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WO2001014562A1 - Toxines insecticides hybrides et sequences d'acide nucleique codant pour ces toxines - Google Patents

Toxines insecticides hybrides et sequences d'acide nucleique codant pour ces toxines Download PDF

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Publication number
WO2001014562A1
WO2001014562A1 PCT/EP2000/008042 EP0008042W WO0114562A1 WO 2001014562 A1 WO2001014562 A1 WO 2001014562A1 EP 0008042 W EP0008042 W EP 0008042W WO 0114562 A1 WO0114562 A1 WO 0114562A1
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Prior art keywords
toxin
seq
hybrid
cryl
nucleic acid
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Rudolf A. De Maagd
Hendrik Jan Bosch
Nadine Barbara Carozzi
Gregory Wayne Warren
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Syngenta Participations AG
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Syngenta Participations AG
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Priority to AU67023/00A priority Critical patent/AU6702300A/en
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Anticipated expiration legal-status Critical
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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • 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
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to novel hybrid insecticidal toxins derived from Bacillus thuringiensis insecticidal crystal proteins, nucleic acid sequences whose expression results in said toxins, and methods of making and methods of using the toxins and corresponding nucleic acid sequences to control insects.
  • Insect pests are a major cause of crop losses. Solely in the US, billions of dollars are lost every year due to infestation by various genera of insects. In addition to losses in field crops, insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and they are a nuisance to gardeners and home owners.
  • Insect pests are mainly controlled by intensive applications of chemical insecticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or death of the insects. Good insect control can thus be reached, but these chemicals can sometimes also affect other, beneficial insects.
  • Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect varieties. This has been partially alleviated by various resistance management strategies, but there is an increasing need for alternative pest control agents.
  • Bacillus thuringiensis strains expressing insecticidal toxins have also been applied with satisfactory results, offering an alternative or a complement to chemical insecticides.
  • Bacillus thuringiensis belongs to the large group of gram-positive, aerobic, endospore-forming bacteria. Unlike other very closely related species of Bacillus such as ⁇ . cereus or B. anthracis, the majority of the hitherto known Bacillus thuringiensis species produce in the course of their sporulation a parasporal inclusion body which, on account of its crystalline structure, is generally referred to also as a crystalline body. This crystalline body is composed of insecticidally active crystalline protoxin proteins, the so-called ⁇ -endotoxins.
  • the ⁇ -endotoxin does not exhibit its insecticidal activity until after oral intake of the crystalline body, when the latter is dissolved in the intestinal juice of the target insects. In most cases the actual toxic component is released from the protoxin as a result of proteolytic cleavage caused by the action of proteases from the digestive tract of the insects.
  • the ⁇ -endotoxins of the various Bacillus thuringiensis strains are characterized by high specificity with respect to certain target insects, especially with respect to various Lepidoptera, Coleoptera and Diptera larvae, and by a high degree of activity against these larvae.
  • a further advantage in using ⁇ -endotoxins of Bacillus thuringiensis resides in the fact that the toxins are harmless to humans, other mammals, birds and fish.
  • Bacillus thuringiensis crystal proteins have been categorized into different classes. Best studied are the Cry1 class of proteins, which are produced as 140 kDa pro-toxins and are active towards lepidopterans. To some extent the mode of action of crystal proteins has been elucidated. After oral uptake the crystals dissolve in the alkaline environment of the larval midgut. The solubilized proteins are subsequently processed by midgut proteinases to a proteinase- resistant toxic fragment of about 65 kDa that binds to receptors on epithelial cells of the insect midgut and penetrates the cell membrane. This eventually leads to bursting of the cells and death of the larvae.
  • the activity spectrum of a particular crystal protein is to a large extent determined by the occurrence of receptors on the midgut epithelial cells of susceptible insects.
  • the spectrum is co-determined by the efficiency of solubilization of the crystal protein and its proteolytic activation in vivo.
  • the importance of the binding of the crystal protein to midgut epithelial receptors is further demonstrated where insects have developed resistance to one of the crystal proteins in that the binding of crystal proteins to midgut epithelial cells in resistant insects is significantly reduced.
  • chimeric insecticidal proteins can be constructed having novel sequences not found in nature by combining the toxin portion from one ⁇ -endotoxin with the protoxin (tail) portion of a different ⁇ -endotoxin. See, for example, WO 98/15170, incorporated herein by reference.
  • Toxic fragments of crystal proteins are thought to be composed of three distinct structural domains. Domain I, the most N-terminal domain, consists of 7 a-helices and probably is partially or entirely inserted in the target cell membrane. Domain II comprises 3 ⁇ -sheets in a so-called Greek key-conformation. Domain II is believed by most researchers to interact with receptors and to thereby determine toxin specificity.
  • domain II residues in specific toxicity and in high affinity binding.
  • Domain III the most C-terminal domain, consists of two ⁇ -sheets in a so-called jellyroll conformation and has also been implicated in determining specificity. Swapping domain III between toxins, such as by in vivo recombination between the coding regions, can result in changes in specific activity. Binding experiments using such hybrids have shown that domain III is involved in binding to putative receptors of target insects, suggesting that domain III may exert its role in specificity through a role in receptor recognition. If projected on Cry1 sequences, domain I runs from about amino acid residue 28 to 260, domain II from about 260 to 460 and domain III from about 460 to 600.
  • H04 is reportedly highly toxic to Spodoptera exigua (beet armyworm) compared with the parental CrylAb toxin and significantly more toxic than the CrylC parental toxin. It has also been shown that substitution of domain III of toxins, which are inactive against the beet armyworm, Spodoptera exigua, such as Cryl E and CrylAb, by domain III of CrylC, which is active, can produce hybrid toxins that are active against this insect. These results have identified domain III of Cryl C as an important determinant of specificity for Spodoptera exigua.
  • control agents that are targeted to economically important insect pests such as Spodoptera exigua (beet armyworm), Manduca sexta (tobacco homworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm) and that efficiently control insect species resistant to existing insect control agents. Furthermore, agents whose application minimizes the burden on the environment are desirable.
  • the present invention thus provides: Nucleic acid molecules comprising:
  • nucleic acid molecule (b) a nucleotide sequence isocoding with the nucleotide sequence of (a); wherein expression of said nucleic acid molecule results in a hybrid insecticidal toxin that is active against insects.
  • nucleic acid molecules are provided wherein said nucleotide sequence is
  • nucleotides 1-1860 of SEQ ID NO:1 nucleotides 1-1965 of SEQ ID NO:3, or nucleotides 1-1965 of SEQ ID NO:5
  • nucleotides 1-1860 of SEQ ID NO:1 is substantially similar to nucleotides 1-1860 of SEQ ID NO:1 , nucleotides 1-1965 of SEQ ID NO:3, or nucleotides 1-1965 of SEQ ID NO:5
  • nucleotides 1-1860 of SEQ ID NO:1 nucleotides 1-1965 of SEQ ID NO:3, or nucleotides 1-1965 of SEQ ID NO:5
  • the invention further provides chimeric genes comprising a heterologous promoter sequence operatively linked to the nucleic acid molecules of the invention, as well as recombinant vectors and host cells comprising the chimeric gene, in particular, wherein the host cell is a
  • the invention further provides plants comprising the plant cell as mentioned hereinbefore, in particular, wherein said plant is maize.
  • the invention provides hybrid toxins produced by expression of a DNA molecule according to the invention, in particular, wherein said toxin
  • • is active against an insect selected from the group consisting of Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm)
  • compositions comprising an insecticidally effective amount of a hybrid toxin according to the invention are also provided.
  • nucleic acid molecule in said cell, which results in at least one hybrid toxin that is active against insects • methods of producing an insect-resistant plant, comprising introducing a nucleic acid molecule according to the invention into said plant, wherein said nucleic acid molecule is expressible in said plant in an effective amount to control an insect
  • the insect is selected from the group consisting of Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm)
  • a hybrid toxin comprising delivering to the insect an effective amount of a hybrid toxin according to the invention, in particular wherein the insect is selected from the group consisting of Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm)
  • Spodoptera exigua beet armyworm
  • Manduca sexta tobacco hornworm
  • Plutella xylostella diamondback moth
  • Ostrinia nubilalis European corn borer
  • Spodoptera frugiperda fall armyworm
  • Heliothis virescens tobacco budworm
  • the invention further provides hybrid insecticidal toxins, comprising: a) domains I and II from a Cryl F toxin joined in the amino to carboxy direction to domain III from a Cryl C toxin, wherein the juncture between the Cryl F toxin domains and the CrylC toxin domain corresponds to amino acids 446-454 of SEQ ID NO:2; b) domains I and II from a Cryl B toxin joined in the amino to carboxy direction to domain III from a CrylC toxin, wherein the juncture between the Cryl B toxin domains and the CrylC toxin domain corresponds to amino acids 482-488 of SEQ ID NO:4; or c) domains I and II from a Cryl B toxin joined in the amino to carboxy direction to domain III from a Cryl C toxin, wherein the juncture between the Cryl B toxin domains and the CrylC toxin domain corresponds to amino acids 491-494 of SEQ ID NO:
  • the invention further provides methods of controlling an insect comprising delivering to the insect an effective amount of a hybrid insecticidal toxin as mentioned hereinbefore, in particular, wherein the insect is selected from the group consisting of Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm).
  • the insect is selected from the group consisting of Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm).
  • the invention further provides DNA molecules comprising a nucleotide sequence that encodes a hybrid insecticidal toxin according to the invention.
  • the present invention addresses a long-standing need for novel insect control agents. Particularly needed are control agents that are targeted to economically important insect pests and that efficiently control insect strains resistant to existing insect control agents. Furthermore, agents whose application minimizes the burden on the environment are desirable.
  • the present invention addresses the aforementioned needs by providing novel gene sequences that encode hybrid Bacillus thuringiensis toxins, including synthetic nucleotide sequences optimized for expression in plants.
  • the present invention particularly concerns hybrids of Cryl toxins with (at least part of) domain III of Cryl Ca produced by a combination of cloning and in vivo recombination.
  • the hybrid Cryl toxins of the invention become more toxic to, for example, Spodoptera exigua, when domain III of Cryl C, e.g. Cryl Ca, replaces their native domain III.
  • Preferred Cryl toxins are Cryl F, e.g. Cryl Fa, and Cryl B, e.g. Cryl Ba.
  • the present invention provides nucleotide sequences whose expression results in hybrid insecticidal toxins that are highly toxic to economically important pests, particularly plant pests such as Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm).
  • plant pests such as Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm).
  • the invention is further drawn to the hybrid insecticidal toxins resulting from the expression of the nucleotide sequences, and to compositions and formulations containing the hybrid insecticidal toxins, which are capable of inhibiting the ability of insect pests to survive, grow or reproduce, or of limiting insect-related damage or loss in crop plants.
