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WO2001021821A2 - Plants de riz resistants aux insectes - Google Patents

Plants de riz resistants aux insectes Download PDF

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Publication number
WO2001021821A2
WO2001021821A2 PCT/EP2000/008877 EP0008877W WO0121821A2 WO 2001021821 A2 WO2001021821 A2 WO 2001021821A2 EP 0008877 W EP0008877 W EP 0008877W WO 0121821 A2 WO0121821 A2 WO 0121821A2
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WIPO (PCT)
Prior art keywords
rice
amino acid
protein
plant
seq
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PCT/EP2000/008877
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English (en)
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WO2001021821A3 (fr
Inventor
Frank Michiels
Stefan Jansens
Harish Kumar
David Lobo
Jan Samson
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Aventis Cropscience N.V.
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Priority to AU75181/00A priority Critical patent/AU7518100A/en
Publication of WO2001021821A2 publication Critical patent/WO2001021821A2/fr
Publication of WO2001021821A3 publication Critical patent/WO2001021821A3/fr

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    • 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
    • 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

  • This invention relates to a method for controlling rice insect pests, particularly Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, preferably an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius and Parnara guttata, most particularly Chilo suppressali
  • This invention also relates to rice plants with resistance to rice pests by expression of a Cry9C protein and to an insecticide composition that is active against rice pests, particularly Lepidopteran rice stemborers, rice skippers, rice cutworms, rice caseworms or rice leaffolders, preferably an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo
  • This invention further relates to the use of a DNA sequence which encodes an insecticidal Cry9C protein as defined herein for obtaining insect resistance in rice plants.
  • Bacillus thuringiensis (“Bt”) is a Gram-positive bacterium which produces crystals upon sporulation. The crystals are composed of proteins which have been shown to be toxic against insect larvae. These crystal proteins and their corresponding genes have been classified based on their amino acid sequence (Crickmore et al., 1998).
  • the Cry9C protein is an insecticidal protein originally found in Bacillus thuringiensis (Lambert et al., 1996). Because of its interesting activity against corn insect pests, transgenic corn plants expressing a Cry9C protein have been successfully used to protect corn fields from insect infestation (Jansens et al., 1997).
  • Insect resistance in several types of rice to some of the damaging Lepidopteran rice insect pests has been obtained by expressing genes encoding insecticidal Cryl Ab or Cryl Ac proteins of Bacillus thuringiensis (e.g., Fujimoto et al., 1993; Wunn et al., 1996; Wu et al. 1997; Ghareyazie et al., 1997; Nayak et al., 1997; and Cheng et al., 1998), a plant lectin (Rao et al., 1998) or protease inhibitors (Xu et al., 1996; Duan et al., 1996) in rice plants.
  • Toxicity of isolated Bacillus thuringiensis crystal proteins to some Lepidopteran rice insect pests has been evaluated for Cryl Aa, Cryl Ab, Cryl Ac, Cryl B, Cryl C, Cryl D, Cryl E, Cryl F, Cryl G, and Cry2A proteins (Karim et al., 1997; Lee et al., 1997b).
  • This invention provides new rice plants expressing a Cry9C protein which provides a high dose effect on relevant Lepidopteran rice insect pest, allowing the development of new commercial rice plants which are better protected against insect attack and which at the same time prevent or delay the development of resistance in such insects.
  • Such rice plants contain, stably integrated in their genome, a plant-expressible DNA sequence encoding a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a DNA sequence encoding a protein comprising the amino acid sequence of SEQ ID NO: 4. Further in accordance with this invention, seeds, grain and processed grain of these rice plants are provided, comprising a DNA sequence encoding a Cry9C protein.
  • these rice plants also comprise in their genome a DNA encoding a protein selected from the group consisting of: Cryl B, Cryl C, Cryl D, Cryl E, Cryl Aa, Cryl Ab, Cryl Ac, Cryl I, Cryl J, Cry2A, Cry6B, Cry3A, a protease inhibitor, a cowpea trypsin inhibitor, protease inhibitor II, cystatin, GNA lectin, an insecticidal protein of Xhenorhabdus or Photorhabdus spp., and a protein conferring resistance to glufosinate ammonium.
  • a protein selected from the group consisting of: Cryl B, Cryl C, Cryl D, Cryl E, Cryl Aa, Cryl Ab, Cryl Ac, Cryl I, Cryl J, Cry2A, Cry6B, Cry3A, a protease inhibitor, a cowpea trypsin inhibitor, protease inhibitor II, cystatin, GNA lectin
  • Also provided in this invention is a process for producing rice plants and reproduction material thereof, such as seeds, which are resistant to 1 ) Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, 2) an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and
  • a process for controlling 1 ) Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, 2) an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Chilo zacconius and Parnara guttata, or 3) an insect selected from the group consisting of: Chilo suppressalis, Marasmia patnalis, Scirpophaga incertulas and
  • new transgenic rice plants particularly Oryza sp., preferably Oryza sativa
  • the Cry9C protein of this invention can also be used to control other relevant insect pests, e
  • the Cry9C protein is used to control rice insect pests, particularly Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Marasmia patnalis, , Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Parnara guttata, preferably Chilo suppressalis, Scirpophaga incertulas, Marasmia patnalis and Cnaphalocrocis medinalis
  • the Cry9C protein can be isolated in a conventional manner from several Bt strains or can be recombinantly expressed, as described in PCT patent publications WO 94/05771 , WO 94/24264, WO 99/00407 and in US Patents 5,885,571 and 5,861 ,543.
