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US20130167268A1 - COMBINATIONS INCLUDING CRY34AB/35AB AND CRY3Aa PROTEINS TO PREVENT DEVELOPMENT OF RESISTANCE IN CORN ROOTWORMS (DIABROTICA SPP.) - Google Patents

COMBINATIONS INCLUDING CRY34AB/35AB AND CRY3Aa PROTEINS TO PREVENT DEVELOPMENT OF RESISTANCE IN CORN ROOTWORMS (DIABROTICA SPP.) Download PDF

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US20130167268A1
US20130167268A1 US13/643,047 US201113643047A US2013167268A1 US 20130167268 A1 US20130167268 A1 US 20130167268A1 US 201113643047 A US201113643047 A US 201113643047A US 2013167268 A1 US2013167268 A1 US 2013167268A1
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protein
plants
seeds
plant
refuge
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Kenneth E. Narva
Thomas Meade
Kristin J. Fencil
Huarong Li
Timothy D. Hey
Aaron T. Woosley
Monica Britt Olson
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Corteva Agriscience LLC
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Dow AgroSciences LLC
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01G1/001—
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
    • 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
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
    • 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
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/50—1,3-Diazoles; Hydrogenated 1,3-diazoles
    • 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
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B32—LAYERED PRODUCTS
    • B32B—LAYERED PRODUCTS, i.e. PRODUCTS BUILT-UP OF STRATA OF FLAT OR NON-FLAT, e.g. CELLULAR OR HONEYCOMB, FORM
    • B32B1/00—Layered products having a non-planar shape
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
    • B65D85/00—Containers, packaging elements or packages, specially adapted for particular articles or materials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • 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
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/13—Hollow or container type article [e.g., tube, vase, etc.]

Definitions

  • corn rootworm species complex includes the northern corn rootworm ( Diabrotica barberi ), the southern corn rootworm ( D. undecimpunctata howardi ), and the western corn rootworm ( D. virgifera virgifera ).
  • Diabrotica barberi the southern corn rootworm
  • D. undecimpunctata howardi the southern corn rootworm
  • D. virgifera virgifera the western corn rootworm
  • Diabrotica virgifera zeae Maexican corn rootworm
  • Diabrotica balteata Brazilian corn rootworm
  • Brazilian corn rootworm complex Diabrotica viridula and Diabrotica speciosa
  • Diabrotica The soil-dwelling larvae of these Diabrotica species feed on the root of the corn plant, causing lodging. Lodging eventually reduces corn yield and often results in death of the plant. By feeding on cornsilks, the adult beetles reduce pollination and, therefore, detrimentally affect the yield of corn per plant. In addition, adults and larvae of the genus Diabrotica attack cucurbit crops (cucumbers, melons, squash, etc.) and many vegetable and field crops in commercial production as well as those being grown in home gardens.
  • Synthetic organic chemical insecticides have been the primary tools used to control insect pests but biological insecticides, such as the insecticidal proteins derived from Bacillus thuringienses (Bt), have played an important role in some areas.
  • Bacillus thuringienses Bacillus thuringienses
  • Insecticidal crystal proteins from some strains of Bacillus thuringienses are well-known in the art. See, e.g., Hofte et al., Microbial Reviews, Vol. 53, No. 2, pp. 242-255 (1989). These proteins are typically produced by the bacteria as approximately 130 kDa protoxins that are then cleaved by proteases in the insect midgut, after ingestion by the insect, to yield a roughly 60 kDa core toxin. These proteins are known as crystal proteins because distinct crystalline inclusions can be observed with spores in some strains of B.t. These crystalline inclusions are often composed of several distinct proteins.
  • delta-endotoxins from Bacillus thuringienses (B.t.). Delta-endotoxins have been successfully expressed in crop plants such as cotton, potatoes, rice, sunflower, as well as corn, and have proven to provide excellent control over insect pests.
  • B.t. Bacillus thuringienses
  • Bt proteins have been used to create the insect-resistant transgenic plants that have been successfully registered and commercialized to date. These include Cry1Ab, Cry1Ac, Cry1F, Cry3Aa, and Cry3Bb in corn, Cry1Ac and Cry2Ab in cotton, and Cry3A in potato. There is also SMART STAX in corn, which comprises Cry1A.105 and Cry2Ab.
  • the commercial products expressing these proteins express a single protein except in cases where the combined insecticidal spectrum of 2 proteins is desired (e.g., Cry1Ab and Cry3Bb in corn combined to provide resistance to lepidopteran pests and rootworm, respectively) or where the independent action of the proteins makes them useful as a tool for delaying the development of resistance in susceptible insect populations (e.g., Cry1Ac and Cry2Ab in cotton combined to provide resistance management for tobacco budworm).
