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 a wound-induced promoter inducing local expression, preferably the wound-inducible TR2' promoter, to the chimeric genes used in this method and the plants obtained thereby, and to the processes for obtaining resistance to insects feeding on plants by localized expression of an insecticidal protein induced on wounding of plants by insect feeding.
• a wound-induced promoter inducing local expression preferably the wound-inducible TR2' promoter
• 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 crylAbi 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 Cryl Ab 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, 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).
• Agrobacterium mannopine synthase promoters have been characterized as being constitutive, root-specific, and tissue-specific promoters, e.g., see US patents 6291745, 6320100 and 63133378. In US patent 5641664, it is believed that various constitutive and organ- and tissue-specific promoters that are used to direct expression of genes in transformed dicotyledonous p lants w ill a lso b e s Menble for u se i n t ransformed m onocots.
• TR1' and the TR2' promoters which drive the expression of the 1' and 2' genes, respectively, of the T-DNA of Agrobacterium, and are said to be wound-induced promoters.
• This patent shows no monocot plant transformed with the TR2' or TR1 ' promoter, nor i s t here a ny s uggestion t hat t hese p romoters a re w ound-induced p romoters i n monocot plants or are useful for expression of an insecticidal protein in a monocot plant.
• 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 b urden o n functions i mportant for m aintaining t he agronomic qualities of the engineered crop.
• 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 CrylAb, CrylF, Cry2Ae, Cry9C or Cry2Ab protein or an insecticidal fragment or mutant thereof.
• a preferred embodiment of the present invention relates to a method for obtaining insect resistance, preferably high dose insect resistance, in plants, plant cells or plant tissues, more particularly in monocotyledonous, especially gramineae, particularly corn, plants, plant cells or plant tissues, by providing the plants, plant cells or tissue 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 is low (i.e., below 0.005% total soluble protein (mean value of multiple measurements taken from several, preferably at least 3, particularly at least 5, plants of the same transformation event) in leaves, in the absence of wounding or infestation and is increased, preferably at least doubled, most preferably increased 5 to 100 fold, in the wounded or infested tissues, particularly leaves, 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 com, 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 is low or not detectable (less than 0.005% of total soluble protein (mean value of multiple measurements taken from several, preferably at least 3, particularly at least 5, plants of the same event)) in leaves of non-wounded, non-infected plants, and, upon infection, increased levels of insecticidal protein (at least double, preferably at least from 5 to 100 fold increase or more) are induced locally in the infected tissues, within 18 hours.
• plants more particularly monocotyledonous p lants a re p rovided t hat a re i nsect r esistant d ue t o t he p resence i n 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.
• 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.
• 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.
• 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 to ECB larvae, including particularly to ECB larvae of the fourth stage as can be determined by insect efficacy assays described herein.
• mortality rates of ECB fourth instar larvae of at least 97 %, preferably at least 99 %, most preferably of 100%, are obtained.
• 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 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).
• 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, preferably in leaves and pollen, particularly in leaves, 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 r egion, 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.
• the 3' UTR comprising a polyadenylation site need not be present in the transferred gene itself, but can be found in the upstream plant DNA sequences after insertion of a gene not containing a 3' UTR comprising a polyadenylation site.
• a coding sequence of this invention can be inserted in the plant genome downstream of an existing plant promoter so that expression of the insecticidal protein of the invention occurs from such reconstituted chimeric gene in the plant (e.g., as in promoter tagging experiments).
• chimeric w hen referring to a gene or DNA s equence 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 genome of a plant, plant tissue or plant cell refers to any genetic material in the plant, plant tissue or plant cell, and includes both the nuclear and the plastid and mitochondrial genome.
• 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 sequence) referred to or a synthetic version thereof (such as a sequence w hich 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” or “mutant'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%, preferably is higher than 85%, more preferably is higher than 90%, especially preferably is higher than 95%, most preferably is between 96 and 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 nubilalis (European corn borer or ECB), Sesamia nonagrioides (Mediterranean Stalk borer) and Helicoverpa z ea (corn e arworm), a nd m ajor c oleopteran p ests, s uch as D iabrotica s pp. insects, particularly Diabrotica virgifera virgifera and Diabrotica undecimpunctata howardi (Corn rootworm).
