EP0989998A1 - Verbessertes bacillus thuringiensis-toxins - Google Patents

Verbessertes bacillus thuringiensis-toxins

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Publication number
EP0989998A1
EP0989998A1 EP98939581A EP98939581A EP0989998A1 EP 0989998 A1 EP0989998 A1 EP 0989998A1 EP 98939581 A EP98939581 A EP 98939581A EP 98939581 A EP98939581 A EP 98939581A EP 0989998 A1 EP0989998 A1 EP 0989998A1
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EP
European Patent Office
Prior art keywords
amino acid
protein
cry9c
leu
thr
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EP98939581A
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English (en)
French (fr)
Inventor
Sandra De Roeck
Jeroen Van Rie
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Bayer CropScience AG
Bayer CropScience SA
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Aventis CropScience GmbH
Aventis CropScience SA
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Publication of EP0989998A1 publication Critical patent/EP0989998A1/de
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • 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 present invention provides new improved proteins derived from a Bacillus thuringiensis Cry9C crystal protein.
  • amino acid positions in a Cry9C protein were identified as involved in insect toxicity.
  • modified Cry9C proteins with increased of decreased toxicity to an insect species and DNA sequences encoding such modified Cry9C proteins. Plants can be protected from insect damage by expressing a chimeric gene encoding an improved Cry9C protein with an increased toxicity to an insect species.
  • Bacillus thuringiensis (Bt)-derived proteins are currently widely used to protect plants from insects by expression of such proteins in transgenic plants. Concerns of insect resistance development and the desire to achieve the optimum toxicity and control of additional insect species resulted in efforts to modify existing Bt-derived proteins so as to increase their toxicity or alter their mode of action. Most studies on the mode of action of Bacillus thuringiensis toxins have focused on lepidopteran-specific Cry1 insecticidal crystal proteins ("ICPs"). The following picture has emerged from these studies (Gill et al., 1992, Annu. Rev. Entomol. 37, 615-36; Knowles, 1993, BioEssays, 15, 469-476).
  • ICPs lepidopteran-specific Cry1 insecticidal crystal proteins
  • the crystals Following ingestion of the crystals by a susceptible insect, they are dissolved in the alkaline reducing environment of the insect midgut lumen. The liberated proteins, the protoxins, are then proteolytically processed by insect midgut proteases to a protease-resistant fragment. This active fragment, the toxin, then passes through the peritrophic membrane and binds to specific receptors located on the brush border membrane of gut epithelial cells. Subsequent to binding, the toxin or part thereof inserts in the membrane resulting in the formation of pores. These pores lead to colloid osmotic swelling and ultimately lysis of the midgut cells, causing death of the insect.
  • the Cry proteins have been found to have three structural domains: the N-terminal domain I consists of 7 alpha helices, domain II contains three beta-sheets and the C-terminal domain III is a beta- sandwich. Based on this structure, a hypothesis has been formulated regarding the structure-function relationships of ICPs. The bundle of long, hydrophobic and amphipathic helices (domain I) is equipped for pore formation in the insect membrane, and regions of the three-sheet domain (domain II) are probably responsible for receptor binding (Li et al, 1991 , supra). The function of domain III is less clear. When different ICP amino acid sequences are aligned, five conserved sequence blocks are evident (H ⁇ fte & Whiteley, 1989, Microbiol. Revs.
  • cry9Ca1 (abbreviated as Cry9C) (Peferoen et al., 1997, in Advances in Insect Control: The role of transgenic plants, pp. 21-48, Taylor & Francis Ltd., London). This protein was found to have a broad insect target range within the group of lepidopteran pest insects making it interesting for insect control applications in agriculture. De Roeck et al. (1995, the 28th annual meeting of the Society for Invertebrate Pathology, Cornell University, Ithaca, New York, p.
  • Cry9C in the new nomenclature (Crickmore et al., 1995, 28 th annual meeting of the Society for Invertebrate Pathology, Georgia University, Ithaca, New York, p.14.). De Roeck et al. (1997, the 6th International Conference on Perspectives in Protein Engineering, John Innes Centre, Norwich, UK, June 28-July 1 , p. 34) determined the likely position of residues in the loops at the apex of the molecule in domain II of the Cry9C protein.
  • This invention provides a modified Cry9C protein with an improved toxicity to an insect species, comprising the amino acid sequence of SEQ ID No. 2 or an insecticidally-effective fragment thereof, wherein at least one amino acid in the following regions in SEQ ID No. 2 is replaced by another amino acid: 313-334, 358- 369, 418-425, 480-492.