  • Cry9C protein refers to an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 2 from amino acid position 44 to amino acid position 658 or an insecticidal protein comprising the amino acid sequence of SEQ ID NO: 4 from amino acid position 1 to amino acid position 625 and variants thereof retaining insecticidal activity such as the protease-resistant Alanine variant disclosed in PCT patent publication WO 94/24264.
  • This term includes the crystal protein, protoxin or insecticidally effective forms thereof such as the toxin form obtained by protease-digestion from the protoxin or the protein with the amino acid sequence of SEQ ID NO: 2 from an amino acid position between amino acid position 1 and amino acid position 44 and an amino acid position between amino acid position 658 and amino acid position 666, as well as fusion proteins or protease-resistant variants thereof. It is known that some amino acids can be substituted by other equivalent amino acids in a Cry9C protein while still retaining all or some insecticidal activity, see, e.g., PCT patent application WO 99/00407, incorporated herein by reference. These proteins are included within the scope of the term "Cry9C protein".
  • Cry9C protein is a protein comprising the amino acid sequence from amino acid position 44 to amino acid position 658 of SEQ ID NO: 2, or a protein comprising the amino acid sequence of SEQ ID NO: 4.
  • the "cry9C DNA”, as used herein, refers to any DNA sequence encoding a Cry9C protein and includes naturally occurring, recombinant, semi-synthetic or synthetic DNA sequences and can include regulatory or other regions not coding for an amino acid sequence such as introns, leader or trailer sequences, promoters and 3' transcription termination and polyadenylation sequences.
  • the "cry9C coding region” refers to any nucleotide sequence which starts with a start codon and ends with a stop codon and encodes a Cry9C protein. Because of the degeneracy of the genetic code, some amino acid codons can be replaced with others without changing the amino acid sequence of the protein.
  • cry9C DNA in accordance with the invention is the DNA of Sequence ID No. 3.
  • the "chimeric cry9C gene”, as used herein, refers to a cry9C DNA consisting of a cry9C coding region flanked by regulatory elements, such as a promoter and a transcription termination and polyadenylation region which allow expression of a Cry9C protein in the cells of a plant, particularly a rice plant.
  • regulatory elements such as a promoter and a transcription termination and polyadenylation region which allow expression of a Cry9C protein in the cells of a plant, particularly a rice plant.
  • the "Cry” nomenclature is used, as described by Crickmore et al. (1998). Whenever only one number and one character is used after "Cry”, e.g.
  • Cryl B this refers to all related sequences starting with the listed number and character, the protoxin form as well as any insecticidally effective fragments thereof such as the toxin form obtained after protease treatment (e.g., “Cryl B” includes all Cryl B forms such as “Cryl Ba1 " and “Cryl Bb1 ").
  • Plants "resistant to” or with “resistance against” insects, or “resistant to” or with “resistance against” insect infestation or attack, as used herein, refers to plants which, by action of man, particularly by transformation with a DNA in accordance with this invention, or by spraying with a composition in accordance with this invention, exhibit a significantly increased control of insects which otherwise damage plants of that species. This includes significantly increased feeding inhibition, growth inhibition or mortality of insects feeding on the plants.
  • resistance against insects of the transgenic plants of the invention refers to complete protection to the insects which can be determined by the minor damage caused by the insects to such plants (e.g., for stemborers, no tunneling in the rice stem) or refers to the achievement of substantially the same yields as would have been obtained under the same conditions but in the absence of attack of that insect pest.
  • resistance against insects of a rice plant in accordance with the invention refers to the killing of at least 90 % of the insects, preferably at least 95 % of the insects, which attempt to feed on the Cry9C rice plants of the invention, this preferably within 10 days, most preferably within 5 days.
  • a "plant-expressible DNA sequence”, in accordance with this invention, is a DNA sequence capable of expressing in a plant a gene product, preferably a protein. In the most preferred embodiment, this is a DNA sequence comprising regulatory elements allowing proper transcription and translation of a desired protein from a coding sequence introduced in the plant's genome.
  • a plant-expressible DNA sequence in accordance with this invention comprises a promoter region capable of transcription in plant cells, particularly rice.
  • a plant-expressible DNA sequence can also contain other elements such as a 3' transcription termination and polyadenylation sequence or introns.
  • genome of a plant or plant cell refers to the totality of genetic material present in a plant or plant cell, and includes but is not limited to mitochondrial, chloroplast and nuclear DNA.
  • DNA "stably integrated” in the genome refers to a DNA which is integrated in such a manner that it can be passed on to the progeny of a plant, preferably by integration of such DNA in the genome, particularly nuclear DNA, of a plant.