  • the invention is further drawn to a method of making the hybrid toxins and to methods of using the nucleotide sequences, for example in microorganisms to control insects or in transgenic plants to confer insect resistance, and to a method of using the toxins, and compositions and formulations comprising the toxins, for example applying the toxins, composition, or formulation to insect infested areas, or to prophylactically treat insect susceptible areas or plants to confer protection or resistance against harmful insects.
  • the hybrid toxins can be used in multiple insect control strategies, resulting in maximal efficiency with minimal impact on the environment.
  • the present invention provides a nucleic acid molecule comprising: (a) a nucleotide sequence substantially similar to nucleotides 1 -1860 of SEQ ID NO:1 , nucleotides 1-1965 of SEQ ID NO:3, or nucleotides 1-1965 of SEQ ID NO:5; or (b) a nucleotide sequence isocoding with the nucleotide sequence of (a); wherein expression of said nucleic acid molecule results in at least one toxin that is active against insects.
  • the nucleotide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 1-1860 of SEQ ID NO:1 , nucleotides 1-1965 of SEQ ID NO:3, or nucleotides 1-1965 of SEQ ID NO:5.
  • the nucleotide sequence encodes an amino acid sequence selected from the group consisting of amino acids 1-620 of SEQ ID NO:2, amino acids 1-655 of SEQ ID NO:4, and amino acids 1-655 of SEQ ID NO:6, or an amino acid sequence substantially similar thereto.
  • the nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6, or an amino acid sequence substantially similar thereto.
  • the nucleotide sequence comprises SEQ ID NO:1 , SEQ ID NO:3, or SEQ ID NO:5.
  • the toxins resulting from expression of the nucleic acid molecules of the invention have activity against an insect selected from the group consisting of Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm).
  • an insect selected from the group consisting of Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm).
  • the present invention also provides a chimeric gene comprising a heterologous promoter sequence operatively linked to a nucleic acid molecule of the invention. Further, the present invention provides a recombinant vector comprising such a chimeric gene. Still further, the present invention provides a host cell comprising such a chimeric gene.
  • a host cell according to this aspect of the invention may be a bacterial cell, a yeast cell, or a plant cell, preferably a plant cell. Even further, the present invention provides a plant comprising such a plant cell. Preferably, the plant is maize.
  • the present invention provides hybrid toxins produced by the expression of DNA molecules of the present invention.
  • the hybrid toxins of the invention have activity against Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm).
  • the present invention provides a hybrid insecticidal toxin comprising: a) domains I and II from a Cryl F toxin joined in the amino to carboxy direction to domain III from a CrylC toxin, wherein the juncture between the Cryl F toxin domains and the Cryl C toxin domain corresponds to amino acids 446-454 of SEQ ID NO:2; b) domains I and II from a Cryl B toxin joined in the amino to carboxy direction to domain III from a CrylC toxin, wherein the juncture between the Cryl B toxin domains and the CrylC toxin domain corresponds to amino acids 482-488 of SEQ ID NO:4; or c) domains I and II from a Cryl B toxin joined in the amino to carboxy direction to domain III from a CrylC toxin, wherein the juncture between the Cryl B toxin domains and the CrylC toxin domain corresponds to amino acids 491 -4
  • a hybrid insecticidal toxin of the invention comprises domains I and II from a Cryl F toxin and domain III from a CrylC toxin, wherein said hybrid insecticidal toxin comprises amino acids 441-459 of SEQ ID NO:2.
  • a hybrid insecticidal toxin according to this embodiment comprises domains I and II from Cryl Fa and domain III from CrylCa, wherein the juncture between the Cryl Fa toxin domains and the Cryl Ca toxin domain corresponds to amino acids 446-454 of SEQ ID NO:2.
  • a hybrid insecticidal toxin according to this embodiment comprises amino acids 1-620 of SEQ ID NO:2.
  • a hybrid insecticidal toxin of the invention comprises domains I and II from a CrylB toxin and domain III from a CrylC toxin, wherein said hybrid insecticidal toxin comprises amino acids amino acids 477-493 of SEQ ID NO:4.
  • a hybrid insecticidal toxin according to this embodiment comprises domains I and II from Cryl Ba and domain III from CrylCa, wherein the juncture between the Cryl Ba toxin domains and the CrylCa toxin domain corresponds to amino acids 482-488 of SEQ ID NO:4.
  • a hybrid insecticidal toxin according to this embodiment comprises amino acids 1-655 of SEQ ID NO:4.
  • a hybrid insecticidal toxin of the invention comprises domains I and II from a Cryl B toxin and domain III from a CrylC toxin, wherein said hybrid insecticidal toxin comprises amino acids 486-499 of SEQ ID NO:6.
  • a hybrid insecticidal toxin according to this embodiment comprises domains I and II from Cryl Ba and domain III from CrylCa, wherein the juncture between the Cryl Ba toxin domains and the Cryl Ca toxin domain corresponds to amino acids 491-494 of SEQ ID NO:6.
  • a hybrid insecticidal toxin according to this embodiment comprises amino acids 1-655 of SEQ ID NO:6.
  • a hybrid toxin of the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:2, 4, and 6 or an amino acid sequence substantially similar thereto.
  • Another aspect of the present invention is the provision of a DNA molecule comprising a nucleotide sequence that encodes any embodiment of a hybrid insecticidal toxin according to the invention.
  • the present invention also provides a composition comprising an insecticidally effective amount of a hybrid toxin according to the invention.
  • the present invention provides a method of producing a hybrid toxin that is active against insects, comprising: (a) obtaining a host cell comprising a chimeric gene, which itself comprises a heterologous promoter sequence operatively linked to the nucleic acid molecule of the invention; and (b) expressing the nucleic acid molecule in the cell, which results in a hybrid toxin that is active against insects.
  • the present invention provides a method of producing an insect- resistant plant, comprising introducing a nucleic acid molecule of the invention into the plant, wherein the nucleic acid molecule is expressible in the plant in an effective amount to control insects.
  • the insects are Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm).
  • the present invention provides a method of controlling insects comprising delivering to the insects an effective amount of a hybrid toxin according to the present invention.
  • the insects are Spodoptera exigua (beet armyworm), Manduca sexta (tobacco hornworm), Plutella xylostella (diamondback moth), Ostrinia nubilalis (European corn borer), Spodoptera frugiperda (fall armyworm), and Heliothis virescens (tobacco budworm).
  • the hybrid toxin is delivered to the insects orally.
  • SEQ ID NO:1 shows the nucleotide sequence encoding a Cry1 Fa/Cry1Ca (FFC1) hybrid toxin according to the invention.
  • SEQ ID NO:2 shows the amino acid sequence of the FFC1 hybrid toxin encoded by the nucleotide sequence depicted in SEQ ID NO:1.
  • SEQ ID NO:3 shows the nucleotide sequence encoding a Cry1 Ba/Cry1Ca (BBC13) hybrid toxin according to the invention.
  • SEQ ID NO:4 shows the amino acid sequence of the BBC13 hybrid toxin encoded by the nucleotide sequence depicted in SEQ ID NO:3.
  • SEQ ID NO:5 shows the nucleotide sequence encoding a Cry1Ba/Cry1Ca (BBC15) hybrid toxin according to the invention.
  • SEQ ID NO:6 shows the amino acid sequence of the BBC15 hybrid toxin encoded by the nucleotide sequence depicted in SEQ ID NO:5.
  • activity of the toxins of the invention is meant that the toxins function as orally active insect control agents, have a toxic effect, or are able to disrupt or deter insect feeding, which may or may not cause death of the insect.
  • a toxin of the invention is delivered to the insect, the result is typically death of the insect, or the insect does not feed upon the source that makes the toxin available to the insect.
  • Associated with / operatively linked refer to two nucleic acid sequences that are related physically or functionally.
  • a promoter or regulatory DNA sequence is said to be “associated with” a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
  • Binding site means a site on a molecule wherein the binding between site and toxin is reversible such that the Ka between site and toxin is on the order of at least 10 4 dm 3 mole "1 .
  • a “chimeric gene” is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA or which is expressed as a protein, such that the regulator nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid sequence.
  • the regulator nucleic acid sequence of the chimeric gene is not normally operatively linked to the associated nucleic acid sequence as found in nature.
  • a "coding sequence” is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.
  • Complementary refers to two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences.
  • Consatively modified variations of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein.
  • nucleic acid variations are "silent variations" which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein which encodes a protein also describes every possible silent variation, except where otherwise noted.
  • each codon in a nucleic acid except ATG, which is ordinarily the only codon for methionine
  • each "silent variation" of a nucleic acid which encodes a protein is implicit in each described sequence.
  • control insects means to inhibit, through a toxic effect, the ability of insect pests to survive, grow, feed, and/or reproduce, or to limit insect-related damage or loss in crop plants.
  • To "control” insects may or may not mean killing the insects, although it preferably means killing the insects.
  • nucleic acid coding sequences or amino acid sequences of different ⁇ -endotoxins of Bacillus thuringiensis are aligned with each other, the nucleic or amino acids that "correspond to" certain enumerated positions are those that align with these positions but that are not necessarily in these exact numerical positions relative to the particular ⁇ -endotoxin's respective nucleic acid coding sequence or amino acid sequence.
  • the nucleic acids or amino acids in the Cryl B sequence that "correspond to" certain enumerated positions of the CrylAb sequence are those that align with these positions of the CrylAb sequence, but are not necessarily in these exact numerical positions of the Cryl B toxin's respective nucleic acid coding sequence or amino acid sequence.
  • a toxin means that the toxin comes in contact with an insect, resulting in toxic effect and control of the insect.
  • the toxin can be delivered in many recognized ways, e.g., orally by ingestion by the insect or by contact with the insect via transgenic plant expression, formulated protein composition(s), sprayable protein composition(s), a bait matrix, or any other art-recognized toxin delivery system.
  • “Expression cassette” as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue, or organ, or stage of development.
  • a “gene” is a defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion.
  • a gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.
  • Gene of interest refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capability.
  • the "gene of ausf may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant.
  • Heterologous nucleic acid sequence The terms “heterologous nucleic acid [or DNA] sequence”, “exogenous nucleic acid [or DNA] segment” or “heterologous gene,” as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of codon optimization. The terms also includes non-naturally occurring multiple copies of a naturally occurring sequence.
  • the terms refer to a nucleic acid segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous nucleic acid segments are expressed to yield exogenous polypeptides.
  • a "homologous" nucleic acid [or DNA] sequence is a nucleic acid [or DNA] sequence naturally associated with a host cell into which it is introduced.