  • This invention further provides improved Cry9C proteins comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658, wherein at least one of the amino acids at the following positions in SEQ ID No. 2 have been replaced by another amino acid: 313, 316, 317, 318, 319, 321 , 323, 325, 329, 330, 368, 369, 418, 420, 421 , 422, 480, 481 , 483, 484, 485, 487, 488, 490 and 491.
  • Preferred improved Cry9C proteins comprise the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658, wherein at least one of the amino acids at the following positions are replaced by other amino acids: 316, 317, 319, 321 , 329, 330, 369, 422, and 488.
  • This invention also provides a modified Cry9C protein with improved toxicity to Ostrinia nubilalis, comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658, wherein at least the amino acids at position 488 or at least at positions 364 and 488 are replaced by other amino acids, preferably by alanine.
  • modified Cry9C proteins with improved toxicity to Heliothis virescens comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658, wherein the amino acid at position 321 or position 329, is replaced by another amino acid, preferably by alanine.
  • This invention further provides modified Cry9C proteins with improved toxicity to Diatraea grandiosella, comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658, wherein the amino acid at any or all of positions 316, 317, 319, 321 , 330, 369, or 422 is replaced by another amino acid, preferably by alanine.
  • DNA sequences encoding the modified Cry9C proteins, and particularly chimeric genes designed for expression in plants comprising these DNA sequences.
  • a plant transformed with a DNA sequence encoding a modified Cry9C protein is provided, so that the plant acquires increased resistance to insects, particularly a corn plant transformed with a modified Cry9C protein yielding increased toxicity towards Heliothis virescens, Ostrinia nubilalis, or Diatraea grandiosella insects.
  • amino acid residues important for toxicity of the Cry9C protein have been identified. These amino acid residues can be replaced by other amino acids to increase the toxicity to a specific insect species.
  • Cry9C protein refers to an insecticidal protein characterized by the amino acid sequence of SEQ ID No. 2 or any equivalents thereof such as the insecticidally effective truncated proteins or the fusion proteins of the Cry9C protein described in PCT patent publications WO 94/05771 and WO 94/24264.
  • Particularly preferred Cry9C proteins are proteins containing at least the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658.
  • Cry9C protein variants for a particular insect species, are insecticidal proteins that differ from but are indirectly or directly derived from the Cry9C protein. Indeed, several variants of a Bt protein in which some amino acids are changed into others without significantly changing activity and/or specificity to a particular insect species can be found in nature (H ⁇ fte & Whiteley, 1989, supra) or can be made by recombinant DNA techniques.
  • Variants of a Cry9C protein also include proteins containing the specificity- or toxicity-determining domain or region of the Cry9C protein, e.g., in a hybrid with another protein, such as another Bt ICP, a membrane-permeating protein domain, a cytotoxin or an antibody fragment, provided that the Cry9C specificity- or toxicity-determining domain or region contributes to the toxicity or specificity of the hybrid protein.
  • Particularly preferred Cry9C protein variants are those proteins comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658 wherein the arginine at position 164 has been replaced by another amino acid, preferably alanine or lysine.
  • protease-resistant variants Like here for the protease-resistant variants, whenever reference to a particular region or position in SEQ ID No. 2 is made, this does not necessarily imply that the protein referred to is the full-length protein of SEQ ID No. 2; this statement merely refers to the position corresponding to the particular position in the reference Cry9C protein in SEQ ID No. 2. Indeed, improved Cry9C proteins of the invention can be truncated so that the actual position of an amino acid in that protein will differ but nevertheless reference will be made throughout this invention to the positions in the full-length reference protein, shown in SEQ ID No. 2.
  • Modified Cry9C protein refers to a Cry9C protein or its protease-resistant variant wherein amino acids have been modified to analyse the contribution of amino acid positions in toxicity, particularly a Cry9C protein or its protease-resistant variant wherein amino acids have been modified in the regions at the following positions in SEQ ID No. 2: 313-334, 358-369, 418-425, 480-492.
  • Improved Cry9C protein refers to a Cry9C protein or its protease-resistant variant wherein at least one amino acid has been replaced, so that the toxicity of this improved protein towards an insect species is significantly increased.
  • the at least one amino acid change is located in domain II of the Cry9C protein, particularly in the regions of the Cry9C protein characterized by the following positions in SEQ ID No. 2: 313-334, 358-369, 418-425, 480-492.
  • a modified Cry9C protein differing in one amino acid from the native protein or its protease-resistant variant and being significantly less toxic towards the target insect, allows the direct identification of this amino acid position as involved in toxicity (provided no gross structural changes are introduced), and thus has considerable value in improving toxicity.