  • Progeny of a plant refers to further generations of a plant. This includes seeds, offspring plants and cells, tissues or whole plants obtainable from a plant or taken from a plant to obtain more plants or further generations with the same or different genetic material, including vegetative propagation.
  • Processed grain refers to grain which has been treated using one or several processes, particularly to grain processed to be used as feed or food. Processing of grain includes but is not limited to polishing, milling, parboiling, dehusking and the like. Processed and unprocessed grain derived from the Cry9C rice plants of the invention, and comprising the cry9C chimeric gene of the invention, is included within the scope of the invention.
  • the Cry9C protein is combined with other insecticidal proteins for providing better protection against relevant rice insect pests and for preventing or delaying insect resistance development.
  • Preferred proteins are selected from the group consisting of: Cryl B, Cryl C, Cryl D, Cryl E, Cryl Aa, Cryl Ab, Cryl Ac, Cryl I, Cryl J, Cry2A, Cry6B, Cry3A, protease inhibitors such as cowpea trypsin inhibitor (CpTI, Xu et al., 1996), protease inhibitor II (pinll, Duan et al., 1996) or cystatin (Irie et al., 1996), the GNA lectin (Galanthus nivalis agglutinin, Rao et al., 1998), an insecticidal protein of Xhenorhabdus or Photorhabdus spp.
  • the Cry9C protein is combined with at least one of the proteins selected from the Cryl Ab, Cryl B, Cry2A and Cryl C (preferably Cryl Ca4) proteins or active fragments thereof, particularly by combined expression in a transgenic rice plant.
  • the use of a DNA sequence encoding a protein conferring resistance to glufosinate ammonium is preferred so that the plant cell or plant containing it can grow normally in otherwise toxic concentrations of this molecule, particularly a phosphinothricin acetyl transferase from Streptomyces hygroscopicus or viridochromogenes, as described in PCT patent publication WO 87/05629, US patent 5,561 ,236, and US patent 5,276,268.
  • suitable restriction sites can be introduced, flanking the DNA. This can be done by site- directed mutagenesis, using well-known procedures (Stanssens et al., 1989; White et al., 1989).
  • the Cry9C protein can bind to a receptor different from the Cryl A type Bt proteins in insect gut membranes, it is useful to cross transgenic rice plants expressing the Cry9C protein with transgenic rice plants expressing a protein with a different binding site in the target insect compared to the Cry9C protein, i.e., a Cryl A or Cry2A protein.
  • rice can be transformed with the cry9C DNA and a DNA encoding a protein with a different binding site in the target insect compared to the Cry9C protein, i.e., a cryl A DNA such as a DNA encoding a truncated insecticidally-effective Cryl Ab5 protein, so as to obtain a transgenic rice plant or progeny thereof expressing these two insect control proteins, or a DNA encoding a Cry2Aa or Cry2Ab protein.
  • a DNA encoding a cryl A protein can be selected from any of the DNA sequences which have already been successfully expressed in plants, particularly in rice or corn, and encoding any of the proteins listed as Cryl A by Crickmore et al.
  • the cry9C DNA preferably the cry9C chimeric gene
  • the cry9C DNA can be stably inserted in a conventional manner into the nuclear genome of a rice cell, and the so-transformed cell can be regenerated in a conventional manner to produce a transformed rice plant and progeny (seed and further generations) thereof that is insect-resistant.
  • a disarmed Ti-plasmid, containing the insecticidally effective cry9C gene part, in Agrobacterium tumefaciens can be used to transform the rice cell, and thereafter, a transformed rice plant can be regenerated from the transformed plant cell using the procedures described, for example, in PCT patent publication WO 92/09696.
  • Preferred tissues for transformation with Agrobacterium include but are not limited to mature seed-derived callus, immature embryo-derived callus and immature embryos. These tissues can be wounded prior to co-cultivation with Agrobacterium, and can be pre-induced with acetosyringone or other plant phenolic compounds as described in PCT patent publication WO 98/3721 2.
  • Preferred Ti-plasmid vectors each contain the cry9C chimeric 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.
  • vectors can be used to transform plant cells or plants, using procedures such as direct gene transfer (as described, for example in EP 0,233,247), pollen mediated transformation (as described, for example in EP 0,270,356, PCT publication WO 85/01 856, and US Patent 4,684,61 1 ), plant RNA virus-mediated transformation (as described, for example in EP 0,067,553 and US Patent 4,407,956), liposome- mediated transformation (as described, for example in US Patent 4,536,475), and other methods such as the rice transformation method of Shimamoto et al. (1989) and Datta et al. (1990) and the method for transforming monocots generally, as described in PCT publications WO 93/21 335 and WO 92/09696. Any known procedures for transforming rice plants are suitable for obtaining the transgenic rice plants in accordance with this invention.
  • the resulting transformed rice plant can be used in a conventional plant breeding scheme to produce more transformed rice plants with the same characteristics or to introduce the cry9C DNA in other rice varieties or in related plant species.