  • the proteins selected for use in an Insect Resistance Management (IRM) stack should be active such that resistance developed to one protein does not confer resistance to the second protein (i.e., there is not cross resistance to the proteins). If, for example, a pest population selected for resistance to “Protein A” is sensitive to “Protein B”, one would conclude that there is not cross resistance and that a combination of Protein A and Protein B would be effective in delaying resistance to Protein A alone.
  • IRM Insect Resistance Management
  • RNAi approaches have also been proposed. See e.g. Baum et al., Nature Biotechnology, vol. 25, no. 11 (November 2007) pp. 1322-1326.
  • the subject invention relates in part to Cry34Ab/35Ab in combination with Cry3Aa.
  • the subject invention relates in part to the surprising discovery that Cry34Ab/Cry35Ab and Cry3Aa are useful for preventing development of resistance (to either insecticidal protein system alone) by a corn rootworm ( Diabrotica spp.) population.
  • plants producing these insecticidal Cry proteins will be useful to mitigate concern that a corn rootworm population could develop that would be resistant to either of these insecticidal protein systems alone.
  • the subject invention is supported in part by the discovery that components of these Cry protein systems do not compete with each other for binding corn rootworm gut receptors.
  • the subject invention also relates in part to triple stacks or “pyramids” of three (or more) toxin systems, with Cry34Ab/Cry35Ab and Cry3Aa being the base pair.
  • plants (and acreage planted with such plants) that produce these two insecticidal protein systems are included within the scope of the subject invention.
  • 125 I-Cry35Ab1 and 125 I-Cry3Aa were able to specifically bind to the BBMV ( FIGS. 1A and 1B ).
  • FIG. 2 Binding of 125 I-Cry3Aa to BBMV prepared from western corn rootworm larvae at different concentrations of non-labeled competitor. Error bars for the homologous competition binding represent standard deviation, and the error bars for heterologous competition are not shown.
  • SEQ ID NO:1 Full length, native Cry3Aa protein sequence
  • SEQ ID NO:2 Trypsin-truncated Cry3Aa core protein sequence
  • SEQ ID NO:3 Full length, native Cry34Ab1 protein sequence
  • SEQ ID NO:4 Full length, native Cry35Ab1 protein sequence
  • SEQ ID NO:5 Chymotrypsin-truncated Cry35Ab1 core protein sequence
  • SEQ ID NO:6 cry34Ab DNA
  • SEQ ID NO:8 cry3Aa DNA
  • Sequences for the Cry34Ab/35Ab protein are obtainable from Bacillus thuringienses isolate PS149B1, for example.
  • PS149B1 Bacillus thuringienses isolate
  • the subject invention includes the use of Cry34Ab/35Ab insecticidal proteins in combination with a Cry3Aa toxin to protect corn from damage and yield loss caused by corn rootworm feeding by corn rootworm populations that might develop resistance to either of these Cry protein systems alone (without the other).
  • the subject invention thus teaches an Insect Resistance Management (IRM) stack to prevent the development of resistance by corn rootworm to Cry3Aa and/or Cry34Ab/35Ab.
  • IRM Insect Resistance Management
  • compositions for controlling rootworm pests comprising cells that produce a Cry3Aa toxin protein and a Cry34Ab/35Ab toxin system.
  • the invention further comprises a host transformed to produce both a Cry3Aa protein and a Cry34Ab/35Ab binary toxin, wherein said host is a microorganism or a plant cell.
  • the invention provides a method of controlling rootworm pests comprising contacting said pests or the environment of said pests with an effective amount of a composition that contains a Cry3Aa protein and further contains a Cry34Ab/35Ab binary toxin.
  • An embodiment of the invention comprises a maize plant comprising a plant-expressible gene encoding a Cry34Ab/35Ab binary toxin and a plant-expressible gene encoding a Cry3Aa protein, and seed of such a plant.
  • a further embodiment of the invention comprises a maize plant wherein a plant-expressible gene encoding a Cry34Ab/35Ab binary toxin and a plant-expressible gene encoding a Cry3Aa protein have been introgressed into said maize plant, and seed of such a plant.
  • Cry34Ab/35Ab and Cry3Aa proteins can be used to produce IRM combinations for prevention or mitigation of resistance development by CRW.
  • Other proteins can be added to this combination to expand insect-control spectrum, for example.
  • the subject pair/combination can also be used in some preferred “triple stacks” or “pyramid” in combination with yet another protein for controlling rootworms, such as Cry3Ba and/or Cry6Aa.
  • RNAi against rootworms is a still further option. See e.g. Baum et al., Nature Biotechnology, vol. 25, no. 11 (November 2007) pp. 1322-1326.
  • Deployment options of the subject invention include the use of Cry3Aa and Cry34Ab/35Ab proteins in corn-growing regions where Diabrotica spp. are problematic. Another deployment option would be to use one or both of the Cry3Aa and Cry34Ab/35Ab proteins in combination with other traits.