• major lepidopteran pests such as Ostrinia nubilalis (European corn borer or ECB), Sesamia nonagrioides (Mediterranean Stalk borer) and Helicoverpa z ea (corn e arworm), a nd m ajor c oleopteran p
• An 'insecticidal protein' or 'toxin' as used herein should be understood as a protein, polypeptide or peptide which is toxic to insects.
• insecticidal protein examples include the Bt Cry toxins, mutants or insecticidal fragments thereof (such as those reviewed by H ⁇ fte and Whiteley, 1989, in Crickmore et al.
• insecticidal proteins are for instance the V IPs, p articularly V IP3A, more p articularly VIP3Aa, VIP3Ab and VIP3Ac proteins or insecticidal fragments thereof (Estruch et al., 1996, WO 96/10083, US patents 6, 429,360, 5,877,012), or the proteins encoded by the mis, war and sup sequences (WO98/18932, WO99/57282), and 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 which have insecticidal activity.
• a Bt toxin, or Bt protein 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 which is essentially identical to the toxic fragment of a naturally produced Bt toxin, or at least a domain thereof.
• a B t toxin or B t protein, as used herein can be a crystal protein or an insecticidal part thereof, or can be a non- crystal protein such as a secreted protein or a protein produced mainly during the vegetative phase of a Bt strain, or can be an insecticidal mutant or part thereof.
• 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 cryl Ab DNA sequence which encodes at least part of the CrylAb5 protein described by H ⁇ fte et al. (1986), preferably a DNA sequence encoding a protein comprising the amino acid sequence from an amino acid position between amino acid positions 1-28 to an amino acid between amino a cid p ositions 607-725 t hereof, m ost p referably comprising amino acids 1- 616.
• the encoded modified CrylAb protein has an insertion of an alanine codon (GCT) behind the ATG start codon (AlaAsp2...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 (Bradford, 1976)). A preferred ELISA in the current invention is a sandwich ELISA (Clark et al., 1986).
• 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 intended to mean either mechanical damage or perforation of at least the plant epidermis or outer cell layer by any kind of insect feeding, wounding as used herein can also be simulated by cutting a leaf with a sharp instrument such as a scalpel.
• wound-inducible expression of an insecticidal protein, preferably a Bt 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 (mean value of measurements taken from several plants of one transformation event), and, upon wounding rises, preferably to a level above 0,01 % of total soluble protein, more preferably to a level of 0.04% to 0.5% total soluble protein content or higher as measured using a leaf part that was detached after wounding and then incubated in vitro for several hours, e.g., about 12 to about 60 hours, preferably about 18 to about 20 hours.
• basal expression i.e., in the absence of wounding, preferably as measured in the greenhouse
• total soluble protein content mean value of measurements taken from several plants of one transformation event
• Measurements made using an excised in vitro leaf assay in such assay an excised leaf or part thereof is incubated in vitro (e.g., in a petri dish on humid filter paper) in a greenhouse or growth chamber at room temperature for several hours, preferably about 12 to 60 hours, more preferably about 18 to 20 hours, after wounding (to better reflect the conditions upon insect feeding) typically will give a higher induction of expression than measurements after wounding in planta without in vitro incubation of a detached leaf or a part thereof.
• Wound-induced protein levels are typically lower (but still significantly higher than non-induced background levels) when measured around 12 to 60 hours, more preferably around 18 to 20 hours, after wounding of a plant leaf without excising the leaf part for in vitro incubation.
• expression of the insecticidal protein at least in the wounded leaves rises to 0.1% total soluble protein content at the site of wounding. It is believed that the percentage of insecticidal protein per total soluble protein in accordance with the invention that is actually ingested by an insect upon insect feeding is higher than the percentage measured in the plant or part thereof, due to the localized expression and the fact that only induced (wounded) tissue will be ingested by the insect.
• a certain minimum amount of plant tissue is excised (typically about 2-3 mm around the wound is excised for leaves), to run the insecticidal protein content analysis, hence the presence of unwounded cells is believe to decrease the percentage of insecticidal protein per total soluble protein extracted from the sample.