  • Homologous recombination is the reciprocal exchange of nucleic acid fragments between homologous nucleic acid molecules.
  • Homoplastidic refers to a plant, plant tissue or plant cell wherein all of the plastids are genetically identical. This is the normal state in a plant when the plastids have not been transformed, mutated, or otherwise genetically altered. In different tissues or stages of development, the plastids may take different forms, e.g., chloroplasts, proplastids, etioplasts, amyloplasts, chromoplasts, and so forth.
  • nucleic acid or protein sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below or by visual inspection.
  • “Insecticidal” is defined as a toxic biological activity capable of controlling insects, preferably by killing them.
  • a nucleic acid sequence is "isocoding with" a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.
  • nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.
  • a "juncture" between toxin domains in a hybrid toxin, i.e., between domains II and III of a hybrid insecticidal toxin according to the invention, is the homologous crossover region or site in the hybrid. Amino acids to the left of the crossover site are from one parental toxin, whereas amino acids to the right of the crossover site are from the other parental toxin.
  • Mature Protein protein that is normally targeted to a cellular organelle and from which the transit peptide has been removed.
  • Minimal Promoter promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.
  • Native refers to a gene that is present in the genome of an untransformed cell.
  • Naturally occurring is used to describe an object that can be found in nature as distinct from being artificially produced by man.
  • a protein or nucleotide sequence present in an organism which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally occurring.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ai, Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et ai, J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et ai., Mol. Cell. Probes 8: 91 -98 (1994)).
  • the terms "nucleic acid” or “nucleic acid sequence” may also be used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • part of a protein is meant a peptide comprised by said protein and having at least 80% of the consecutive sequence thereof.
  • a "plant” is any plant at any stage of development, particularly a seed plant.
  • a "plant cell” is a structural and physiological unit of a plant, comprising a protoplast and a cell wall.
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
  • Plant cell culture means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
  • Plant material refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
  • a "plant organ” is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
  • Plant tissue as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
  • a “promoter” is an untranslated DNA sequence upstream of the coding region that contains the binding site for RNA polymerase II and initiates transcription of the DNA.
  • the promoter region may also include other elements that act as regulators of gene expression.
  • a "protoplast” is an isolated plant cell without a cell wall or with only parts of the cell wall.
  • purified when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as poiyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified.
  • purified denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.
  • Two nucleic acids are “recombined” when sequences from each of the two nucleic acids are combined in a progeny nucleic acid.
  • Two sequences are “directly” recombined when both of the nucleic acids are substrates for recombination.
  • Two sequences are "indirectly recombined” when the sequences are recombined using an intermediate such as a cross-over oligonucleotide.
  • no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination.
  • Regulatory elements refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
  • substantially identical in the context of two nucleic acid or protein sequences, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90, even more preferably 95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • substantially identical nucleic acid or protein sequences perform substantially the same function.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981 ), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by visual inspection (see generally, Ausubel et ai., infra).
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01 , and most preferably less than about 0.001.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Bod(s) substantially refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
  • Stringent hybridization conditions and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize to its target subsequence, but to no other sequences.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42 C C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.1 5M NaCI at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1x SSC at 45°C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 1X SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodec
  • nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the protein encoded by the second nucleic acid.
  • a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised to the protein with the amino acid sequence encoded by any of the nucleic acid sequences of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins except for polymorphic variants.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York "Harlow and Lane"), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • a “subsequence” refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., protein) respectively.
  • Synthetic refers to a nucleotide sequence comprising structural characters that are not present in the natural sequence. For example, an artificial sequence that resembles more closely the G+C content and the normal codon distribution of dicot and/or monocot genes is said to be synthetic.
  • Transformation is a process for introducing heterologous nucleic acid into a host cell or organism.
  • transformation means the stable integration of a DNA molecule into the genome of an organism of interest.
  • Transformed / transgenic / recombinant refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • non-transformed refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
  • Nucleotides are indicated by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G).
  • Amino acids are likewise indicated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y),
  • Novel Nucleic Acid Sequences Encoding Hybrid Insecticidal Toxins This invention relates to nucleic acid sequences whose expression results in novel toxins, and to the making and using of the toxins to control insect pests.
  • the present invention concerns hybrids of Cryl toxins with (at least part of) domain III of CrylCa produced by a combination of cloning and in vivo recombination.
  • the hybrid Cryl toxins of the invention become more toxic to, for example, Spodoptera exigua, when domain III of CrylCa replaces their native domain III.
  • Preferred Cryl toxins are Cryl Fa and Cryl Ba.
  • a Cryl Fa/Cry1 Ca domain Ill-hybrid is constructed by replacing the domains I and II encoding fragment of the cty 7/ cty 7 Ca-hybrid gene H04 (SEQ ID NO: 7 of US Pat. No. 5,736,131) by the homologous fragment from crylFa.
  • Cryl Fb see, e.g., Lambert, B., Genbank accession Z22512 (1993), incorporated herein by reference
  • CrylCb see, e.g., Kalman, S.S., Appl. Environ. Microbiol. 59: 1 131-1137 (1993), incorporated herein by reference
  • Cryl Fa may be used instead of Cryl Fa and CrylCa to construct similar FFC hybrids.
  • FFC hybrids constructed according to the present invention preferably comprise domains I and II from a Cryl F toxin joined in the amino to carboxy direction to domain III from a CrylC toxin, wherein the juncture between the Cryl F toxin domains and the Cryl C toxin domain corresponds to amino acids 446-454 of SEQ ID NO:2. More preferably, FFC hybrids constructed according to the present invention comprise amino acids 441-459 of SEQ ID NO:2. Still more preferably, FFC hybrids according to the invention comprise amino acids 1-620 of SEQ ID NO:2. Most preferably, FFC hybrid protoxins according to the invention comprise SEQ ID NO:2.
  • cryl Ba-cry1 Ca tandem plasmid which contains the toxin encoding part (bases 1-1853) of a cryWa gene (see, e.g., Brizzard and Whiteley, Nucleic Acids Res. 16(6): 2723-2724 (1988), incorporated herein by reference) followed by a polylinker, and most (bases 220 to 3570) of a crylCa gene (see, e.g., Honee, et al., Nucleic Acids Res. 16(13): 6240 (1988)).
  • Recombination in this plasmid is selected for by digestion with ⁇ /ofl and Ssu36l, which have a unique site in the polylinker, and in the crylCa gene at position 1242, respectively.
  • Ssu36l which cuts in the 3' end of the domain II encoding part of the crylCa gene, recombination events can be effectively selected for behind this position, i.e. in or close to domain III.
  • Digested DNA is retransformed to E. coli XL-1. Restriction analysis of transformants confirms that most of them represent recombination events in the targeted area.
  • Several recombinants produce soluble protoxins. Sequencing subsequently shows that these have different crossover sites at the approximate border between domain II and domain III.
  • Recombinants BBC13 and BBC15, which produce a soluble protoxins with different crossover sites, are selected for toxicity studies.
  • Cryl Ba shows no activity against either S. exigua or M. sexta, either as a truncated toxin or full-length protoxin. Trypsin treatment of Cryl Ba and Cry1 Ba/Cry1Ca- hybrid toxins results in production of only a stable product of approximately 55 kDa in size, as opposed to the expected size of approximately 65 kDa for an activated toxin. This is probably due to trypsin cleavage within domain I, between alpha helices 3 and 4, as reported earlier for Cryl B, as well as for Cry3A (Carroll, et al., J. Invertebr. Pathol. 70: 41 -49 (1997)).
  • a 65 kDa intermediary product is frequently observed during trypsin treatment, but can not be purified in large quantities as a stable product.
  • trypsinization of Cryl Ba has been shown to reduce its activity against Lepidopterans (Bradley, et al., J. Invertebr. Pathol. 65: 162-173 (1995); H ⁇ fte, et ai., Appl. Environ. Microbiol. 54: 2010-2017 (1988)).
  • the purified 55 kDa protein fractions from Cryl Ba and the Cn/1 Ba/Cry1 Ca-hybrids show no or little toxicity to either S. exigua or to M.
  • Cryl Bb (see, e.g., Donovan, W.P., genbank accession L32020 (1994), incorporated herein by reference), Cryl Bc (see, e.g., Bishop, et al., genbank accession Z46442 (1994), incorporated herein by reference), and/or Cryl Cb (see, e.g., Kalman, S.S., Appl. Environ. Microbiol. 59: 1131-1137 (1993)) may be used instead of Cryl Ba and Cryl Ca to construct similar BBC hybrids.
  • BBC hybrids constructed according to the present invention preferably comprise domains I and II from a Cryl B toxin joined in the amino to carboxy direction to domain III from a CrylC toxin, wherein the juncture between the Cryl B toxin domains and the Cryl C toxin domain corresponds to amino acids 482-488 of SEQ ID NO:4 or amino acids 491-494 of SEQ ID NO:6. More preferably, BBC hybrids constructed according to the present invention comprise amino acids 477-493 of SEQ ID NO:4 or amino acids 486-499 of SEQ ID NO:6. Still more preferably, BBC hybrids according to the invention comprise amino acids 1-655 of SEQ ID NO:4 or amino acids 1- 655 of SEQ ID NO:6. Most preferably, BBC hybrid protoxins according to the invention comprise SEQ ID NO:4 or SEQ ID NO:6.
  • the present invention encompasses DNA molecules comprising nucleotide sequences that encode the hybrid insecticidal toxins of the invention.
  • the present invention further encompasses recombinant vectors comprising the nucleic acid sequences of this invention.
  • the nucleic acid sequences are preferably comprised in expression cassettes comprising regulatory elements for expression of the nucleotide sequences in a host cell capable of expressing the nucleotide sequences.
  • regulatory elements usually comprise promoter and termination signals and preferably also comprise elements allowing efficient translation of polypeptides encoded by the nucleic acid sequences of the present invention.
  • Vectors comprising the nucleic acid sequences are usually capable of replication in particular host cells, preferably as extrachromosomal molecules, and are therefore used to amplify the nucleic acid sequences of this invention in the host cells.
  • host cells for such vectors are microorganisms, such as bacteria, in particular Bacillus thuringiensis or E. coli.
  • host cells for such recombinant vectors are endophytes or epiphytes.
  • a preferred host cell for such vectors is a eukaryotic cell, such as a yeast, a plant cell, or an insect cell. Plant cells such as maize cells are most preferred host cells.
  • such vectors are viral vectors and are used for replication of the nucleotide sequences in particular host cells, e.g. insect cells or plant cells.
  • Recombinant vectors are also used for transformation of the nucleotide sequences of this invention into host cells, whereby the nucleotide sequences are stably integrated into the DNA of such host cells.