  • some preferred “triple stacks” or “multiple modes of action stacks” of the subject invention include a Cry3Aa protein combined with Cry34Ab/35Ab proteins, together with a Cry6Aa protein and/or a Cry3Ba protein.
  • Transgenic plants, including corn, comprising a cry3Ba gene, cry34Ab/35Ab genes, and a third or fourth toxin system e.g., cry3Aa and/or cry6Aa gene(s)
  • cry3Aa and/or cry6Aa gene(s) are included within the scope of the subject invention.
  • Bt toxins even within a certain class such as Cry3Aa and Cry34Ab/35Ab can vary to some extent.
  • genes and toxins refers to a polynucleotide in a non-naturally occurring construct, or to a protein in a purified or otherwise non-naturally occurring state.
  • the genes and toxins useful according to the subject invention include not only the full length sequences disclosed but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein.
  • the terms “variants” or “variations” of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity.
  • the term “equivalent toxins” refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins. Domains/subdomains of these proteins can be swapped to make chimeric proteins. See e.g. U.S. Pat. No. 7,309,785 and 7,524,810. The '785 patent also teaches truncated Cry35 proteins. Truncated toxins are also exemplified herein.
  • the boundaries represent approximately 95% (Cry3Aa's and Cry34Ab's and Cry35Ab's), 78% (Cry3A's and Cry 34A's and Cry35A's), and 45% (Cry3's and Cry 34's and Cry 35's) sequence identity, per “Revision of the Nomenclature for the Bacillus thuringienses Pesticidal Crystal Proteins,” N. Crickmore, D. R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean. Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813. The same applies to Cry6's if used in triple stacks, for example, according to the subject invention.
  • genes encoding active toxins can be identified and obtained through several means.
  • the specific genes or gene portions exemplified herein may be obtained from the isolates deposited at a culture depository. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Genes that encode active fragments may also be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these protein toxins.
  • Fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the subject invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to “essentially the same” sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity. Fragments of genes encoding proteins that retain pesticidal activity are also included in this definition.
  • a further method for identifying the genes encoding the toxins and gene portions useful according to the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO93/16094. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology. Preferably, hybridization is conducted under stringent conditions by techniques well-known in the art, as described, for example, in Keller, G. H., M. M.
  • DNA Probes Stockton Press, New York, N.Y., pp. 169-170.
  • salt concentrations and temperature combinations are as follows (in order of increasing stringency): 2 ⁇ SSPE or SSC at room temperature; 1 ⁇ SSPE or SSC at 42° C.; 0.1 ⁇ SSPE or SSC at 42° C.; 0.1 ⁇ SSPE or SSC at 65° C.
  • Detection of the probe provides a means for determining in a known manner whether hybridization has occurred.
  • Such a probe analysis provides a rapid method for identifying toxin-encoding genes of the subject invention.
  • the nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures. These nucleotide sequences can also be used as PCR primers to amplify genes of the subject invention.
  • Variant toxins Certain toxins of the subject invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin.
  • Equivalent toxins will have amino acid homology with an exemplified toxin. This amino acid identity will typically be greater than 75%, or preferably greater than 85%, preferably greater than 90%, preferably greater than 95%, preferably greater than 96%, preferably greater than 97%, preferably greater than 98%, or preferably greater than 99% in some embodiments.
  • amino acid identity will typically be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity.
  • certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule.
  • amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound.
  • Table 1 provides a listing of examples of amino acids belonging to each class.
  • non-conservative substitutions can also be made.
  • the critical factor is that these substitutions must not significantly detract from the biological activity of the toxin.
  • Recombinant hosts The genes encoding the toxins of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. Conjugal transfer and recombinant transfer can be used to create a Bt strain that expresses both toxins of the subject invention. Other host organisms may also be transformed with one or both of the toxin genes then used to accomplish the synergistic effect. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested. The result is control of the pest.
  • suitable microbial hosts e.g., Pseudomonas
  • the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell.
  • the treated cell which retains the toxic activity, then can be applied to the environment of the target pest.
  • Non-regenerable/non-totipotent plant cells from a plant of the subject invention (comprising at least one of the subject IRM genes) are included within the subject invention.
  • a preferred embodiment of the subject invention is the transformation of plants with genes encoding the subject insecticidal protein or its variants.
  • the transformed plants are resistant to attack by an insect target pest by virtue of the presence of controlling amounts of the subject insecticidal protein or its variants in the cells of the transformed plant.
  • genetic material that encodes the insecticidal properties of the B.t. insecticidal toxins into the genome of a plant eaten by a particular insect pest, the adult or larvae would die after consuming the food plant. Numerous members of the monocotyledonous and dicotyledonous classifications have been transformed. Transgenic agronomic crops as well as fruits and vegetables are of commercial interest.