• a selection procedure is preferred to select the best plants for further development (i.e., for crossing into suitable plant lines adapted to a certain growing region).
• plants are selected showing low or undetectable protein levels in plant tissue, preferably in leaves and p ollen, most preferably in leaves, which plants upon wound-induction show at least a 5-fold increase in expression in leaves.
• a “greenhouse”, as used herein, refers to a relatively stable growing environment for plants which shields plants from normal field conditions, with no or little infestation by insects, rabbits, birds or other animals or external factors (such as wind or storms) which can damage a crop plant in the field.
• a typical greenhouse is largely made out of transparent materials such as glass o r p lastic, so as to allow natural day-light to reach the plants, and can have regulated light, growth medium (soil or an artificial medium), water and nutrient supply and/or temperature control.
• a greenhouse, as used herein, also includes rooms or boxes with no daylight, as long as a light source and a growth medium is provided so that normal plant growing conditions are established.
• a greenhouse is the most suitable place to measure basal or background expression levels in leaves for the wound-inducible constructs of this invention (in a non-induced state), for comparative purposes. While similar basal or background expression values of insecticidal protein can be found in leaves of plants in a field, it is expected that infestation by insects or mechanical damage, e.g., by vehicles, rabbits or birds to the plants in a field will increase the expression level by induction of the wound-inducible promoter of this invention, and hence not a real basal level will typically be measured in all plants in a field.
• 'High dose' expression, or 'high dose' insect resistance refers to a concentration of the insecticidal protein in a plant (measured by ELISA as a percentage of the total soluble protein, which total soluble protein is measured after extraction of soluble proteins in an extraction buffer (e.g., the extraction buffer described in Jansens et al., 1997) using Bradford analysis (Bio-Rad, Richmond, CA; Bradford, 1976)) 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, most preferably a high d ose i nsect r esistance i s t he obtaining of at least 97 percent, preferably at least 99 percent, most preferably 100 percent, mortality for the fourth larval instar (
• a target insect species i.e., an insect species which can cause commercially significant damage to a plant species or variety, and which is typically an insect for which a transgenic plant is developed
• a transformed plant according to this i nvention p rovides a high dose insect resistance is sufficient for a plant to be designated as giving "high dose” expression in accordance with this invention.
• plant cells or plant tissues particularly corn plant cells or plant tissues, whether contained in a plant or present in an in vitro culture, transformed with the wound-induced chimeric gene of the invention, that have a high dose insect resistance as described above using insect bio-assays.
• High dose when referring to ECB control in co as used herein refers to the production of insecticidal protein by the plant in mid-whorl stage in an amount which is toxic to ECB larvae of the L4 stage (European Com 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 90%, preferably at least 97, more preferably at least 99 %, most preferably 100% of the L4 ECB larvae is obtained in a test 14 days, preferably 10 days, after infestation of the plants with L4 larvae.
• a high dose expression of an insecticidal protein is obtained in the com plants of the invention using the TR2' promoter to drive wound-induced expression, even when expression is low or undetectable by sensitive ELISA protocols in an non-induced state, and expression is only induced upon insect feeding.
• suitable plant lines with low basal or background expression and high dose wound-induced expression will need to be selected from the transformation events obtained, and preferably such a plant 1 ine c ontains o nly one i nserted DNA encoding an insecticidal protein.
• commercially acceptable plant lines are obtained from over one hundred, preferably from several hundreds, of initial transformed plants.
• 'wound-inducible' expression is furthermore preferably characterized in that the effect of the promoter is local, i.e., is confined essentially to those cells or 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 defense mechanisms of the plant.
• 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 (mean value of multiple measurements taken from several, preferably at least 3, particularly at least 5, plants from one transformation event), as measured by ELISA (see above) in greenhouse-grown plants.
• wound- inducible p romoter a ctivity i n 1 eaves, p articularly co 1 eaves, i s 1 ocalized t o t he r egion of wounding, and will not be detected (using ELISA assays) in a region of the plant, particularly the leaf, more than 10cm, particularly more than 2 cm, distant of the wounding site (e.g., not in a leaf lower or higher than the wounded leaf in the same plant, nor in the same leaf more then 10 cm, preferably more than 2 cm, away from the wounded site on the leaf).