  • host cells are prokaryotic cells.
  • host cells are eukaryotic cells, such as yeast cells, insect cells, or plant cells.
  • the host cells are plant cells, such as maize cells.
  • the insecticidal toxins are produced by expression of the nucleotide sequences in heterologous host cells capable of expressing the nucleotide sequences.
  • at least one of the nucleotide sequences of the invention is inserted into an appropriate expression cassette, comprising a promoter and termination signals. Expression of the nucleotide sequence is constitutive, or an inducible promoter responding to various types of stimuli to initiate transcription is used.
  • the cell in which the toxin is expressed is a microorganism, such as a virus, a bacteria, or a fungus.
  • a virus such as a baculovirus
  • a virus contains a nucleotide sequence of the invention in its genome and expresses large amounts of the corresponding insecticidal toxin after infection of appropriate eukaryotic cells that are suitable for virus replication and expression of the nucleotide sequence.
  • the insecticidal toxin thus produced is used as an insecticidal agent.
  • baculoviruses engineered to include the nucleotide sequence are used to infect insects in-vivo and kill them either by expression of the insecticidal toxin or by a combination of viral infection and expression of the insecticidal toxin.
  • Bacterial cells are also hosts for the expression of the nucleotide sequences of the invention.
  • non-pathogenic symbiotic bacteria which are able to live and replicate within plant tissues, so-called endophytes, or non-pathogenic symbiotic bacteria, which are capable of colonizing the phyllosphere or the rhizosphere, so-called epiphytes, are used.
  • Such bacteria include bacteria of the genera Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia, Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas.
  • Symbiotic fungi such as Trichoderma and Gliocladium are also possible hosts for expression of the inventive nucleotide sequences for the same purpose.
  • the expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in E. coll, either in transcriptional or translational fusion, behind the tac or trc promoter.
  • the simplest procedure is to insert the operon into a vector such as pKK223- 3 in transcriptional fusion, allowing the cognate ribosome binding site of the heterologous genes to be used.
  • Techniques for overexpression in gram-positive species such as Bacillus are also known in the art and can be used in the context of this invention (Quax et al.
  • At least one of the described nucleotide sequences is transferred to and expressed in Pseudomonas fluorescens strain CGA267356 (described in the published application EP 0 472 494 and in WO 94/01561) which has biocontrol characteristics.
  • a nucleotide sequence of the invention is transferred to Pseudomonas aureofaciens strain 30-84 which also has biocontrol characteristics. Expression in heterologous biocontrol strains requires the selection of vectors appropriate for replication in the chosen host and a suitable choice of promoter. Techniques are well known in the art for expression in gram-negative and gram- positive bacteria and fungi.
  • At least one of the insecticidal toxins of the invention is expressed in a higher organism, e.g., a plant.
  • transgenic plants expressing effective amounts of the toxins protect themselves from insect pests.
  • the insect starts feeding on such a transgenic plant, it also ingests the expressed toxins. This will deter the insect from further biting into the plant tissue or may even harm or kill the insect.
  • a nucleotide sequence of the present invention is inserted into an expression cassette, which is then preferably stably integrated in the genome of said plant.
  • the nucleotide sequence is included in a non-pathogenic self- replicating virus.
  • Plants transformed in accordance with the present invention may be monocots or dicots and include, but are not limited to, maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugarbeet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous and deciduous trees.
  • nucleotide sequence Once a desired nucleotide sequence has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.
  • a nucleotide sequence of this invention is preferably expressed in transgenic plants, thus causing the biosynthesis of the corresponding toxin in the transgenic plants. In this way, transgenic plants with enhanced resistance to insects are generated.
  • the nucleotide sequences of the invention may require modification and optimization. Although in many cases genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants may result from microbial nucleotide sequences having codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in this invention can be changed to conform with plant preferences, while maintaining the amino acids encoded thereby.
  • coding sequences that have at least 35% about GC content, preferably more than about 45%, more preferably more than about 50%, and most preferably more than about 60%.
  • Microbial nucleotide sequences which have low GC contents may express poorly in plants due to the existence of ATTTA motifs which may destabilize messages, and AATAAA motifs which may cause inappropriate polyadenylation.
  • preferred gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res.
  • nucleotide sequences are screened for the existence of illegitimate splice sites that may cause message truncation. All changes required to be made within the nucleotide sequences such as those described above are made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods described in the published patent applications EP 0 385 962 (to Monsanto), EP 0 359 472 (to Lubrizol, and WO 93/07278 (to Ciba-Geigy).
  • sequences adjacent to the initiating methionine may require modification.
  • they can be modified by the inclusion of sequences known to be effective in plants.
  • Joshi has suggested an appropriate consensus for plants (NAR 15: 6643-6653 (1987)) and Clontech suggests a further consensus translation initiator (1993/1994 catalog, page 210).
  • These consensuses are suitable for use with the nucleotide sequences of this invention.
  • the sequences are incorporated into constructions comprising the nucleotide sequences, up to and including the ATG (whilst leaving the second amino acid unmodified), or alternatively up to and including the GTC subsequent to the ATG (with the possibility of modifying the second amino acid of the transgene).
  • expression of the nucleotide sequences in transgenic plants is driven by promoters shown to be functional in plants.
  • the choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species.
  • expression of the nucleotide sequences of this invention in leaves, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/or seedlings is preferred.
  • inflorescences e.g. spikes, panicles, cobs, etc.
  • promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons.
  • Preferred promoters that are expressed constitutively include promoters from genes encoding actin or ubiquitin and the CaMV 35S and 19S promoters.
  • the nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated.
  • a preferred category of promoters is that which is wound inducible. Numerous promoters have been described which are expressed at wound sites and also at the sites of phytopathogen infection. Ideally, such a promoter should only be active locally at the sites of infection, and in this way the insecticidal toxins only accumulate in cells which need to synthesize the insecticidal toxins to kill the invading insect pest.
  • Preferred promoters of this kind include those described by Stanford et al., Mol. Gen. Genet. 215: 200-208 (1989), Xu et ai., Plant Molec. Biol.
  • Preferred tissue specific expression patterns include green tissue specific, root specific, stem specific, and flower specific. Promoters suitable for expression in green tissue include many which regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons.
  • a preferred promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)).
  • a preferred promoter for root specific expression is the maize metallothionein-like (MTL) promoter described by de Framond (FEBS 290: 103-106 (1991); EP 0 452 269 to Ciba-Geigy).
  • a preferred stem specific promoter is that described in US patent 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene.
  • transgenic plants expressing at least one of the nucleotide sequences of the invention in a root-preferred or root-specific fashion. Further preferred embodiments are transgenic plants expressing the nucleotide sequences in a wound-inducible or pathogen infection-inducible manner.
  • constructions for expression of an insecticidal toxin in plants require an appropriate transcription terminator to be attached downstream of the heterologous nucleotide sequence.
  • an appropriate transcription terminator e.g. tm1 from CaMV, E9 from rbcS. Any available terminator known to function in plants can be used in the context of this invention.
  • sequences which have been shown to enhance expression such as intron sequences (e.g. from Adh1 and bronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).
  • intron sequences e.g. from Adh1 and bronzel
  • viral leader sequences e.g. from TMV, MCMV and AMV.
  • nucleotide sequences of the present invention may be target expression of different cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle may be preferred. Subcellular localization of transgene encoded enzymes is undertaken using techniques well known in the art. Typically, the DNA encoding the target peptide from a known organelle-targeted gene product is manipulated and fused upstream of the nucleotide sequence. Many such target sequences are known for the chloroplast and their functioning in heterologous constructions has been shown. The expression of the nucleotide sequences of the present invention is also targeted to the endoplasmic reticulum or to the vacuoles of the host cells. Techniques to achieve this are well-known in the art.
  • Vectors suitable for plant transformation are described elsewhere in this specification.
  • binary vectors or vectors carrying at least one T-DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construction of interest may be preferred.
  • direct gene transfer transformation with a single DNA species or co-transformation can be used (Schocher et al. Biotechnology 4: 1093-1096 (1986)).
  • ⁇ gro__>acter/ut77-mediated transfer transformation is usually (but not necessarily) undertaken with a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (basta).
  • markers are neomycin phosphotransferase, hygromycin phosphotransferase, dihydrofolate reductase, phosphinothricin acetyltransferase, 2, 2-dichloroproprionic acid dehalogenase, acetohydroxyacid synthase, 5-enolpyruvyl-shikimate-phosphate synthase, haloarylnitrilase, protoporhyrinogen oxidase, acetyl-coenzyme A carboxylase, dihydropteroate synthase, chloramphenicol acetyl transferase, and ⁇ -glucuronidase.
  • MPI/PMI mannose-6-phosphate isomerase
  • the recombinant DNA described above can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al., BioTechniques 4:320-334 (1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci.
  • a particularly preferred set of embodiments for the introduction of recombinant DNA molecules into maize by microprojectile bombardment can be found in Koziel et al., Biotechnology 11: 194-200 (1993), Hill et al., Euphytica 85:119-123 (1995) and Koziel et al., Annals of the New York Academy of Sciences 792:164-171 (1996).
  • An additional preferred embodiment is the protoplast transformation method for maize as disclosed in EP 0 292 435. Transformation of plants can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with a coding sequence of the invention.
  • a nucleotide sequence of the present invention is directly transformed into the plastid genome.
  • a major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification, and plastids are capable of expressing multiple open reading frames under control of a single promoter. Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451 ,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride etai. (1994) Proc. Natl. Acad. Sci. USA 91 , 7301-7305.
  • the basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation).
  • a suitable target tissue e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation).
  • the 1 to 1.5 kb flanking regions termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome.
  • a nucleotide sequence of the present invention is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence.
  • compositions comprising at least one of the insecticidal toxins of the present invention.
  • such compositions preferably contain sufficient amounts of toxin. Such amounts vary depending on the crop to be protected, on the particular pest to be targeted, and on the environmental conditions, such as humidity, temperature or type of soil.
  • compositions comprising the insecticidal toxins comprise host cells expressing the toxins without additional purification.
  • the cells expressing the insecticidal toxins are lyophilized prior to their use as an insecticidal agent.
  • the insecticidal toxins are engineered to be secreted from the host cells. In cases where purification of the toxins from the host cells in which they are expressed is desired, various degrees of purification of the insecticidal toxins are reached.
  • the present invention further embraces the preparation of compositions comprising at least one insecticidal toxin of the present invention, which is homogeneously mixed with one or more compounds or groups of compounds described herein.
  • the present invention also relates to methods of treating plants, which comprise application of the insecticidal toxins or compositions containing the insecticidal toxins, to plants.