  • the identification of these amino acid positions involved in toxicity allows the construction of modified proteins having increased toxicity to the target insect by amino acid randomization at these positions.
  • Preferred modified Cry9C proteins in accordance with this invention are the modified Cry9C proteins having altered toxicity to Ostrinia nubilalis, Heliothis virescens or Diatraea grandiosella as shown in Table 1 , as well as combinations of those modifications in one modified protein.
  • An example of an improved Cry9C protein in accordance with this invention is a protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658 or 666 wherein an amino acid in at least one of the following amino acid positions of SEQ ID No.
  • a preferred improved Cry9C protein in accordance with this invention is the protein of SEQ ID No.
  • V319A or "Cry9C(V319A)" means a change of the valine amino acid at position 319 in SEQ ID No. 2 to an alanine amino acid.
  • Preferred improved Cry9C proteins also include Cry9C proteins having also the arginine amino acid at position 164 in SEQ ID No. 2 altered into another amino acid, particularly alanine or lysine, to enhance stability upon protease, particularly trypsin, cleavage.
  • a preferred Cry9C protein for the control of Ostrinia nubilalis insects in accordance with this invention is a protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658 wherein an amino acid in at least one of the following amino acid positions in SEQ ID No. 2 has been replaced by another amino acid: 325, 364, 418, 421 , 485, and 488.
  • a particularly preferred improved Cry9C protein for the control of Ostrinia nubilalis insects is a protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658 wherein the amino acids in at least position 364 or at least in positions 364 and 488 of SEQ ID No.
  • a preferred Cry9C protein for the control of Heliothis virescens insects in accordance with this invention is a protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658 wherein an amino acid in at least one of the following amino acid positions in SEQ ID No.
  • a particularly preferred improved Cry9C protein for the control of Heliothis virescens insects is a protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658 wherein the amino acids in at least one of the amino acid positions 321 and 329 of SEQ ID No.
  • a preferred Cry9C protein for the control of Diatraea grandiosella insects in accordance with this invention is a protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658 wherein an amino acid in at least one of the following amino acid positions in SEQ ID No. 2 has been replaced by another amino acid: 316, 317, 319, 321 , 325, 330, 369, 421 , 422, 480, 483, 484, 485, 487, 488, 490, and 491 ; particularly at least one of the following amino acid positions: 480, 484, 485, 487, and 490.
  • a particularly preferred improved Cry9C protein for the control of Diatraea grandiosella insects is a protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 1 or 44 to amino acid position 658 wherein the amino acids in at least one of the amino acid positions 316, 317, 319, 321 , 330, 369 and 422 of SEQ ID No. 2 are replaced by another amino acid, particularly alanine or valine (for 317).
  • a “cry9C gene”, as used herein, is a DNA sequence comprising a DNA encoding a Cry9C protein (a coding region), and includes necessary regulatory sequences so that a Cry9C protein can be expressed in a cell, preferably a plant or bacterial cell.
  • a cry9C gene does not necessarily need to be expressed everywhere at all times, expression can be periodic (e.g. at certain stages of development in a plant) and/or can be spatially restricted (e.g. in certain cells or tissues in a plant), mainly depending on the activity of regulatory elements provided in the chimeric gene or in the site of insertion in the plant genome.
  • a cry9C gene can be naturally- occurring or can be a hybrid or synthetic DNA and the regulatory elements can be from prokaryotic or eucaryotic origin.
  • modified cry9C gene is a DNA sequence comprising a DNA encoding a modified Cry9C protein (a modified coding region), and includes necessary regulatory sequences so that a Cry9C protein can be expressed in a cell, preferably a plant or bacterial cell.
  • An example of a modified cry9C coding region is the cry9C coding region of SEQ ID No. 3 wherein the valine codon at nucleotide positions 844-846 of SEQ ID No. 3 has been replaced by an alanine codon.
  • Substantial sequence homology refers to DNA sequences differing in some, most or all of their codons from another DNA sequence but encoding the same or substantially the same protein. Indeed, because of the degeneracy of the genetic code, the codon usage of a particular DNA coding region can be substantially modified, e.g., so as to more closely resemble the codon usage of the genes in the host cell, without changing the encoded protein. Changing the codon usage of a DNA coding region to that of the host cell has been described to be desired for gene expression in foreign hosts (e.g. Bennetzen & Hall, 1982, J. Biol. Chem.
  • a modified cry9C gene has all appropriate control regions so that the modified Cry9C protein can be expressed in a host cell, e.g. for expression in plants, a plant-expressible promoter and a 3' termination and polyadenylation region active in plants.