• the wound-induced expression of an insecticidal protein in accordance with this invention is characterized by a quick induction of expression, rising from basal or background level to a higher protein level (at least twice the amount in the controls, preferably at least 5 to 100 times the amount in the controls) at around 18 to 24 hours, preferably at 18, 20 or 24 hours, after wounding using an assay wherein a leaf part is wounded, cut out of the leaf and incubated in vitro for that period of time (the basal or background level is measured in freshly excised and not previously wounded leaf parts, which are not incubated in vitro).
• TR2' promoter as used herein relates to any promoter comprising the TR2' (or mannopine synthase, abbreviated as mas) functional part of the TR1 '-TR2' dual promoter element from Agrobacterium (Velten et al. 1984; Langridge et al. 1989).
• TR2' element either alone or in combination with the divergent TRl ' element (Guevara-Garcia et al., 1998) or other (regulatory) elements, including but not limited to enhancer regions, introns and the like, as long as the wound-induction promoter characteristics in accordance with the present invention are substantially retained.
• transcription is directed from the TR2' promoter region (and the coding sequence is hence operably linked to and downstream of the TR2' promoter sequence), even if the TR1'-TR2' dual promoter (or any part thereof retaining the TR2' promoter e lement) is used.
• the TR2' promoter refers to a promoter region comprising a fragment of SEQ ID NO:l spanning from a nucleotide position between nucleotide positions 1 and 336 to nucleotide position 483, preferably comprising the sequence of nucleotides 96 to 483 of SEQ ID NO:l, most preferably comprising SEQ ID NO: 1 or a functional equivalent thereof, i.e., 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:l 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:l and includes hybrid promoters comprising the functional TR2' part of SEQ ID NO:l. Such promoter sequences 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.
• controlling is meant a toxic (lethal) or combative (sub-lethal) amount.
• a high dose is produced.
• the p lants should b e mo ⁇ ho logically 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 targeted by the insecticidal protein).
• 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 (see, e.g., Jansens et al., 1997 for stalk tunnel length analysis).
• 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 c an 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 (4X the normal field rate), does not kill the plants.
• g.a.i./ha grams active ingredient/hectare
• a broadcast application should be applied at a rate of 28-34 oz LibertyTM + 31b 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 are the genes encoding resistance to phenmedipham (such as the pmph gene, US 5,347,047; US 5,543,306), the genes encoding resistance to glyphosate (such as the EPSPS genes, US 5,510,471), genes encoding bromoxynil resistance (such as described in US 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).
• PPT such as described in EP-A-0 242 236, EP-A-0 242 246,
• 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, so as to obtain different traits in the plant (also referred to as 'stacking').
• the plant of the invention containing a chimeric gene 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 traits 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 crylAb coding sequence
• Example 1 Generation of Events with a foreign gene under control of the TR2' promoter.
• a construct was made comprising a promoter region comprising the TR2' promoter (Velten et al. 1984) directing the expression of a modified crylAb protein.
• the plasmid pTSVH0212 containing the genes of interest placed between the T-DNA borders (also referred to as 'TR2'-CrylAb') was used for Agrobacterium-mediated transformation (WO 98/37212).
• the structure of the PTSVH0212 c onstruct i s p rovided i n T able 1 .
• transformations were performed with constructs comprising a DNA sequence encoding the modified CrylAb protein under control of either the 35S promoter from Cauliflower Mosaic Virus (Franck et al. 1980)(referred to as 35S-crylAb), or the promoter of the GOS2 gene from rice (de Pater et al., 1992) with the cab22 leader from Petunia (Harpster et al.
• Regenerated plantlets were selected based on tolerance to Liberty.
• the Agrobacterium transformants were c homeed 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 (TO). Example 2. Wound-induced expression of an insecticidal protein.