  • the insecticidal toxins can be applied to the crop area in the form of compositions or plant to be treated, simultaneously or in succession, with further compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth.
  • Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.
  • a preferred method of applying insecticidal toxins of the present invention is by spraying to the environment hosting the insect pest like the soil, water, or foliage of plants.
  • the number of applications and the rate of application depend on the type and intensity of infestation by the insect pest.
  • the insecticidal toxins can also penetrate the plant through the roots via the soil (systemic action) by impregnating the locus of the plant with a liquid composition, or by applying the compounds in solid form to the soil, e.g. in granular form (soil application).
  • the insecticidal toxins may also be applied to seeds (coating) by impregnating the seeds either with a liquid formulation containing insecticidal toxins, or coating them with a solid formulation. In special cases, further types of application are also possible, for example, selective treatment of the plant stems or buds.
  • the insecticidal toxins can also be provided as bait located above or below the ground.
  • insecticidal toxins are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances.
  • the methods of application such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.
  • compositions or preparations containing the insecticidal toxins and, where appropriate, a solid or liquid adjuvant are prepared in known manner, for example by homogeneously mixing and/or grinding the insecticidal toxins with extenders, for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).
  • extenders for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).
  • Suitable solvents include aromatic hydrocarbons, preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl formamide, as well as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil or water.
  • aromatic hydrocarbons preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate,
  • the solid carriers used e.g. for dusts and dispersible powders are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite.
  • Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand.
  • a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.
  • Suitable surface-active compounds are nonionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties.
  • surfactants will also be understood as comprising mixtures of surfactants.
  • Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds.
  • Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon atoms), for example the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained for example from coconut oil or tallow oil.
  • the fatty acid methyltaurin salts may also be used.
  • so-called synthetic surfactants are used, especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.
  • the fatty sulfonates or sulfates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and have a 8 to 22 carbon alkyl radical which also includes the alkyl moiety of alkyl radicals, for example, the sodium or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture of fatty alcohol sulfates obtained from natural fatty acids.
  • These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts.
  • the sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 8 to 22 carbon atoms.
  • alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnapthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product.
  • corresponding phosphates e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.
  • Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.
  • non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit.
  • non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol.
  • Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.
  • Cationic surfactants are preferably quaternary ammonium salts which have, as N- substituent, at least one C8-C22 alkyl radical and, as further substituents, lower unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals.
  • the salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium bromide.
  • Plasmid pRM8, encoding Cryl Ab/Cry1 Ca-hybrid H04, is constructed as described in de Maagd et al., Appl. Environ. Microbiol. 62: 1537-1543 (1996), incorporated herein by reference. Wild-type protoxins described herein are cloned in an E. coli expression vector based on pBD10, a derivative of pKK233-2 (Bosch, et al., Bio/technology 12:915-918 (1994), incorporated herein by reference). All contain full-length protoxin encoding genes with a ⁇ /col-site at the start.
  • pBD140 containing crylAb
  • pBD150 containing crylCa
  • pBD160 containing crylEa
  • Cryl Fa For production of Cryl Fa, a Dra ⁇ -Kpn ⁇ (base pairs 44-2153) fragment of crylEa in pBD160 is replaced by the corresponding fragment of crylFa (see, e.g., Chambers et ai., Journal of Bacteriology 173: 3966-3976 (1991), incorporated herein by reference), resulting in Cryl Fa expression vector pMH21.
  • pMH23 containing a cry 1 Fa/cry 1 Ca-hyb ⁇ d gene, is constructed by replacing the Nco ⁇ -Sac ⁇ fragment of crylAb (bases 1-1348) present in hybrid H04 encoding plasmid pRM8, by the corresponding fragment from crylFa (bases 1-1327). This results in a hybrid gene encoding protoxin FFC1, containing domains I and II from Cryl Fa and domain III from Cryl Ca.
  • the nucleotide sequence encoding the Cry1 Fa/Cry1Ca (FFC1) hybrid toxin is depicted in SEQ ID NO:1.
  • SEQ ID NO:2 shows the amino acid sequence of the FFC1 hybrid toxin encoded by the nucleotide sequence depicted in SEQ ID NO:1.
  • the homologous crossover region of FFC1 between domain II from Cryl Fa and domain III from Cryl Ca is located at amino acids 446-454 of SEQ ID NO:2.
  • Example 2 Cryl Ba/Cry1 Ca Hybrids cry1Ba/cry1Ca- ⁇ andem plasmid pMH22 is made by replacing the ⁇ /col-Sa l fragment containing crylEa in crylaE/crylCa tandem plasmid pBD650 (Bosch, et al., Bio/technology 12:915-918 (1994)) by a corresponding Nco ⁇ -Stu ⁇ fragment (bases 1-1853) from cryl Ba (see, e.g., Brizzard and Whiteley, Nucleic Acids Research 16: 2723-2724 (1988), incorporated herein by reference), and a synthetic Stu ⁇ -Sac ⁇ linker fragment.
  • a synthetic Stu ⁇ -Sac ⁇ linker fragment See, e.g., Brizzard and Whiteley, Nucleic Acids Research 16: 2723-2724 (1988), incorporated herein by reference
  • coli strain JM101 (recA + ) is transformed with pMH22 and plasmid DNA is purified.
  • the DNA is digested with ⁇ /ofl and _3su36l, which have a unique site in the polylinker region between the crylBa and crylCa parts, and in crylCa (position 1241), respectively.
  • _3su36l which cuts in the 3' end of the domain II encoding part of the crylCa gene, recombination events can effectively selected for behind this position, i.e. in or close to domain III.
  • Digested DNA is transformed to E. coli strain XL-1 and transformants are screened for production of soluble protoxin, as described in de Maagd et al., Appl. Environ. Microbiol. 62: 1537-1543 (1996). Restriction analysis and DNA sequencing determine the locations of the crossover points in the hybrids. Recombinants BBC13 and BBC15, which have different crossover points, both produce soluble protoxins and are therefore selected for toxicity studies.
  • SEQ ID NO:3 shows the amino acid sequence of the BBC13 hybrid toxin encoded by the nucleotide sequence depicted in SEQ ID NO:3.
  • the homologous crossover region of BBC13 between domain II from Cryl Ba and domain III from CrylCa is located at amino acids 482-488 of SEQ ID NO:4.
  • the nucleotide sequence encoding the Cryl Ba/Cry1 Ca (BBC15) hybrid toxin is depicted in SEQ ID NO:5.
  • SEQ ID NO:6 shows the amino acid sequence of the BBC15 hybrid toxin encoded by the nucleotide sequence depicted in SEQ ID NO:5.
  • the homologous crossover region of BBC15 between domain II from Cryl Ba and domain III from CrylCa is located at amino acids 491-494 of SEQ ID NO:6.
  • Protoxins are produced in E. coli strain XL-1. Protoxin isolation, and trypsin-treatment and FLPC-purification of activated toxin, are performed as described in Bosch, et al., Bio/technology 12:915-918 (1994).
  • the FFC1 toxin fragment produced by trypsin-treatment desirably comprises amino acid 1 to approximately amino acid 620 of SEQ ID NO:2.
  • the BBC13 and BBC15 toxin fragments produced by trypsin-treatment desirably comprise approximately amino acid 1 to approximately amino acid 655 of SEQ ID NO:4 and SEQ ID NO:6, respectively.
  • Toxicity of proteins is tested by spreading activated toxin dilutions on artificial diet.
  • CrylBa and the Cryl Ba/Cry1 Ca hybrids (BBC13 and BBC15) are also tested as full-length protoxins.
  • Neonate larvae of Spodoptera exigua (beet armyworm) are used, and mortality is scored after 6 days at 28°C.
  • LC 50 concentration with 50% mortality
  • PoloPC computer program Russel, et al., ESA Bulletin 23: 209-213 (1977)
  • the toxicity of the BBC protoxin is tested against Heliothis virescens (tobacco budworm), Spodoptera frugiperda (fall armyworm), Ostrinia nubilalis (European corn borer), Agrotis ipsilon (black cutworm), and Helicove ⁇ a zea (corn earworm).
  • 200 ⁇ l of a 0.13mg/ml solution of purified BBC protoxin is spread over the 18.08cm 3 surface of a dish containing diet. The solution is allowed to dry and 10 insects are applied. The samples are incubated at 27°C for five days, at which time readings are taken.
  • BBC shows 100% activity against European corn borer, but none against the other four insects.
  • ECB LC50 151 ng/cm 2
  • FAW LC 50 53 ng/cm 2
  • TBW LC ⁇ 659 ng/cm 2 .
  • Microorganisms which are suitable for the heterologous expression of the nucleotide sequences of the invention are all microorganisms which are capable of colonizing plants or the rhizosphere. As such they will be brought into contact with insect pests. These include gram-negative microorganisms such as Pseudomonas, Enterobacter and Serratia, the gram-positive microorganism Bacillus and the fungi Trichoderma, Gliocladium, and Saccharomyces cerevisiae.
  • heterologous hosts are Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonas aurantiaca, Enterobacter cloacae, Serratia marscesens, Bacillus subtilis, Bacillus cereus, Trichoderma viride, Trichoderma harzianum, Gliocladium virens, and Saccharomyces cerevisiae.
  • Expression vector pKK223-3 (Pharmacia catalogue # 27-4935-01) allows expression in E. coli. This vector has a strong tac promoter (Brosius, J. et al., Proc. Natl. Acad. Sci. USA 81) regulated by the lac repressor and induced by IPTG. A number of other expression systems have been developed for use in E. coli.
  • _ (Pharmacia #27-4946-01) uses a tightly regulated bacteriophage ⁇ promoter which allows for high level expression of proteins.
  • the lac promoter provides another means of expression but the promoter is not expressed at such high levels as the tac promoter.
  • expression of the nucleotide sequence in closely related gram negative- bacteria such as Pseudomonas, Enterobacter, Serratia and Erwinia is possible.
  • pLRKD211 Kaiser & Kroos, Proc. Natl. Acad. Sci. USA 81.: 5816-5820 (1984)
  • induction by IPTG is required for expression of the tac (i.e. t ⁇ -lac) promoter.
  • tac i.e. t ⁇ -lac
  • this same promoter e.g. on wide-host range plasmid pLRKD211
  • This tip- lac promoter can be placed in front of any gene or operon of interest for expression in Pseudomonas or any other closely related bacterium for the purposes of the constitutive expression of such a gene.
  • a nucleotide sequence whose expression results in an insecticidal toxin can therefore be placed behind a strong constitutive promoter, transferred to a bacterium which has plant or rhizosphere colonizing properties turning this organism to an insecticidal agent.