  • a "chimeric improved cry9C gene”, as used herein, refers to a chimeric gene comprising a DNA sequence encoding the improved Cry9C protein inserted in between controlling elements of different origin, e.g. a DNA sequence encoding the improved Cry9C protein under the control of a promoter transcribing the DNA in the plant cell, and fused to 3' transcription termination sequences active in plant cells.
  • Protection of a plant, preferably a corn or cotton plant, against an insect species which is known to feed on said plant is preferably accomplished by expressing an improved Cry9C protein in the cells of the plant.
  • This is preferably accomplished by expressing a chimeric improved cry9C gene encoding such an improved Cry9C protein in the cells of a plant, preferably a corn or cotton plant.
  • An improved Cry9C protein of this invention preferably only has a small number, particularly less than 20, more particularly less than 15, preferably less than 10 amino acids replaced by other amino acids as compared to the Cry9C protein, preferably as compared to the region from between amino acid positions 1 and 45 to amino acid position 658 of the Cry9C protein of SEQ ID No. 2.
  • a significant increase in toxicity can already be obtained by replacing only 1 amino acid, but it is preferred that more than one amino acid is changed to improve toxicity.
  • the following steps are followed to construct the new modified Cry9C proteins: amino acids in domain II of the Cry9C protein from amino acid positions 313-334, 358-369, 418-425, and 480-492 were chosen for modification, using alanine- scanning mutagenesis (Cunningham & Wells, 1989, Science 244, 1081-85). In case the original position is alanine, a substitution by valine is done.
  • mutant ICPs can be analysed by a variety of methods including toxicity to another target insect, crystal formation, solubilization, monoclonal antibody binding analysis, protease resistance, fluorometric monitoring of unfolding and circular dichroism spectrum analysis.
  • structural distortion it is impossible to determine the functional role of this position by alanine replacement.
  • a more conservative amino acid substitution may yield a correctly folded mutant protein which allows to determine the functional role of this position.
  • mutants which yield modified proteins with significantly decreased toxicity
  • This method is further referred to as "amino acid randomization”.
  • Such mutants may be generated by a variety of methods, e.g. following the PCR overlap extension method (Ho et al., 1989, Gene 77, 51-59). These mutant proteins are then tested in toxicity assays on the target insect. Mutants at each position which are more toxic, e.g., yield higher mortality than the wild type protein, are selected. Such mutants with improved toxicity are termed "up-mutants”.
  • protruding regions of domain II are meant the solvent-exposed regions organized in loops, alpha helices or beta-strands which are protruding from domain II and are located at or towards the apex of the molecule.
  • This invention is particularly suited for improving the toxicity to an insect species for which the Cry9C protein has a rather weak toxicity.
  • the toxicity of this improved Cry9C protein can be increased by combining amino acid mutations in the protein, each yielding an increased toxicity when compared to the amino acid present in the native Cry9C protein.
  • Insect species for which improved Cry9C proteins can be made also include Spodoptera frugiperda, Heliothis zea, Heliothis armigera, and Agrotis ipsilon.
  • this invention is suited to increase toxicity of a Cry9C protein or its protease-resistant variant to one insect species and to decrease toxicity of the same protein to another insect species by making the proper amino acid substitutions in the protein. This may be advantageous, e.g., to limit the likelihood of insect resistance occurrence to the protein in a particular insect species.
  • An insecticidally effective part of the modified cry9C gene of this invention encoding an insecticidally effective portion of the modified Cry9C protein can be made in a conventional manner.
  • An "insecticidally effective part" of the modified cry9C gene refers to a gene comprising a DNA coding region encoding a polypeptide with fewer amino acids than the full length modified Cry9C protein but that still retains toxicity to insects.
  • a preferred insecticidally effective part of the Cry9C protein is the part from amino acid position 1 or 44 to amino acid position 658 in SEQ ID No. 2.
  • suitable restriction sites can be introduced, flanking each gene or gene part. This can be done by site-directed mutagenesis, using well-known procedures (Stanssens et al., 1989, Nucl. Acids Res. 12, 4441-4454; white et al., 1989, Trends in Genet. 5, 185-189).
  • Stanssens et al., 1989, Nucl. Acids Res. 12, 4441-4454; white et al., 1989, Trends in Genet. 5, 185-189 In order to improve expression in foreign host cells such as plant cells, it may be preferred to alter the improved cry9C coding region or its insecticidally effective part to form an equivalent, artificial improved cry9C coding region. Expression is improved by selectively inactivating certain cryptic regulatory or processing elements present in the native sequence as described in PCT publications WO 91/16432 and WO 93/09218.