  • Preferred rice plants include plants from the Oryza species, particularly Oryza sativa, preferably japonica, indica or javanica rice, whether soil, water, upland, rainfed shallow, deep water, floating or irrigated rice.
  • Seeds, which are obtained from the transformed rice plants contain the cry9C DNA as a stable genomic insert, either in the nuclear, mitochondrial or chloroplast genome.
  • Cells of the transformed rice plant can be cultured in a conventional manner to produce the insecticidally effective Cry9C protein.
  • the cry9C DNA is inserted in the rice cell genome so that the inserted DNA is downstream (i.e., 3 1 ) of, and under the control of, a promoter which can direct the expression of the DNA in the plant cell. This is preferably accomplished by inserting the cry9C chimeric gene 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 ), CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., 1992, see also Cornejo et al., 1993), the gos2 promoter (de Pater et al., 1992), the emu promoter (Last et al., 1990), rice actin promoters such as the promoter described by Zhang et al.
  • the TR1 ' promoter and the TR2 1 promoter (the "TR1 ' promoter” and “TR2 1 promoter”, respectively) which drive the expression of the 1 ' and 2' genes, respectively, of the T-DNA (Velten et al., 1984).
  • a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (e.g., leaves, stem, pith and/or root tissue) whereby the inserted cry9C gene part is expressed only in cells of the specific tissue(s) or organ(s).
  • the insecticidally effective cry9C gene part could be selectively expressed in rice leaves and stem or could be expressed by a promoter not active in pollen.
  • promoters include light-inducible promoters such as the promoter of the rice ribulose-1 ,5-bisphosphate carboxylase small subunit gene or of another plant such as pea as disclosed in US Patent 5,254,799.
  • Another alternative is to use a promoter whose expression is inducible (e.g., by wounding caused by feeding insects, by temperature or by chemical factors).
  • any promoter known to be highly expressed in rice plants, particularly in leaves or stem can be used in accordance with this invention.
  • the cry9C DNA is inserted in the rice genome so that the inserted gene part is upstream (i.e., 5 1 ) of suitable 3' end transcription regulation signals (i.e., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the cry9C chimeric gene in the rice genome.
  • suitable 3' end transcription regulation signals i.e., transcript formation and polyadenylation signals.
  • Preferred polyadenylation and transcript formation signals include those of the octopine synthase gene (Gielen et al., 1984) and the T-DNA gene 7 (Velten and Schell, 1985), which act as 3 '-untranslated DNA sequences in transformed plant cells.
  • cry9C DNA can optionally be inserted in the plant genome as a hybrid gene (Vaeck et al., 1987) under the control of the same promoter as a selectable marker gene, so that the plant expresses a fusion protein retaining the activity of both proteins.
  • high expression levels of Cry9C protein can be selected by increasing the concentration of selectable marker agent in the culture medium.
  • a process for making a rice plant resistant to Lepidopteran insects, particularly Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Parnara guttata, preferably Chilo spp., Cnaphalocrocis spp., Marasmia spp., and Scirpophaga s
  • This process is characterized by transforming rice plants with a DNA sequence encoding a Cry 9C protein using known procedures.
  • rice plants are rendered resistant to Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders, by transforming rice plants with a DNA sequence encoding a Cry 9C protein.
  • Another embodiment in accordance with the invention is a process for producing rice plants and reproduction material thereof, such as seeds, which are resistant to infestation with an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Parnara guttata, preferably Chilo spp., Cnaphalocrocis spp., Marasmi
  • a process for producing rice plants and reproduction material thereof, such as seeds, which are resistant to infestation with a Lepidopteran rice leaffolder, rice skipper, rice cutworm, rice armyworm, rice caseworm or rice stemborer, which process comprises transforming rice cells with a DNA sequence encoding a Cry9C protein and regenerating plants and reproduction material thereof comprising said DNA sequence.
  • a method for controlling Lepidopteran insect pests particularly Lepidopteran rice leaffolders, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice stemborers, more particularly Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, or Parnara guttata, preferably Chilo spp.
  • such insects are contacted with the Cry9C protein by spraying compositions comprising insecticidally effective amounts of Cry9C protein to the rice plants.
  • This composition can be formulated in a conventional manner using the Cry9C protein as an active ingredient, together with suitable carriers, diluents, emulsifiers and/or dispersants (e.g., as described by Bernhard and Utz, 1 993).
  • This insecticide composition can be formulated as a wettable powder, pellets, granules or dust or as a liquid formulation with aqueous or non-aqueous solvents as a foam, gel, suspension, concentrate, etc.
  • the concentration of the Cry9C protein in such a composition will depend upon the nature of the formulation and its intended mode of use.
  • an insecticide composition of this invention can be used to protect a rice field for 1 or 2 weeks against Lepidoptera with each application of the composition. For more extended protection (e.g., for a whole growing season), additional amounts of the composition should be applied periodically in case a sprayable composition is used.
  • Bt cells or crystals, crystal proteins, protoxins, toxins, and insecticidally effective protoxin portions and other insecticides, as well as fungicides, biocides, herbicides and fertilizers, can be employed along with the Cry9C protein to provide additional advantages or benefits.