  • Other possible promoters can be used for the constitutive expression of the nucleotide sequence in gram-negative bacteria. These include, for example, the promoter from the Pseudomonas regulatory genes gafA and lemA (WO 94/01561) and the Pseudomonas savastanoi IAA operon promoter (Gaffney et al., J. Bacteriol. 172: 5593-5601 (1990).
  • Heterologous expression of the nucleotides sequence in gram-positive bacteria is another means of producing the insecticidal toxins.
  • Expression systems for Bacillus and Streptomyces are the best characterized.
  • the promoter for the erythromycin resistance gene (ermR) from Streptococcus pneumoniae has been shown to be active in gram-positive aerobes and anaerobes and also in E.coli (Trieu-Cuot et al., Nucl Acids Res 18: 3660 (1990)).
  • a further antibiotic resistance promoter from the thiostreptone gene has been used in Streptomyces cloning vectors (Bibb, Mol Gen Genet 199: 26-36 (1985)).
  • the shuttle vector pHT3101 is also appropriate for expression in Bacillus (Lereclus, FEMS Microbiol Lett 60: 211 -218 (1989)).
  • a significant advantage of this approach is that many gram- positive bacteria produce spores which can be used in formulations that produce insecticidal agents with a longer shelf life. Bacillus and Streptomyces species are aggressive colonizers of soils.
  • Trichoderma harzianum and Gliocladium virens have been shown to provide varying levels of biocontrol in the field (US 5,165,928 and US 4,996,157, both to Cornell Research Foundation).
  • a nucleotide sequence whose expression results in an insecticidal toxin could be expressed in such a fungus. This could be accomplished by a number of ways which are well known in the art.
  • particle bombardment can be used to transform protoplasts or other fungal cells with the ability to develop into regenerated mature structures.
  • the vector pAN7-1 originally developed for Aspergillus transformation and now used widely for fungal transformation (Curragh et al., Mycol. Res. 97(3): 313-317 (1992;; Tooley et ai., Curr. Genet. 27: 55-60 (1992); Punt et ai., Gene 56: 117-124 (1987)) is engineered to contain the nucleotide sequence.
  • This plasmid contains the E. coli the hygromycin B resistance gene flanked by the Aspergillus nidulans gpd promoter and the trpC terminator (Punt et al., Gene 56: 117-124 (1987)).
  • the nucleic acid sequences of the invention are expressed in the yeast Saccharomyces cerevisiae.
  • each of the two ORF's from pCIB9369, pCIB9381 , pCIB9354, or pCIB9383 are cloned into individual vectors with the GAL1 inducible promoter and the CYC1 terminator.
  • Each vector has ampicillin resistance and the 2 micron replicon.
  • the vectors preferably differ in their yeast growth markers.
  • the constructs are transformed into S. cerevisiae independently and together.
  • the ORFs are expressed together and tested for protein expression and insecticidal activity.
  • Insecticidal formulations are made using active ingredients which comprise either the isolated toxin or alternatively suspensions or concentrates of cells which produce it and which are described in the examples above.
  • active ingredients which comprise either the isolated toxin or alternatively suspensions or concentrates of cells which produce it and which are described in the examples above.
  • Bt or E. coli cells expressing the insecticidal toxin may be used for the control of the insect pests.
  • Formulations are made in liquid or solid form and are described below.
  • Example 7 Liquid Formulation of Insecticidal Compositions
  • Emulsifiable concentrates a b c
  • Emulsions of any required concentration can be produced from such concentrates by dilution with water.
  • the active ingredient is dissolved in methylene chloride, the solution is sprayed onto the carrier, and the solvent is subsequently evaporated off in vacuo.
  • the active ingredient is thoroughly mixed with the adjuvants and the mixture is thoroughly ground in a suitable mill, affording wettable powders which can be diluted with water to give suspensions of the desired concentrations.
  • Emulsions of any required concentration can be obtained from this concentrate by dilution with water.
  • Ready-to-use dusts are obtained by mixing the active ingredient with the carriers, and grinding the mixture in a suitable mill.
  • the active ingredient is mixed and ground with the adjuvants, and the mixture is subsequently moistened with water.
  • the mixture is extruded and then dried in a stream of air.
  • the finely ground active ingredient is uniformly applied, in a mixer, to the kaolin moistened with polyethylene glycol. Non-dusty coated granulates are obtained in this manner.
  • the finely ground active ingredient is intimately mixed with the adjuvants, giving a suspension concentrate from which suspensions of any desire concentration can be obtained by dilution with water.
  • insecticidal formulations described above are applied to the plants according to methods well known in the art, in such amounts that the insect pests are controlled by the insecticidal toxin.
  • nucleic acid sequences described in this application can be incorporated into plant cells using conventional recombinant DNA technology. Generally, this involves inserting a coding sequence of the invention into an expression system to which the coding sequence is heterologous (i.e., not normally present) using standard cloning procedures known in the art.
  • the vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • a large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses.
  • Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems ⁇ gth , ⁇ gtIO and Charon 4; plasmid vectors such as pBI121 , pBR322, pACYC177, PACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101 , pCDNAII; and other similar systems.
  • the components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed.
  • the expression systems described herein can be used to transform virtually any crop plant cell under suitable conditions. Transformed cells can be regenerated into whole plants such that the nucleotide sequence of the invention confer insect resistance to the transgenic plants.
  • the nucleotide sequences described in this application can be modified for expression in transgenic plant hosts.
  • a host plant expressing the nucleotide sequences and which produces the insecticidal toxins in its cells has enhanced resistance to insect attack and is thus better equipped to withstand crop losses associated with such attack.
  • the transgenic expression in plants of genes derived from microbial sources may require the modification of those genes to achieve and optimize their expression in plants.
  • bacterial ORFs which encode separate enzymes but which are encoded by the same transcript in the native microbe are best expressed in plants on separate transcripts.
  • each microbial ORF is isolated individually and cloned within a cassette which provides a plant promoter sequence at the 5' end of the ORF and a plant transcriptional terminator at the 3' end of the ORF.
  • the isolated ORF sequence preferably includes the initiating ATG codon and the terminating STOP codon but may include additional sequence beyond the initiating ATG and the STOP codon.
  • the ORF may be truncated, but still retain the required activity; for particularly long ORFs, truncated versions which retain activity may be preferable for expression in transgenic organisms.
  • plant promoter and “plant transcriptional terminator” it is intended to mean promoters and transcriptional terminators which operate within plant cells. This includes promoters and transcription terminators which may be derived from non-plant sources such as viruses (an example is the Cauliflower Mosaic Virus).
  • modification to the ORF coding sequences and adjacent sequence is not required. It is sufficient to isolate a fragment containing the ORF of interest and to insert it downstream of a plant promoter.
  • Gaffney et al. (Science 261 : 754- 756 (1993)) have expressed the Pseudomonas nahG gene in transgenic plants under the control of the CaMV 35S promoter and the CaMV tml terminator successfully without modification of the coding sequence and with x bp of the Pseudomonas gene upstream of the ATG still attached, and y bp downstream of the STOP codon still attached to the nahG ORF.
  • Preferably as little adjacent microbial sequence should be left attached upstream of the ATG and downstream of the STOP codon. In practice, such construction may depend on the availability of restriction sites.
  • genes derived from microbial sources may provide problems in expression. These problems have been well characterized in the art and are particularly common with genes derived from certain sources such as Bacillus. These problems may apply to the nucleotide sequence of this invention and the modification of these genes can be undertaken using techniques now well known in the art. The following problems may be encountered:
  • Codon Usage The preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. Comparison of the usage of codons within a cloned microbial ORF to usage in plant genes (and in particular genes from the target plant) will enable an identification of the codons within the ORF which should preferably be changed. Typically plant evolution has tended towards a strong preference of the nucleotides C and G in the third base position of monocotyledons, whereas dicotyledons often use the nucleotides A or T at this position. By modifying a gene to incorporate preferred codon usage for a particular target transgenic species, many of the problems described below for GC/AT content and illegitimate splicing will be overcome.
  • Plant genes typically have a GC content of more than 35%.
  • ORF sequences which are rich in A and T nucleotides can cause several problems in plants. Firstly, motifs of ATTTA are believed to cause destabilization of messages and are found at the 3' end of many short-lived mRNAs. Secondly, the occurrence of polyadenylation signals such as AATAAA at inappropriate positions within the message is believed to cause premature truncation of transcription. In addition, monocotyledons may recognize AT-rich sequences as splice sites (see below).
  • Plants differ from microorganisms in that their messages do not possess a defined ribosome binding site. Rather, it is believed that ribosomes attach to the 5' end of the message and scan for the first available ATG at which to start translation. Nevertheless, it is believed that there is a preference for certain nucleotides adjacent to the ATG and that expression of microbial genes can be enhanced by the inclusion of a eukaryotic consensus translation initiator at the ATG.
  • Clontech (1993/1994 catalog, page 210, incorporated herein by reference) have suggested one sequence as a consensus translation initiator for the expression of the E. coli uidA gene in plants.
  • This analysis can be done for the desired plant species into which the nucleotide sequence is being incorporated, and the sequence adjacent to the ATG modified to incorporate the preferred nucleotides.
  • Genes cloned from non-plant sources and not optimized for expression in plants may also contain motifs which may be recognized in plants as 5' or 3' splice sites, and be cleaved, thus generating truncated or deleted messages. These sites can be removed using the techniques well known in the art.
  • Coding sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants.
  • the expression cassettes may also comprise any further sequences required or selected for the expression of the transgene.
  • Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments.
  • the selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The following are non- limiting examples of promoters that may be used in expression cassettes.
  • Ubiquitin is a gene product known to accumulate in many cell types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower - Binet et al. Plant Science 79: 87-94 (1991 ); maize - Christensen et al. Plant Molec. Biol. 12: 619- 632 (1989); and Arabidopsis - Norris et ai., Plant Mol. Biol. 21 :895-906 (1993)).
  • the maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0 342 926 (to Lubrizol) which is herein incorporated by reference.
  • pAHC25 a vector that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile bombardment.
  • the Arabidopsis ubiquitin promoter is ideal for use with the nucleotide sequences of the present invention.
  • the ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons.
  • Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this application, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences.
  • pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone.
  • a derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761 ENX.
  • pCGN1761 ENX is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polylinker for the purpose of their expression under the control of the 35S promoter in transgenic plants.
  • the entire 35S promoter-coding sequence-tm/ terminator cassette of such a construction can be excised by Hindlll, Sphl, Sail, and Xbal sites 5' to the promoter and Xbal, BamHI and Bgll sites 3' to the terminator for transfer to transformation vectors such as those described below.