  • the chimeric improved cry9C gene can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so-transformed plant cell can be used in a conventional manner to produce a transformed plant that is insect-resistant.
  • Particularly preferred plants in accordance with this invention are corn plants.
  • Corn cells can be stably transformed (e.g. by electroporation) using wounded or enzyme-degraded intact tissues capable of forming compact embryogenic callus (such as corn immature embryos), or the embryogenic callus (such as type I callus in corn) obtained thereof, as described in PCT patent publication WO 92/09696 or US Patent 5,641 ,664.
  • Other methods for transformation of com include the methods by Fromm et al. (1990, Bio/Technology 8, 833-839), Gordon-Kamm et al. (1990, The Plant Cell 2, 603-618) and Ishida et al. (1996, Nature Biotechnology 14, 745-750).
  • a disarmed Ti plasmid, containing the insecticidally effective chimeric improved cry9C gene, in Agrobacterium tumefaciens can be used to transform the plant cell, preferably the corn or cotton cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0116718, EP 0270822, PCT publication WO 84/02913 and EP 0242246 (which are also incorporated herein by reference), and in Gould et al. (1991 , Plant Physiol. 95, 426-434) or Ishida et al. (1996, supra), particularly the method described in PCT publication WO 94/00977.
  • Preferred Ti- plasmid vectors each contain the insecticidally effective chimeric improved cry9C gene between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid.
  • other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0233247), pollen mediated transformation (as described, for example in EP 0270356, PCT publication WO 85/01856, and US Patent 4,684,611 ), plant RNA virus-mediated transformation (as described, for example in EP 0067553 and US Patent 4,407,956), and liposome-mediated transformation (as described, for example in US Patent 4,536,475).
  • a resulting transformed plant such as a transformed corn or cotton plant
  • seeds which are obtained from the transformed plants, contain the chimeric improved cry9C gene or its insecticidally effective part as a stable genomic insert.
  • Cells of the transformed plant can be cultured in a conventional manner to produce the improved Cry9C protein or insecticidally effective portions thereof, which can be recovered for use in conventional insecticide compositions against insects, particularly lepidopteran insects (U.S. Patent 5,254,799).
  • Preferred plants in accordance with this invention include rice, plants of the genus Brassica such as oilseed rape, cauliflower and broccoli, and also soybean, tomato, tobacco, potato, eggplant, beet, oat, pepper, gladiolus, dahlia, chrysanthemum, sorghum, and garden peas.
  • the improved cry9C coding region or its insecticidally effective part is inserted in a plant cell genome so that the inserted coding region is downstream (i.e., 3') of, and under the control of, a promoter which can direct the expression of the gene part in the plant cell. This is preferably accomplished by inserting the chimeric improved cry9C gene or its insecticidally effective part in the plant cell genome.
  • Preferred promoters include: the strong constitutive 35S promoters (the "35S promoters") of the cauliflower mosaic virus of isolates CM 1841 (Gardner et al., 1981 , Nucleic Acids Research 9, 2871-2887), CabbB-S (Franck et al., 1980, Cell 21, 285-294) and CabbB-JI (Hull and Howell, 1987, Virology 86, 482-493); the ubiquitin promoter (EP 0342926), and the TR1 ' promoter and the TR2' promoter which drive the expression of the 1 ' and 2' genes, respectively, of the T-DNA (Velten et al., 1984, EMBO J. 3, 2723-2730).
  • a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant, preferably leaf and stem tissue, whereby the inserted chimeric improved cry9C gene or its insecticidally effective part is expressed only in cells of the specific tissue(s) or organ(s).
  • a promoter whose expression is inducible (e.g., by insect feeding or by chemical factors).
  • Known wound-induced promoters inducing systemic expression of their gene product throughout the plant are also of particular interest.
  • the improved cry9C coding region or its insecticidally effective part, is inserted in the plant genome so that the inserted coding region is upstream (i.e., 5') of suitable 3' end transcription regulation signals (i.e., transcript termination and polyadenylation signals).
  • suitable 3' end transcription regulation signals i.e., transcript termination and polyadenylation signals.
  • Preferred polyadenylation and transcript formation signals include those of the 35S gene (Mogen et al., 1990, The Plant Cell 2, 1261-1272), the octopine synthase gene (Gielen et al., 1984, EMBO J. 3, 835-845) and the T-DNA gene 7 (Velten and Schell, 1985, Nucl. Acids Res. 13, 6981-6998), which act as 3'-untranslated DNA sequences in transformed plant cells.