  • the concentration of the Cry9C protein in a composition will be at least about 0.1 % by weight of the formulation to about 100% by weight of the formulation, more often from about 0.1 5% to about 0.8% by weight of the formulation.
  • a method for producing processed or unprocessed rice grain comprising planting Cry9C rice of the invention in the field, harvesting and treating seeds from such rice to obtain processed or unprocessed grain. An improved yield is obtainable in rice plants transformed or treated in accordance with the invention.
  • Toxicity of a protein to a Lepidopteran rice insect such as a Lepidopteran rice leaffolder, rice armyworm, rice cutworm, rice caseworm, rice skipper, rice stemborer, Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda,
  • Mythimna unipuncta, Chilo zacconius, or Parnara guttata can be measured using established procedures.
  • a suitable procedure in accordance with this invention is drenching a solution containing the protein (at determined concentration) on a layer of rice seeds or leaves. After addition of larvae to these seeds or leaves, the number of dead larvae are counted and the larvae still alive are weighed after determined time periods. Appropriate control samples are run in the same test so as to determine the background mortality and standard larval growth under the same conditions. Similar assays using artificial diet with protein applied thereon or incorporated therein or rice seeds or rice leaf material with protein applied thereon can be used as described, e.g., in Lee et al.
  • Toxicity to insects of transgenic rice expressing a protein in accordance with the invention can be measured following routine protocols with appropriate controls such as by using a cut stem in vitro test or a whole plant assay as described, e.g., by Nayak et al. (1997) or Ghareyazie et al. (1997).
  • Rice pests can be caught in a rice field and can be cultivated following known procedures (see, e.g., Kamano and Sato, 1985, for Chilo suppressalis cultivation).
  • Scirpophaga incertulas e.g., the method as described by Yannian et al. (1988) can be used.
  • the taxonomy of a Lepidopteran rice insect pest can be determined by procedures known to a person of ordinary skill in the art, see, e.g. Barrion and Litsinger (1994).
  • a method for controlling Lepidopteran rice insect pests particularly Lepidopteran rice stem borers, rice caseworms, rice cutworms, rice armyworms or rice leaffolders, more particularly an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipuncta, Chilo zacconius, and Parnara guttata, preferably Chilo spp., Cn
  • SEQ ID NO: 2 amino acid sequence of the Cry9C protein of SEQ ID NO: 1
  • SEQ ID NO: 3 cry9C DNA and Cry9C amino acid sequence for expression in rice.
  • SEQ ID NO: 4 amino acid sequence of the Cry9C protein of SEQ ID NO: 3.
  • Example 1 Insecticidal activity of Cry9C protein to rice insect pests. Using insect feeding assays, the toxicity of the Cry9Ca protein to selected rice insects was evaluated.
  • the rice leaffolders Cnaphalocrocis medinalis and Marasmia patnalis were tested using a rice leaf dip assay.
  • Whole rice leaves of a leaffolder-susceptible rice variety (Taichung native 1 variety (TN1 )) were used to determine the LC50 concentration of purified Cry9Ca(R1 64K) protein (a Cry9Ca protein wherein the amino acid at position 1 64 (arginine) has been replaced by lysine to obtain improved stability to proteases).
  • the leaves were dipped in a serial solution of 0, 0.025, 0.1 , 0.39, 1 .56, 6.25, 25 and 100 microgram/ml Cry9C(R1 64K) trypsin- treated active toxin.
  • Leaf sheaths were dipped in a serial dilution of Cry9C(R163K) tryps in-treated active toxin. Five concentrations were tested, the negative control was: 0 microgram/ml Cry9Ca protein, all dilutions were made with sterile distilled water with 0.01 % Triton X- 100. After drying the leaf sheath was placed in a Petri dish and five neonate larvae were used per concentration. Mortality was scored after 96 hours. The bioassay was replicated ten times.
  • the LC50 values were calculated by the Probit- Least Square Method employing the Field Trial System/Agricultural Research Manager Software (ARM version 6.0.2; Gylling Data Management (GDM), 1999). An LC50 value of 6.44 microgram/ml was obtained (95 % confidence level fiducial limits: 5.74-7.23). Thus, the Cry9C protein was found to be highly toxic to the striped stem borer.
  • Example 2 Transformation of rice with a cry9C chimeric gene.
  • the vector system For transformation of rice plants, the vector system as described by Deblaere et al. (1985, 1987) was used.
  • the vector system consists of an Agrobacterium strain and two plasmid components: 1 ) an intermediate cloning vector, and 2) a non- oncogenic Ti-plasmid.
  • the intermediate cloning vector was essentially derived from pGSC1 700 (Cornelissen and Vandewiele, 1 989). It contained an artificial T-region consisting of the left and right border sequences of the TL-DNA from pTiB6S3 and multilinker cloning sites allowing the insertion of chimeric genes between the T-DNA border repeats.
  • the DNA inserted between the T-DNA border repeats encodes two genes, a chimeric bar gene and a chimeric cry9C gene.