  • the double 35S promoter fragment can be removed by 5' excision with Hindlll, Sphl, Sail, Xbal, or Pstl, and 3' excision with any of the polylinker restriction sites (EcoRI, Notl or Xhol) for replacement with another promoter.
  • modifications around the cloning sites can be made by the introduction of sequences that may enhance translation. This is particularly useful when overexpression is desired.
  • pCGN1761 ENX may be modified by optimization of the translational initiation site as described in Example 37 of U.S. Patent No. 5,639,949, incorporated herein by reference.
  • actin promoter is a good choice for a constitutive promoter.
  • the promoter from the rice Actl gene has been cloned and characterized (McElroy et al. Plant Cell 2: 163-171 (1990)).
  • a 1.3kb fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts.
  • numerous expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons (McElroy et ai. Mol. Gen. Genet. 231 : 150-160 (1991)).
  • promoter- containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761 ENX, which is then available for the insertion of specific gene sequences.
  • the fusion genes thus constructed can then be transferred to appropriate transformation vectors.
  • the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).
  • the double 35S promoter in pCGN1761 ENX may be replaced with any other promoter of choice that will result in suitably high expression levels.
  • one of the chemically regulatable promoters described in U.S. Patent No. 5,614,395, such as the tobacco PR-1 a promoter may replace the double 35S promoter.
  • the Arabidopsis PR-1 promoter described in Lebel et al., Plant J. 16:223-233 (1998) may be used.
  • the promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites.
  • the chemically/pathogen regulatable tobacco PR-1 a promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104, which is hereby incorporated by reference) and transferred to plasmid pCGN1761 ENX (Uknes et al., 1992).
  • pCIB1004 is cleaved with Ncol and the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase.
  • the fragment is then cleaved with Hindlll and the resultant PR-1 a promoter-containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed. This is done by cleavage with Xhol and blunting with T4 polymerase, followed by cleavage with Hindlll and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761 ENX derivative with the PR-1 a promoter and the tml terminator and an intervening polylinker with unique EcoRI and Notl sites.
  • the selected coding sequence can be inserted into this vector, and the fusion products (i.e.
  • promoter-gene-terminator can subsequently be transferred to any selected transformation vector, including those described infra.
  • Various chemical regulators may be employed to induce expression of the selected coding sequence in the plants transformed according to the present invention, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U.S. Patent Nos. 5,523,311 and 5,614,395.
  • a promoter inducible by certain alcohols or ketones, such as ethanol, may also be used to confer inducible expression of a coding sequence of the present invention.
  • a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et al. (1998) Nat. Biotechnol 16:177-180).
  • the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer.
  • the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter are replaced by a coding sequence of the present invention to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods well known in the art.
  • glucocorticoid- mediated induction system is used (Aoyama and Chua (1997) The Plant Journal 11 : 605- 612) and gene expression is induced by application of a glucocorticoid, for example a synthetic glucocorticoid, preferably dexamethasone, preferably at a concentration ranging from 0.1 mM to 1mM, more preferably from 10mM to 100mM.
  • the luciferase gene sequences are replaced by a nucleic acid sequence of the invention to form an expression cassette having a nucleic acid sequence of the invention under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter.
  • the trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al. (1986) Science 231 : 699-704) fused to the transactivating domain of the herpes viral protein VP16 (Triezenberg et al. (1988) Genes Devel.
  • tissue- or organ-specificity of the fusion protein is achieved leading to inducible tissue- or organ-specificity of the insecticidal toxin.
  • a suitable root promoter is the promoter of the maize metallothionein-like (MTL) gene described by de Framond (FEBS 290: 103-106 (1991)) and also in U.S. Patent No. 5,466,785, incorporated herein by reference.
  • This "MTL" promoter is transferred to a suitable vector such as pCGN1761 ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene- terminator cassette to a transformation vector of interest.
  • Wound-inducible promoters may also be suitable for gene expression. Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell V. 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et ai. Plant Molec. Biol. 22: 129-142 (1993), Warner et ai. Plant J. 3: 191-201 (1993)) and all are suitable for use with the instant invention. Logemann et al. describe the 5' upstream sequences of the dicotyledonous potato wunl gene.
  • Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the maize Wipl cDNA which is wound induced and which can be used to isolate the cognate promoter using standard techniques. Similar, Firek et al. and Warner et al. have described a wound- induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to this invention, and used to express these genes at the sites of plant wounding.
  • Pith-Preferred Expression Patent Application WO 93/07278, which is herein incorporated by reference, describes the isolation of the maize trpA gene, which is preferentially expressed in pith cells.
  • the gene sequence and promoter extending up to -1726 bp from the start of transcription are presented.
  • this promoter, or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner.
  • fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utility in transgenic plants.
  • a maize gene encoding phosphoenol carboxylase has been described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.
  • WO 93/07278 describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene which is expressed in pollen cells.
  • CDPK calcium-dependent protein kinase
  • the gene sequence and promoter extend up to 1400 bp from the start of transcription.
  • this promoter or parts thereof can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a nucleic acid sequence of the invention in a pollen-specific manner.
  • transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation.
  • Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
  • a gene's native transcription terminator may be used.
  • intron sequences have been shown to enhance expression, particularly in monocotyledonous cells.
  • the introns of the maize Adhl gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells.
  • Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200 (1987)).
  • the intron from the maize bronzel gene had a similar effect in enhancing expression.
  • Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader.
  • leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells.
  • TMV Tobacco Mosaic Virus
  • MCMV Maize Chlorotic Mottle Virus
  • AMV Alfalfa Mosaic Virus
  • DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized. See also, the section entitled “Expression With Chloroplast Targeting" in Example 37 of U.S. Patent No. 5,639,949.
  • cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting cellular protein bodies has been described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).
  • sequences have been characterized which cause the targeting of gene products to other cell compartments.
  • Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et ai. Plant Molec. Biol. 14: 357-368 (1990)).
  • the transgene product By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organelle or cell compartment.
  • chloroplast targeting for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene.
  • the signal sequence selected should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence.
  • Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions followed by in vitro chloroplast uptake using techniques described by Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986). These construction techniques are well known in the art and are equally applicable to mitochondria and peroxisomes.
  • the above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.
  • transformation vectors available for plant transformation are known to those of ordinary skill in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors.
  • the selection of vector will depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al.
  • vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.
  • pCIB200 and pCIB2001 The binary vectors pclB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner.
  • pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, J. Bacteriol.
  • Xhol linkers are ligated to the EcoRV fragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and the Xhol- digested fragment are cloned into Sa//-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19).
  • pCIB200 contains the following unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sail.
  • pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites.
  • Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, Mlul, Bell, Avrll, Apal, Hpal, and Stul.
  • pCIB2001 in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-med ⁇ ated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the Or/Tand OriV functions also from RK2.
  • the pCIB2001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
  • the binary vector pCIB10 contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E. coli and Agrobacterium. Its construction is described by Rothstein et ai. (Gene 53: 153-161 (1987)). Various derivatives of pCIB10 are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).
  • Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for non-Agrobacterium transformation is described.
  • pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin).
  • the plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT published application WO 93/07278.
  • the 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and Pvull.
  • the new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site.
  • the resultant derivative of pCIB246 is designated pCIB3025.
  • the GUS gene is then excised from pCIB3025 by digestion with Sail and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060.
  • the plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smal fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)).
  • This generated pCIB3064 which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampicillin/resistance (for selection in E. coli) and a polylinker with the unique sites Sphl, Pstl, Hindlll, and BamHI.
  • This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.
  • pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate.
  • DFR E. coli gene dihydrofolate reductase
  • PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adh1 gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOG10. A 250-bp fragment encoding the E.
  • coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pB1221 (Clontech) which comprises the pUC19 vector backbone and the nopaline synthase terminator. Assembly of these fragments generates pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaline synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistance and have Hindlll, Sphl, Pstl and EcoRI sites available for the cloning of foreign substances.
  • MCMV Maize Chlorotic Mottle Virus
  • plastid transformation vector pPH143 (WO 97/32011 , example 36) is used.
  • the nucleotide sequence is inserted into pPH143 thereby replacing the PROTOX coding sequence.
  • This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance.
  • the nucleotide sequence is inserted in pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.
  • nucleic acid sequence of the invention Once a nucleic acid sequence of the invention has been cloned into an expression system, it is transformed into a plant cell.
  • Methods for transformation and regeneration of plants are well known in the art.
  • Ti plasmid vectors have been utilized for the delivery of foreign DNA, as well as direct DNA uptake, liposomes, electroporation, micro- injection, and microprojectiles.
  • bacteria from the genus Agrobacterium can be utilized to transform plant cells. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as well as a representative plastid transformation technique.
  • Transformation techniques for dicotyledons are well known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium.
  • Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, or microinjection. Examples of these techniques are described by Paszkowski et ai., EMBO J 3: 2717-2722 (1984), Potrykus et ai., Mol. Gen. Genet. 199: 169-177 (1985), Reich et ai., Biotechnology 4: 1001 -1004 (1986), and Klein et al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.
  • Agrobacterium-medlated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species.
  • Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of wr genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)).
  • the transfer of the recombinant binary vector to Agrobacterium is accomplished by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain.
  • the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (H ⁇ fgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).
  • Transformation of the target plant species by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T- DNA borders.
  • Transformation of most monocotyledon species has now also become routine.
  • Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co- transformation) and both these techniques are suitable for use with this invention.
  • Co- transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable.
  • a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher etai. Biotechnology 4: 1093-1096 (1986)).
  • Patent Applications EP 0292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts.
  • Gordon-Kamm et al. Plant Cell 2: 603-618 (1990)
  • Fromm et al. Biotechnology 8: 833-839 (1990)
  • WO 93/07278 and Koziel et ai. describe techniques for the transformation of elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics device for bombardment.
  • Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment.
  • Protoplast-mediated transformation has been described for Japon/ca-types and Indica-xypes (Zhang ef al. Plant Cell Rep 7: 379-384 (1988); Shimamoto etai. Nature 338: 274-277 (1989); Datta etai. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).
  • WO 93/21335 describes techniques for the transformation of rice via electroporation.
  • Patent Application EP 0332581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology 11 : 1553-1558 (1993)) and Weeks et ai. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus.
  • a preferred technique for wheat transformation involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery.
  • any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark.
  • MS medium with 3% sucrose
  • 3 mg/l 2,4-D for induction of somatic embryos, which is allowed to proceed in the dark.
  • embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typically 15%).
  • the embryos are allowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical.
  • An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures.
  • Each plate of embryos is shot with the DuPont Biolistics® helium device using a burst pressure of -1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (still on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration.