  • the chimeric improved cry9C gene, or its insecticidally effective gene part, can optionally be inserted in the plant genome as a hybrid gene (EP 0 193 259;
  • the improved cry9C gene is expressed in a plant in combination with another insect control protein, e.g., another Bt-derived crystal protein or an insecticidal fragment thereof, particularly a Cry1 Ab- or Cry1 B-type protein, to prevent or delay the occurrence of insect resistance development (EP 0 408403).
  • another insect control protein e.g., another Bt-derived crystal protein or an insecticidal fragment thereof, particularly a Cry1 Ab- or Cry1 B-type protein, to prevent or delay the occurrence of insect resistance development (EP 0 408403).
  • All or part of the improved cry9C coding region can also be used to transform bacteria, such as a B. thuringiensis which produces other insecticidal toxins (Lereclus et al., 1992, Bio/Technology 10, 418-421 ; Gelemter & Schwab, 1993, In Bacillus thuringiensis, An Environmental Biopesticide: theory and Practice, pp. 89-104, eds. Entwistle, P.F., Cory, J.S., Bailey, M.J. and Higgs, S., John Wiley & Sons Ltd.).
  • bacteria such as a B. thuringiensis which produces other insecticidal toxins (Lereclus et al., 1992, Bio/Technology 10, 418-421 ; Gelemter & Schwab, 1993, In Bacillus thuringiensis, An Environmental Biopesticide: theory and Practice, pp. 89-104, eds. Entwistle, P.F.
  • a transformed Bt strain is produced which is useful for combating a wide spectrum of insect pests or for combating insects in such a manner that insect resistance development is prevented or delayed (EP 0408 403).
  • Preferred promoter and 3' termination and polyadenylation sequences for the chimeric improved cry9C gene are derived from Bacillus thuringiensis genes, such as the native ICP genes.
  • the improved coding region of the invention can be inserted and expressed in endophytic and/or root-colonizing bacteria, such as bacteria of the genus Pseudomonas or Clavibacter, e.g., under the control of a Bt ICP gene promoter and 3' termination sequences.
  • the improved Cry9C-producing strain can also be transformed with all or an insecticidally effective part of one or more DNA sequences encoding a Bt protein or an insecticidally effective part thereof, such as: a DNA encoding the Bt2 or Cry1 Ab protein (US patent 5,254,799; EP 0 193 259) or the Bt109P or Cry3C protein (PCT publication WO 91/16433), or another DNA coding for an anti-lepidoptera or an anti- Coleoptera protein.
  • a transformed Bt strain can be produced which is useful for combating an even greater variety of insect pests (e.g., Coleoptera and/or additional lepidoptera) or for preventing or delaying the development of insect resistance.
  • any DNA sequence encoding any of the above described improved Cry9C proteins can be used.
  • SEQ ID No. 1 Nucleotide sequence of the Bacillus thuringiensis cry9C gene, showing the coding region and flanking 5' and 3' regions.
  • SEQ ID No. 2 Amino acid sequence of the full length Bacillus thuringiensis
  • SEQ ID No. 3 Nucleotide sequence of a codon-optimized DNA sequence encoding a truncated Cry9C protein wherein the arginine at amino acid position 123 (corresponding to amino acid position 164 in the protein of SEQ ID No. 2) has been replaced by lysine.
  • SEQ ID No. 4 Amino acid sequence of the modified Cry9C protein encoded by the DNA of SEQ ID No. 3.
  • each of the amino acids in these identified regions was changed into alanine, one by one, using splice overlap extension PCR (Ho et al., 1989, supra) on the protease-resistant form of the native cry9C gene wherein the arginine codon at position 164 was replaced by an alanine codon.
  • the codon most preferred in the cry9C native gene for alanine, GCA was used for these modifications.
  • GTA valine codon
  • the obtained PCR fragments were ligated in pUC19-derived vectors. If not present, suitable unique restriction sites were created in the cry9C DNA. All plasmids containing modified DNA sequences were controlled by sequencing the relevant portions and were found to be correctly constructed.
  • the modified cry9C genes were expressed in transformed WK6 cells. Every mutant protein was expressed in these E. coli cells at least twice. Mutants causing problems in expression, probably caused by structural changes in these mutants, were discarded.
  • Bio assays on the modified Cry9C proteins obtained in Example 1 were carried out with first instar larvae of the Southeastern corn borer, Diatraea grandiosella (family Pyralidae); the European corn borer, Ostrinia nubilalis (family Pyralidae); and the tobacco budworm, Heliothis virescens (family Noctuidae). A dilution series of each protein was surface-layered on the artificial diet to determine the LC 50 value.
