformed)st. dev.0.0000.0000000000.0020Control 2Mean0.0000.00000000000(untransformed)st. dev.0.0000.00000000000CE0104-0202Mean0.0220.0230.0080.0170.0230.0160.0001\\\(Gos/cab-Cry1Ab)st. dev.0.0090.0050.0050.0040.0120.0050.000CE1014-0402Mean0.0250.0170.0070.0100.0280.0340.0000.0100.0400.002(Gos/cab-Cry1Ab)0.0050.0030.0010.0030.0170.0130.0000.0030.0200.001

[0048] Expression of the Cry1Ab protein was again either absent or around the detection limit in leaves, stalk and kernels of the different TR2′-cry1Ab events tested. No significant expression was found in leaves and pollen in V4 and flowering plant stage. Constitutive expression of around 0.02-0.03% total soluble protein was seen in the roots for these plants. When leaves are mechanically damaged, expression of the Cry1Ab protein is induced and goes up to 0.05-0.1%. The 35S-cry1Ab events showed constitutive expression of the Cry1Ab protein of around 0.5% in the leaves of V3 plants, irrespective of wounding. During flowering and harvest, expression levels of around 0.1-0.2% were measured before and after wounding.

Example 3

[0049] Insect Resistance of Plants With Wound-Inducible Expression

[0050] a) Efficacy Against Controlled Infestation With ECB Fourth Instar Larvae

[0051] Mid whorl corn plants were placed in a Plexiglas cylinder and infested with 10 or 15 fourth instar European corn borer larvae. After 10 days, plants were dissected and percentage survival of larvae was scored per plant. The results are provided in Table 3.

6TABLE 3Efficacy against fourth instar ECB larvaeNumber of plantsAveraged percentageEventtestedsurvival (s.d.)WI602-040260WI602-080260WI604-160260WI606-080260WI606-120660B73 control949.6 (18.8)

[0052] The efficacy of the control of fourth instar ECB larvae in controlled infestation was found to be 100% for all TR2′-cry1Ab plants tested. Control plants showed around 50% mortality rates. As the fourth instar ECB larvae are believed to be between 25 and 100 times less susceptible to the modified Cry1Ab protein than first instar larvae, the level of protein produced in the TR2′-cry1Ab plants can be considered ‘high dose’.

[0053] b) Efficacy Against ECB

[0054] Fourteen events were evaluated for ECB efficacy in the greenhouse and in field trials (Table 4). Results are presented as average length of tunnels (with standard deviation value between brackets) over maximum length of tunnel for each plant (average (sd)/max length/pl). In the greenhouse, average length of tunnels were taken of measurements on 10 plants. In the field, ECB efficacy is expressed as the average of 3 values obtained for different groups of 10 plants. Four of the five single-copy events (indicated by an asterisk) gave total ECB2 control, determined as less than 3.5 cm average tunnel length.

7TABLE 4ECB Efficacy in greenhouse and field trialsECB efficacyECB efficacyECB efficacyGreenhouseField 1Field 2EventAverage (sd)/max length/plAverage (sd)/max length/plAverage (sd)/max length/plWI602-0402* 0.1 (0.31)/10.24 (0.41)/20.21 (0.01)/2WI604-1602* 0.2 (0.42)/10.05 (0.071)/1WI606-0406* 51.3 (73.5)/1958.41 (1.8)/327.45 (0.07)/30WI606-0802* 3.3 (2.6)/8 0 (0)/00.05 (0.07)/1WI606-1206* 0.38 (0.74)/20.17 (0.29)/3 0.1 (0.14)/1WI600-0218 2.0 (1.87)/6WI600-1402 29.4 (45.8)/1426.55 (2.47)/28WI602-0202168.5 (26.9)/20012.6 (3.8)/31WI604-0604102.5 (83)/251 3.7 (2.0)/25WI602-0802 0 (0)/00.33 (0.15)/3WI602-0204 66 (68.2)/160WI602-1002102.4 (63.4)/215WI606-0602 45.5 (52.8)/160WI600-0802 0.9 (1.44)/40.06 (0.11)/2 0 (0)/0

[0055] No penalty on agronomic performance was observed for the plants after second selfing (ear to row) of the different TR2′ events in any of the locations tested.

[0056] c) Efficacy Against Sesamia nonagroides

[0057] Five mid whorl corn plants comprising the TR2′-cry1Ab construct were each infested with 2 egg masses. Damage was scored after 14 days and numbers of larvae were counted. Damage ratings (expressed in plant height and length of tunnels per plant in cm) were averaged over the five plants.

8EventNumber of larvae per plantPlant height in cmCm tunnels per plantWI600-08020.6 (1.3)198 (8.4)0B73 Control197.2 (14.0) 84 (5.5)70 (9.4)

[0058] The results indicate that there is also good control of the Mediterranean stalk borer in the TR2′-cry1Ab plants tested.

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[0101]

Wound-inducible expression in plants

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