  • the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mg/liter NAA, 5 mg/liter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35).
  • regeneration medium MS + 1 mg/liter NAA, 5 mg/liter GA
  • selection agent 10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35.
  • GA7s sterile containers which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.
  • Nicotiana tabacum c.v. 'Xanthi nc' are germinated seven per plate in a 1" circular array on T agar medium and bombarded 12-14 days after sowing with 1 ⁇ m tungsten particles (M10, Biorad, Hercules, CA) coated with DNA from plasmids pPH143 and pPH145 essentially as described (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917).
  • Bombarded seedlings are incubated on T medium for two days after which leaves are excised and placed abaxial side up in bright light (350-500 ⁇ mol photons/m 2 /s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990) PNAS 87, 8526-8530) containing 500 ⁇ g/ml spectinomycin dihydrochloride (Sigma, St. Louis, MO). Resistant shoots appearing underneath the bleached leaves three to eight weeks after bombardment are subcloned onto the same selective medium, allowed to form callus, and secondary shoots isolated and subcloned.
  • the plants obtained via tranformation with a nucleic acid sequence of the present invention can be any of a wide variety of plant species, including those of monocots and dicots; however, the plants used in the method of the invention are preferably selected from the list of agronomically important target crops set forth supra.
  • the expression of a gene of the present invention in combination with other characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981); Crop Breeding, Wood D. R.
  • the genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants.
  • said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting.
  • Specialized processes such as hydroponics or greenhouse technologies can also be applied.
  • measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield.
  • Use of the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering.
  • the various breeding steps are characterized by well-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants.
  • different breeding measures are taken.
  • the relevant techniques are well known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc.
  • Hybridization techniques also include the sterilization of plants to yield male or female sterile plants by mechanical, chemical, or biochemical means.
  • Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines.
  • the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow one to dispense with said methods due to their modified genetic properties.
  • new crops with improved stress tolerance can be obtained, which, due to their optimized genetic "equipmenf , yield harvested product of better quality than products that were not able to tolerate comparable adverse developmental conditions.
  • germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important.
  • seed production In seed production, germination quality and uniformity of seeds are essential product characteristics, whereas germination quality and uniformity of seeds harvested and sold by the farmer is not important.
  • Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof.
  • Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD®), methalaxyl (Apron®), and pirimiphos-methyl (Actellic®). If desired, these compounds are formulated together with further carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests.
  • the protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.
  • the seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds.
  • the bag, container or vessel may be designed for either short term or long term storage, or both, of the seed.
  • a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal.
  • the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type.
  • the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed.
  • the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering.
  • water absorbent materials are placed between or adjacent to packaging material layers.
  • the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed.
  • An example of such treatment is sterilization, for example by chemical means or by exposure to radiation.
  • a commercial bag comprising seed of a transgenic plant comprising a gene of the present invention that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with label instructions for the use thereof for conferring broad spectrum disease resistance to plants.

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Abstract

Selon l'invention, on construit, par clonage et recombinaison in vivo des séquences nucléotidiques codant pour les delta-endotoxines hybrides de Bacillus thuringiensis contenant les domaines I et II des toxines Cry1Fa et Cry1Ba combinés au domaine III de Cry1Ca. L'invention concerne également des compositions et des formulations contenant lesdites toxines insecticides hybrides sont efficaces dans la lutte contres les insectes nuisibles. L'invention concerne en outre des méthodes de fabrication de ces toxines hybrides ainsi que des méthodes d'utilisation de ces séquences nucléotidiques, par exemple dans des micro-organismes, afin de lutter contre les insectes nuisibles et, dans les plantes transgéniques, afin de rendre ces dernières résistantes aux insectes.
PCT/EP2000/008042 1999-08-19 2000-08-17 Toxines insecticides hybrides et sequences d'acide nucleique codant pour ces toxines Ceased WO2001014562A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050880A3 (fr) * 2002-11-29 2004-07-29 Univ Sussex Agent insecticide
CN102821596A (zh) * 2009-12-16 2012-12-12 陶氏益农公司 CRY1Ca和CRY1Fa蛋白在昆虫抗性管理中的组合应用
JP2013514768A (ja) * 2009-12-16 2013-05-02 ダウ アグロサイエンシィズ エルエルシー 昆虫抵抗性管理のためのＣＲＹ１ＤａおよびＣＲＹ１Ｆａタンパク質の併用
RU2497830C2 (ru) * 2007-03-28 2013-11-10 Зингента Партисипейшнс Аг Гибридный инсектицидный белок, молекула нуклеиновой кислоты, кодирующая такой белок, трансгенные растения и их семена, содержащие такой белок, способ получения белка и его применение
WO2016061391A3 (fr) * 2014-10-16 2016-07-21 Monsanto Technology Llc Nouvelles protéines insecticides chimères toxiques pour les lélipidoptères nuisibles ou les inhibant
US9522937B2 (en) 2007-03-28 2016-12-20 Syngenta Participations Ag Insecticidal proteins
US9567381B2 (en) 2012-03-09 2017-02-14 Vestaron Corporation Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides
KR101841292B1 (ko) * 2009-12-16 2018-03-22 다우 아그로사이언시즈 엘엘씨 곤충 내성 관리를 위한 ｃｒｙ１ｄａ 및 ｃｒｙ１ｃａ의 병용 용도
EP3207050A4 (fr) * 2014-10-16 2018-09-12 Pioneer Hi-Bred International, Inc. Polypeptides insecticides ayant un spectre d'activité amélioré et leurs utilisations
US10487123B2 (en) 2014-10-16 2019-11-26 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to lepidopteran pests
US11447531B2 (en) 2016-10-21 2022-09-20 Vestaron Corporation Cleavable peptides and insecticidal and nematicidal proteins comprising same
CN116199752A (zh) * 2016-12-12 2023-06-02 先正达参股股份有限公司 工程化的杀有害生物蛋白和控制植物有害生物的方法
US11692016B2 (en) 2012-03-09 2023-07-04 Vestaron Corporation High gene expression yeast strain
US11781151B2 (en) 2016-04-14 2023-10-10 Pioneer Hi-Bred International, Inc. Insecticidal CRY1B variants having improved activity spectrum and uses thereof

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GE A Z ET AL: "FUNCTIONAL DOMAINS OF BACILLUS THURINGIENSIS INSECTICIDAL CRYSTAL PROTEINS REFINEMENT OF HELIOTHIS VIRESCENS AND TRICHOPLUSIA NI SPECIFICITY DOMAINS ON CRYIA(C)", JOURNAL OF BIOLOGICAL CHEMISTRY,US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 266, no. 27, 25 September 1991 (1991-09-25), pages 17954 - 17958, XP000567704, ISSN: 0021-9258 *

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WO2004050880A3 (fr) * 2002-11-29 2004-07-29 Univ Sussex Agent insecticide
US10239921B2 (en) 2007-03-28 2019-03-26 Syngenta Participations Ag Insecticidal proteins
RU2497830C2 (ru) * 2007-03-28 2013-11-10 Зингента Партисипейшнс Аг Гибридный инсектицидный белок, молекула нуклеиновой кислоты, кодирующая такой белок, трансгенные растения и их семена, содержащие такой белок, способ получения белка и его применение
RU2532838C2 (ru) * 2007-03-28 2014-11-10 Зингента Партисипейшнс Аг Инсектицидные белки
US11746129B2 (en) 2007-03-28 2023-09-05 Syngenta Participations Ag Insecticidal proteins
US9522937B2 (en) 2007-03-28 2016-12-20 Syngenta Participations Ag Insecticidal proteins
CN102821596A (zh) * 2009-12-16 2012-12-12 陶氏益农公司 CRY1Ca和CRY1Fa蛋白在昆虫抗性管理中的组合应用
JP2013514769A (ja) * 2009-12-16 2013-05-02 ダウ アグロサイエンシィズ エルエルシー 害虫抵抗性管理のためのＣＲＹ１ＣａおよびＣＲＹ１Ｆａタンパク質の併用
JP2013514768A (ja) * 2009-12-16 2013-05-02 ダウ アグロサイエンシィズ エルエルシー 昆虫抵抗性管理のためのＣＲＹ１ＤａおよびＣＲＹ１Ｆａタンパク質の併用
US9567602B2 (en) 2009-12-16 2017-02-14 Dow Agrosciences Llc Combined use of CRY1Ca and CRY1Fa proteins for insect resistance management
KR101841295B1 (ko) * 2009-12-16 2018-03-22 다우 아그로사이언시즈 엘엘씨 곤충 내성 관리를 위한 ＣＲＹ１Ｃａ 및 ＣＲＹ１Ｆａ 단백질의 조합 용도
KR101841292B1 (ko) * 2009-12-16 2018-03-22 다우 아그로사이언시즈 엘엘씨 곤충 내성 관리를 위한 ｃｒｙ１ｄａ 및 ｃｒｙ１ｃａ의 병용 용도
US11472854B2 (en) 2012-03-09 2022-10-18 Vestaron Corporation Insecticidal peptide production, peptide expression in plants and combinations of cysteine rich peptides
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US11692016B2 (en) 2012-03-09 2023-07-04 Vestaron Corporation High gene expression yeast strain
US9567381B2 (en) 2012-03-09 2017-02-14 Vestaron Corporation Toxic peptide production, peptide expression in plants and combinations of cysteine rich peptides
EP3715363A1 (fr) * 2014-10-16 2020-09-30 Monsanto Technology LLC Nouvelles protéines insecticides chimères toxiques pour les lélipidoptères nuisibles ou les inhibant
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EA034918B1 (ru) * 2014-10-16 2020-04-07 Монсанто Текнолоджи Ллс Новые химерные инсектицидные белки, токсичные или ингибиторные в отношении чешуекрылых-вредителей
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US12391730B2 (en) 2014-10-16 2025-08-19 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides having improved activity spectrum and uses thereof
US11649266B2 (en) 2014-10-16 2023-05-16 Pioneer Hi-Bred International, Inc. Insecticidal polypeptides having improved activity spectrum and uses thereof
US12264182B2 (en) 2014-10-16 2025-04-01 Monsanto Technology Llc Chimeric insecticidal proteins toxic or inhibitory to lepidopteran pests
EP3207050A4 (fr) * 2014-10-16 2018-09-12 Pioneer Hi-Bred International, Inc. Polypeptides insecticides ayant un spectre d'activité amélioré et leurs utilisations
WO2016061391A3 (fr) * 2014-10-16 2016-07-21 Monsanto Technology Llc Nouvelles protéines insecticides chimères toxiques pour les lélipidoptères nuisibles ou les inhibant
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