  • the artificial diet consisted of: agar (20 g), water (1 ,000 ml), corn flour (96 g, ICN Biochemicals), yeast (30 g), wheat germs (64 g, ICN Biochemicals), wesson salt (7.5 g, ICN Biochemicals), casein (15 g), sorbic acid (2 g), aureomycin (0.3 g), nipagin (1 g), wheat germ oil (4 ml), sucrose (15 g), cholesterol (1 g), ascorbic acid (3.5 g), Vanderzand modified vitamin mix (12 g, ICN Biochemicals). Larvae were placed on the diet in multi-well plates, 1 larva per well (2 for Ostrinia nubilalis).
  • the mutant proteins were digested with trypsin to release the toxin fragments.
  • the assays are repeated at least 5 times, using two different protein preparations.
  • control protein the trypsin-digested Cry9C(R164A) protein was used.
  • the Cry9C(R164A) protein has the amino acid sequence of SEQ ID No. 2 wherein the arginine at position 164 was replaced by alanine.
  • This protein was found to be more stable than the wild-type Cry9C toxin while retaining its toxicity to the test insects (see, e.g., PCT patent publication WO 94/24264).
  • the LC 5 o values were calculated with the POLO- program, which is based on the probit analysis (POLO-PC, LeOra Software, 1119 Shattuck Ave., Berkeley California 94707).
  • the results of these assays for those protein mutants which gave an LC 50 value that is significantly different from that of the control protein in repeated bio assays are summarized in Table 1. It is clear that different positions in the Cry9C protein when substituted to alanine cause increased toxicity in each of the tested insects.
  • Binding assays on isolated brush border membrane vesicles of Heliothis virescens and Ostrinia nubilalis performed as described in Van Rie et al. (1990, Appl. Environm. Microbiol. 56, 1378-1385) showed that for all, with the exception of two, of the modified Cry9C proteins with altered toxicity, receptor binding is also altered (e.g., an observed shift in K D value), thus confirming that for most amino acid residues altered toxicity is due to altered receptor binding. Hence, these residues are proper candidates for improvement of toxicity by amino acid randomization at or near the identified critical position.
  • the Cry9C(R164A) protein was tested in competition binding assays using the ECL protein biotinylation system (Amersham Life Sciences, Amersham International pic, UK) as described by Lambert et al. (1996, supra) to determine if competition occurred with other Bt toxins in selected insects.
  • 3ng biotinylated Cry9C(R164A) protein was added to 30 ⁇ g brush border membrane vesicles in PBS buffer (comprising 0,1 % BSA) in the presence of a 300-fold excess of non- biotinylated toxin (homologous competition assays were included in every test as control). Repeated competition tests showed that in both Ostrinia nubilalis and
  • combination of the Cry9C and a selected non- competitively binding Bt toxin with good toxicity to the target insect can be used simultaneously in order to prevent or delay insect resistance development.
  • a particularly interesting combination would be the Cry9C (or its protease-resistant variant) and a Cryl B and/or any of the Cry1 A-type toxins for Ostrinia nubilalis control and the Cry9C (or its protease-resistant variant) and any one of the Cry1 A-type toxins, preferably a Cry1 Ab-type toxin, for D. grandiosella control.
  • the Cry9C (or its protease-resistant variant) and any of the Cry1 A-type toxins are preferred toxins to be co-expressed.
  • the modified position in every mutant protein of Example 2 giving rise to a significantly decreased or increased toxicity to an insect species is altered to all other amino acids and the toxicity is re-evaluated.
  • the amino acids yielding the highest toxicity at a particular position are combined to form an improved Cry9C protein.
  • the alanine mutants yielding an increase in toxicity (up-mutant amino acid positions) are included in such combinations to form improved Cry9C proteins for the selected insect species.
  • Table 1 indeed shows already two up-mutant proteins for every insect tested. Analysis of all these improved Cry9C proteins in the bio assay shows that combinations of up-mutant amino acid positions can substantially increase toxicity of the Cry9C protein towards selected insect species.
  • a modified DNA sequence encoding a truncated Cry9C(R164K) protein for expression in corn and cotton plants is shown in SEQ ID No. 3.
  • This DNA sequence has an optimized codon usage for plants and encodes an N- and C-terminally truncated Cry9C protein wherein an arginine amino acid has been replaced by a lysine (at position 123 in SEQ ID No. 3).
  • DNA sequences are made encoding the above improved Cry9C proteins and comprising amino acids 1 to 666 of the Cry9C(R164K) protein.
  • Preferred codons to encode the amino acid replacements in the improved Cry9C proteins are those which are most preferred by the plant host (see, e.g., Murray, 1989, supra).