• the basal level of expression of the modified CrylAb insecticidal protein was determined by a CrylAb sandwich ELISA with a polycondensated IgG fraction of a polyclonal rabbit antiserum against CrylAb as first antibody and a monoclonal antibody against CrylAb as second antibody, after extraction of soluble protein using the extraction method and buffer described in Jansens et al., 1997. Samples of leaves at V3 stage plants, pollen and leaf of Rl stage plants and leaves, stalk and pollen at harvest w ere taken o f p lants in the greenhouse (com stages are as determined in 'How a Com Plant Develops, Special Report No.
• Results are provided in Table 2 as percentage of detected CrylAb protein per total soluble protein (measured using the Bradford assay (Bio-Rad, Richmond, CA; Bradford, 1976)). The mean value represents the average of 5 samples taken from different plant lines of one transformation event.
• CrylAb protein levels were measured by ELISA (Table 3) on excised leaf parts (leaf parts were cut out 2 to 3 mm around the wound) that were incubated for 18 hours in a growth chamber at 20° C in a petri dish on a filter paper moistened with Murashige and Skoog medium (MS medium, see Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK, for composition) - control leaf pieces of similar size were cut from plants and were put immediately on dry ice for protein measurement (without in vitro incubation). Means represent averages of 5 plants per transformation event .
• Table 3 expression of insecticidal protein in different plant parts before and after mechanical wounding
• WI600-0218 Mean 0.000 0.020 0.000 0.020 0.023 0.036 0 000 0.000 0.000 0.000 0.000
• WI606-0406 Mean 0.005 0.046 0.000 0.007 0.012 0.008 0.000 ⁇ 0.002 0.002
• WI604-1602 Mean 0.002 0.104 0.001 0.020 0.020 0.024 0 000 0.004 0.004 0.001
• WI606-0802 Mean 0.003 0.064 0.001 0.010 0.027 0.037 0.000 0.002 0.004 0.003
• WI606-1206 Mean 0 001 0.081 0.000 0.022 0.032 0.024 0.000 0.004 0.002 0.001
• CE048-2402 Mean 0.83 0.614 0.280 0.397 0.196 0.253 0.000 0.376 1.120 0.047
• CE048-2602 Mean 0.636 0.527 0.007 0.006 0.087 0.045 0 000 0.106 0.040 0.015
• Control 1 Mean 0.000 0.000 0 0 0 0 0 0 0 0.001 0
• Control 2 Mean 0.000 0.000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
• CE0104-0202 Mean 0.022 0.023 0.008 0.017 0.023 0.016 0.0001 ⁇ ⁇ ⁇
• CE1014-0402 Mean 0 025 0 017 0.007 0.010 0.028 0.034 0.000 0.010 0.040 0.002
• Example 3 Insect resistance of plants with wound-inducible expression.
• the performance in the field of the plants of the invention, using the TR2' promoter is compared to commercially-available com plants expressing a CrylAb protein portion.
• com plants obtained from events MON810 and Btl 1 (see published USDA petitions of these approved com events for a detailed description), which use a 35S promoter for high constitutive expression levels, and which are known to provide high dose insect resistance, are used.
• the mean tunnel length (in cm per stalk, measured after stalk splitting as described in Jansens et al. (1997)) for artificial infestation with European com borer is 0, 0, 0.04, 0, and 0,21 cm, respectively, for 5 different CrylAb-TR2' com events, while the mean tunnel length for MON810 com is 0.3 and for Btl 1 com 0.13 (in non-transformed control com lines, tunnel lengths from 12.2 to 39.92 cm are found in the same trial).
• comparative analysis of the wound-induced TR2' promoter in com shows that expression of an insecticidal protein under control of the TR2' promoter in com provides high level insect resistance comparable to that obtainable with commercially-available events with constitutive promoters.
• the mean tunnel length (in cm per stalk, measured after stalk splitting as described in Jansens et al. (1997)) for artificial infestation with Soiled com borer is 0.53, 0.68, 0.3, 0.64, and 0,6 cm, respectively, for the 5 different Cryl Ab-TR2' com events, while the mean length for MON810 com is 0.43 and for Btl 1 com 0.15 (in non-transformed control com lines, tunnel lengths from 32 to 39.6 cm are found in the same trial, some had no plants standing anymore by stalk breakage due to the tunnels).

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