  • the chimeric bar gene which confers resistance to glufosinate ammonium or phosphinothricin comprises the coding region from the phosphinothricin acetyl transferase gene of Streptomyces hygroscopicus (PCT patent publication WO 87/05629), under the control of the CaMV 35S promoter (Odell et al., 1985), and the CaMV 35S 3' transcript termination and polyadenylation region (Mogen et al., 1990).
  • the chimeric bar gene is useful as selectable marker and also confers herbicide resistance in the field to commercial formulations containing as active ingredient glufosinate ammonium.
  • the chimeric cry9C gene comprises the coding region of SEQ ID NO: 3, under the control of the CaMV 35S promoter (Odell et al., 1985), and flanked by the CaMV 35S 3' transcript termination and polyadenylation region (Mogen et al., 1990).
  • the acceptor Agrobacterium strain carries a non-oncogenic (disarmed) Ti plasmid from which the internal T-DNA has been deleted but the normal vir transfer genes have been retained.
  • the plasmid also has a region of homology that allows cointegrate formation with the intermediate cloning vector.
  • the intermediate vector is constructed in E. coli.
  • Agrobacterium-mediated gene transfer of the intermediate cloning vector resulted in transfer to the rice genome of the DNA fragment between the T-DNA border repeats.
  • target tissue for transformation immature embryo or callus derived thereof from japonica and indica rice cultivars were used which had been cut into small pieces, using the technique described in PCT patent publication WO 92/09696. After co-cultivation of this tissue with Agrobacterium, the transformed rice cells were selected by addition of glufosinate ammonium to the rice tissue culture medium.
  • cry9C genes were constructed for expression in rice, including different plant-expressible promoters such as the ubiquitin and gos2 promoter (Christensen et al., 1992, de Pater et al., 1992). These chimeric genes were also used to transform rice plants with the above Agrobacterium transformation protocol. Rice plants having a single copy of the cry9C chimeric gene integrated into their genome were selected by means of Southern blot. Expression levels of the Cry9C protein in leaf and stem material were analyzed using ELISA assays and Western blot. Seeds of successfully transformed plants were grown into plants which were tested for expression of the Cry9C protein and insect resistance. Following the above protocol, also indica rice plants were transformed with the chimeric Cry9C gene. Upon transfer to the greenhouse these plants were evaluated for transformation and insect resistance.
  • plant-expressible promoters such as the ubiquitin and gos2 promoter (Christensen et al., 1992, de Pater et al., 1992). These
  • crosses between the above Cry9C indica rice lines and indica rice plants transformed with a chimeric gene encoding a truncated Cryl Ab5 protein are made to improve the resistance of the plants to the insects and prevent or delay the development of insect resistance.
  • Chimeric genes encoding the truncated Cryl Ab5 protein comprising the ubiquitin or gos2 promoter as described above have been used to obtain the Cryl Ab5-expressing rice plants.
  • plant-expressible chimeric genes encoding an active fragment of the Cryl Ca4 protein are made to transform rice plants in accordance with the above description.
  • Example 3 Insect resistance of transformed Cry9C-expressing japonica type rice plants.
  • Four rice transformation events (ROS-01 2-2805, -2401 , -2702, -3703, having been confirmed as transformed plants expressing Cry9C protein) and one untransformed negative control, Chiyonishiki rice, were evaluated for Chilo suppressalis resistance in two greenhouse experiments. The events were sown in the greenhouse and when reaching the fourth leaf stage were sprayed with glufosinate ammonium to segregate non-transgenic and transgenic plants.
  • ROS1 2-2805, -2401 , -2702 showed a very low plant damage after the vegetative stage infestation with a maximum plant damage score of 0.25. All transgenic Cry9C rice plants showed a lower number of stems with tunnels than Chiyonishiki control plants.
  • ROS1 2-3703 was completely protected and showed no tunnels at all.
  • the transformed plants can also be useful to obtain resistance against other Lepidopteran rice pests such as rice skippers, Sesamia inferens, Spodoptera litura, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, or Parnara guttata.
  • Lepidopteran rice pests such as rice skippers, Sesamia inferens, Spodoptera litura, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, or Parnara guttata.
  • Example 4 Insect resistance of transformed Cry9C-expressing indica type rice plants.
  • Cnaphalocrocis medinalis resistance in a greenhouse experiment (results see in Table 1 ). The events were sown in the greenhouse, transplanted and when reaching the fourth leaf stage were sprayed with glufosinate ammonium to segregate non-transgenic and transgenic plants. Scirpophaga incertulas larvae were collected in the field. They developed to adults in the lab, and eggmasses were collected.
  • ROS045-01001 , ROS046-00401 , ROS048- 00207 showed a very low plant damage after the vegetative stage infestation with each event containing 1 -2 tillers showing deadheart among the total number tillers of all plants together. The other events were completely protected from insect damage by Scirpophaga incertulas.
  • Cnaphalocrocis medinalis Some minor damage is normal, since the insects need to ingest rice plant material in order to experience the toxicity of the Cry9C protein.