  • a chimeric improved cry9C gene comprising the 35S promoter and 35S 3' transcription termination and polyadenylation signal is constructed by routine molecular biology techniques as described in the detailed description.
  • Corn cells are stably transformed by either Agrobacterium-med ⁇ aXed transformation (Ishida et al., 1996, supra and U.S. Patent No. 5,591 ,616) or by electroporation using wounded and enzyme-degraded embryogenic callus, as described in WO 92/09696 or US Patent 5,641 ,664 (incorporated herein by reference).
  • the resulting transformed cells are selected by means of the incorporated selectable marker gene, grown into plants and tested for susceptibility towards insects.
  • Corn plants expressing a truncated improved Cry9C(R164K) protein wherein the amino acids at positions 364, 488, 319 and 321 have been replaced into alanine show a significantly higher protection from Ostrinia nubilalis and Diatraea grandiosella damage in comparative tests against corn plants expressing a truncated Cry9C(R164K) protein.
  • a positive correlation is found between the level of expression, as measured by RNA and protein analysis, and the observed insecticidal effect.
  • Cotton cells are stably transformed by Agrobacterium-me ⁇ a e ⁇ transformation (Umbeck et al., 1987, Bio/Technology 5, 263-266; US Patent 5,004,863, incorporated herein by reference). The resulting transformed cells are selected by means of the incorporated selectable marker gene, grown into plants and tested for susceptibility towards insects.
  • Cotton plants expressing the truncated improved Cry9C(R164K, L321 A, P329A) protein at similar levels than cotton plants expressing the truncated Cry9C(R164K) protein show a significantly higher protection from Heliothis virescens damage.
  • a positive correlation is found between the level of expression, as measured by RNA and protein analysis, and the observed insecticidal effect.
  • Table 1 relative toxicity of modified trypsin-digested Cry9C proteins to different insects when compared with the Cry9C(R164A) trypsin-digested protein (mutant 'F313A': the Cry9C(R164A) trypsin-digested protein wherein also the phenylalanine at position 313 is replaced by alanine; 'down(2x)': mutant protein with a significantly lower toxicity (LC50 value about 2 times higher than the control protein), 'up (2x)': mutant with a significantly higher toxicity (LC50 value about two times lower than that of the control protein), '-': no difference in toxicity found):
  • MOLECULE TYPE DNA (genomic)
  • ATATGTGGAC CATATTTTAA AATATAGCGT CCAACAACTA CCATATTATG TAATTGATGG 360
  • GGTTAATTCA CCATTAACAC AACAATATCG CCTAAGAGTT CGTTTTGCCT CAACAGGAAA 2400 TTTCAGTATA AGGGTACTCC GTGGAGGGGT TTCTATCGGT GATGTTAGAT TAGGGAGCAC 2460
  • GGTACCAAAA CCATGGCTGA CTACCTGCAG ATGACCGACG AGGACTACAC CGACAGCTAC 60

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US6369213B1 (en) 1996-07-01 2002-04-09 Mycogen Corporation Toxins active against pests
US6570005B1 (en) 1996-07-01 2003-05-27 Mycogen Corporation Toxins active against pests
AU7518100A (en) * 1999-09-17 2001-04-24 Aventis Cropscience N.V. Insect-resistant rice plants
CA2395897C (en) * 1999-12-28 2011-11-15 Bayer Cropscience N.V. Insecticidal proteins from bacillus thuringiensis
US20030131378A1 (en) * 2000-08-11 2003-07-10 Aroian Raffi V. Methods for blocking resistance to bt toxins in insects and nematodes
US7230167B2 (en) 2001-08-31 2007-06-12 Syngenta Participations Ag Modified Cry3A toxins and nucleic acid sequences coding therefor
US8268583B2 (en) 2003-12-10 2012-09-18 Novozymes A/S Cell with improved secretion mediated by MrgA protein or homologue
US7629504B2 (en) 2003-12-22 2009-12-08 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry9 nucleic acids
EP2281447B1 (de) 2004-03-25 2016-07-27 Syngenta Participations AG Mais der Linie MIR604
JP4899180B2 (ja) * 2004-12-22 2012-03-21 独立行政法人農業・食品産業技術総合研究機構 核酸検査用プライマーセット及びこれらを用いた検査キット及び検査方法
US9522937B2 (en) 2007-03-28 2016-12-20 Syngenta Participations Ag Insecticidal proteins
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US8319019B2 (en) * 2009-01-23 2012-11-27 Pioneer Hi Bred International Inc Bacillus thuringiensis gene with lepidopteran activity

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