  • this invention is not limited to the above rice plants and the particular Cry9C protein used. Rather, the invention also includes any mutant or variant of the Cry9C protein retaining insecticidal activity or having improved insecticidal activity to Lepidopteran rice insect pests, such as a protein having substantially the amino acid sequence of the Cry9C protein and having substantially the insecticidal activity of the Cry9C protein (e.g., an insecticidal protein with an amino acid sequence identity of at least 90 %, particularly at least 95 %, to the protein of SEQ ID NO: 2 or 4 or insecticidally-effective fragments thereof, and which is insecticidal to Chilo suppressalis and Scirpophaga incertulas).
  • a protein having substantially the amino acid sequence of the Cry9C protein and having substantially the insecticidal activity of the Cry9C protein e.g., an insecticidal protein with an amino acid sequence identity of at least 90 %, particularly at least 95 %, to the protein of SEQ ID NO: 2
  • the invention is not limited to the above exemplified rice plants but includes any rice variety, hybrid, inbred or elite line.
  • any plant which is susceptible to damage by Lepidopteran rice stemborers, rice skippers, rice cutworms, rice armyworms, rice caseworms or rice leaffolders preferably an insect selected from the group consisting of: Chilo suppressalis, Scirpophaga incertulas, Sesamia inferens, Cnaphalocrocis medinalis, Hereitogramma licarisalis, Naranga aenescens, Mycalesis gotama, Marasmia patnalis, Marasmia exigua, Marasmia ruralis, Nymphula depunctalis, Scirpophaga innotata, Spodoptera litura, Chilo polychrysus, Rupela albinella, Diatraea saccharalis, Spodoptera frugiperda, Mythimna unipun.
  • plants which can also be used in accordance with the invention in a method for controlling such insects include but are not limited to other Graminea plants, including water oat, sugarcane, sorghum, wheat, millet, monophagous rice and Indian corn. Also in accordance with the invention a process is provided for protecting a plant other than rice from any of the above listed insects, comprising transforming the plants with a DNA encoding an Cry9C protein or applying the Cry9C protein to such plants.
  • This invention is not limited to the use of Agrobacterium tumefaciens Ti-plasmids for transforming rice cells with a DNA encoding an insecticidally effective Cry9C protein.
  • any method for transforming rice so that it expresses the Cry9C protein at sufficient levels can be used to develop in sect- resistant rice plants.

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Abstract

L'invention concerne de nouveaux plants de riz, et des méthodes de lutte contre les lépidoptères du riz (notamment le Lerodea eufala, le perce-tige ou hespérie, le vers gris, la chenille de Spodoptera mauritia, le Nymphula depunctalis), qui consistent à utiliser une protéine Cry9C contre ces insectes. Dans une forme de réalisation préférée, cette utilisation s'effectue par expression d'une protéine Cry9C dans des plants de riz.
PCT/EP2000/008877 1999-09-17 2000-09-07 Plants de riz resistants aux insectes WO2001021821A2 (fr)

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EP1937057A2 (fr) * 2005-09-09 2008-07-02 Board Of Supervisors Of Louisiana State University Cultivar de riz appele "cl131"
CN100424177C (zh) * 2005-06-17 2008-10-08 中国农业科学院生物技术研究所 融合杀虫基因cryci及其应用
US7541517B2 (en) 2003-12-22 2009-06-02 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis CRY9 nucleic acids
WO2010046422A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides de type auxine sur des plantes cultivées
WO2010046423A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides sulfonylurées sur des plantes cultivées
CN102288769A (zh) * 2011-05-10 2011-12-21 中国检验检疫科学研究院 一种检测Bt cry1 Ac蛋白的液相芯片及其应用
US20120324606A1 (en) * 2009-12-16 2012-12-20 Dow Agrosciences Llc Use of cry 1ab in combination with cry1be for management of resistant insects
US20130310543A1 (en) * 2008-06-25 2013-11-21 Athenix Corporation Toxin genes and methods for their use
WO2014053395A1 (fr) 2012-10-01 2014-04-10 Basf Se Utilisation de composés de n-thio-anthranilamide sur des plantes cultivées
WO2014079820A1 (fr) 2012-11-22 2014-05-30 Basf Se Utilisation de composés d'anthranilamides pour réduire les infections virales véhiculées par les insectes
EP3028573A1 (fr) 2014-12-05 2016-06-08 Basf Se Utilisation d'un triazole fongicide sur des plantes transgéniques
WO2016091674A1 (fr) 2014-12-12 2016-06-16 Basf Se Utilisation de cyclaniliprole sur des plantes cultivées
WO2016162371A1 (fr) 2015-04-07 2016-10-13 Basf Agrochemical Products B.V. Utilisation d'un composé de carboxamide insecticide contre les nuisibles sur des plantes cultivées
EP3338552A1 (fr) 2016-12-21 2018-06-27 Basf Se Utilisation d'un fongicide tetrazolinone sur des plantes transgéniques
US10017827B2 (en) 2007-04-04 2018-07-10 Nidera S.A. Herbicide-resistant sunflower plants with multiple herbicide resistant alleles of AHASL1 and methods of use
CN110452293A (zh) * 2019-08-21 2019-11-15 广东省农业科学院植物保护研究所 一种水稻抗虫害调控蛋白及其编码基因与应用
RU2745322C2 (ru) * 2014-12-12 2021-03-23 Зингента Партисипейшнс Аг Композиции и способы контроля вредителей растений

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UA108733C2 (uk) 2006-12-12 2015-06-10 Толерантна до гербіциду рослина соняшника
CN101880725B (zh) * 2010-07-19 2013-02-27 中国农业科学院植物保护研究所 转cry2A基因水稻品系T2A-1的PCR检测方法及其应用
CN102972243B (zh) * 2012-12-11 2017-05-17 北京大北农科技集团股份有限公司 控制害虫的方法
CN103233022B (zh) * 2013-04-17 2014-12-03 中国农业大学 一种苏云金芽孢杆菌晶体融合蛋白Bt Cry9C的制备方法
CN105219781A (zh) * 2013-04-19 2016-01-06 华中农业大学 抗虫转基因水稻T1c-19的培育方法
CN103718895B (zh) * 2013-11-18 2016-05-18 北京大北农科技集团股份有限公司 控制害虫的方法
CN103718896B (zh) * 2013-11-18 2016-02-10 北京大北农科技集团股份有限公司 控制害虫的方法
UA126966C2 (uk) * 2016-04-14 2023-03-01 Піонір Хай-Бред Інтернешнл, Інк. Інсектицидний поліпептид, що характеризується поліпшеним спектром активності, та його застосування

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US7541517B2 (en) 2003-12-22 2009-06-02 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis CRY9 nucleic acids
US7557080B2 (en) 2003-12-22 2009-07-07 Pioneer Hi-Bred International, Inc. Bacillus Cry9 family members
US7629504B2 (en) 2003-12-22 2009-12-08 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis cry9 nucleic acids
US7790846B2 (en) 2003-12-22 2010-09-07 Pioneer Hi-Bred International, Inc. Bacillus thuringiensis Cry9 toxins
CN100424177C (zh) * 2005-06-17 2008-10-08 中国农业科学院生物技术研究所 融合杀虫基因cryci及其应用
EP1937057A4 (fr) * 2005-09-09 2009-10-21 Univ Louisiana State Cultivar de riz appele "cl131"
EP1937057A2 (fr) * 2005-09-09 2008-07-02 Board Of Supervisors Of Louisiana State University Cultivar de riz appele "cl131"
US10017827B2 (en) 2007-04-04 2018-07-10 Nidera S.A. Herbicide-resistant sunflower plants with multiple herbicide resistant alleles of AHASL1 and methods of use
US20130310543A1 (en) * 2008-06-25 2013-11-21 Athenix Corporation Toxin genes and methods for their use
WO2010046422A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides de type auxine sur des plantes cultivées
WO2010046423A2 (fr) 2008-10-22 2010-04-29 Basf Se Utilisation d'herbicides sulfonylurées sur des plantes cultivées
US20120324606A1 (en) * 2009-12-16 2012-12-20 Dow Agrosciences Llc Use of cry 1ab in combination with cry1be for management of resistant insects
US9663795B2 (en) * 2009-12-16 2017-05-30 Dow Agrosciences Llc Use of Cry1Ab in combination with Cry1Be for management of resistant insects
CN102288769A (zh) * 2011-05-10 2011-12-21 中国检验检疫科学研究院 一种检测Bt cry1 Ac蛋白的液相芯片及其应用
WO2014053395A1 (fr) 2012-10-01 2014-04-10 Basf Se Utilisation de composés de n-thio-anthranilamide sur des plantes cultivées
WO2014079820A1 (fr) 2012-11-22 2014-05-30 Basf Se Utilisation de composés d'anthranilamides pour réduire les infections virales véhiculées par les insectes
EP3028573A1 (fr) 2014-12-05 2016-06-08 Basf Se Utilisation d'un triazole fongicide sur des plantes transgéniques
WO2016091674A1 (fr) 2014-12-12 2016-06-16 Basf Se Utilisation de cyclaniliprole sur des plantes cultivées
RU2745322C2 (ru) * 2014-12-12 2021-03-23 Зингента Партисипейшнс Аг Композиции и способы контроля вредителей растений
US11261459B2 (en) 2014-12-12 2022-03-01 Syngenta Participations Ag Compositions and methods for controlling plant pests
US11680272B2 (en) 2014-12-12 2023-06-20 Syngenta Partcipations Ag Compositions and methods for controlling plant pests
WO2016162371A1 (fr) 2015-04-07 2016-10-13 Basf Agrochemical Products B.V. Utilisation d'un composé de carboxamide insecticide contre les nuisibles sur des plantes cultivées
EP3338552A1 (fr) 2016-12-21 2018-06-27 Basf Se Utilisation d'un fongicide tetrazolinone sur des plantes transgéniques
CN110452293A (zh) * 2019-08-21 2019-11-15 广东省农业科学院植物保护研究所 一种水稻抗虫害调控蛋白及其编码基因与应用

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