  • Such crops include, but are not limited to, maize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like.
  • Genes encoding any of the subject toxins can be inserted into plant cells using a variety of techniques which are well known in the art as disclosed above. For example, a large number of cloning vectors comprising a marker that permits selection of the transformed microbial cells and a replication system functional in Escherichia coli are available for preparation and modification of foreign genes for insertion into higher plants. Such manipulations may include, for example, the insertion of mutations, truncations, additions, or substitutions as desired for the intended use.
  • the vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the Cry protein or variants can be inserted into the vector at a suitable restriction site.
  • the resulting plasmid is used for transformation of cells of E. coli, the cells of which are cultivated in a suitable nutrient medium, then harvested and lysed so that workable quantities of the plasmid are recovered.
  • Sequence analysis, restriction fragment analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each manipulated DNA sequence can be cloned in the same or other plasmids.
  • T-DNA-containing vectors for the transformation of plant cells has been intensively researched and sufficiently described in EP 120516; Lee and Gelvin (2008), Fraley et al. (1986), and An et al. (1985), and is well established in the field.
  • the vector used to transform the plant cell normally contains a selectable marker gene encoding a protein that confers on the transformed plant cells resistance to a herbicide or an antibiotic, such as bialaphos, kanamycin, G418, bleomycin, or hygromycin, inter alia.
  • the individually employed selectable marker gene should accordingly permit the selection of transformed cells while the growth of cells that do not contain the inserted DNA is suppressed by the selective compound.
  • a large number of techniques are available for inserting DNA into a host plant cell. Those techniques include transformation with T-DNA delivered by Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent. Additionally, fusion of plant protoplasts with liposomes containing the DNA to be delivered, direct injection of the DNA, biolistics transformation (microparticle bombardment), or electroporation, as well as other possible methods, may be employed.
  • plants will be transformed with genes wherein the codon usage of the protein coding region has been optimized for plants. See, for example, U.S. Pat. No. 5,380,831, which is hereby incorporated by reference. Also, advantageously, plants encoding a truncated toxin will be used. The truncated toxin typically will encode about 55% to about 80% of the full length toxin. Methods for creating synthetic B.t. genes for use in plants are known in the art (Stewart, 2007).
  • the gene is preferably incorporated into a gene transfer vector adapted to express the B.t insecticidal toxin genes and variants in the plant cell by including in the vector a plant promoter.
  • promoters from a variety of sources can be used efficiently in plant cells to express foreign genes.
  • promoters of bacterial origin such as the octopine synthase promoter, the nopaline synthase promoter, and the mannopine synthase promoter.
  • Non- Bacillus - thuringienses promoters can be used in some preferred embodiments.
  • Promoters of plant virus origin may be used, for example, the 35S and 19S promoters of Cauliflower Mosaic Virus, a promoter from Cassava Vein Mosaic Virus, and the like.
  • Plant promoters include, but are not limited to, ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, phaseolin promoter, ADH (alcohol dehydrogenase) promoter, heat-shock promoters, ADF (actin depolymerization factor) promoter, ubiquitin promoter, actin promoter, and tissue specific promoters. Promoters may also contain certain enhancer sequence elements that may improve the transcription efficiency.
  • Typical enhancers include but are not limited to ADH1-intron 1 and ADH1-intron 6.
  • Constitutive promoters may be used. Constitutive promoters direct continuous gene expression in nearly all cells types and at nearly all times (e.g., actin, ubiquitin, CaMV 35S). Tissue specific promoters are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP (Acyl Carrier Protein) promoters), and these promoters may also be used. Promoters may also be used that are active during a certain stage of the plants' development as well as active in specific plant tissues and organs. Examples of such promoters include but are not limited to promoters that are root specific, pollen-specific, embryo specific, corn silk specific, cotton fiber specific, seed endosperm specific, phloem specific, and the like.
  • an inducible promoter is responsible for expression of genes in response to a specific signal, such as: physical stimulus (e.g. heat shock genes); light (e.g. RUBP carboxylase); hormone (e.g. glucocorticoid); antibiotic (e.g. tetracycline); metabolites; and stress (e.g. drought).
  • a specific signal such as: physical stimulus (e.g. heat shock genes); light (e.g. RUBP carboxylase); hormone (e.g. glucocorticoid); antibiotic (e.g. tetracycline); metabolites; and stress (e.g. drought).
  • Other desirable transcription and translation elements that function in plants may be used, such as 5′ untranslated leader sequences, RNA transcription termination sequences and poly-adenylate addition signal sequences. Numerous plant-specific gene transfer vectors are known to the art.
  • Transgenic crops containing insect resistance (IR) traits are prevalent in corn and cotton plants throughout North America, and usage of these traits is expanding globally.
  • Commercial transgenic crops combining IR and herbicide tolerance (HT) traits have been developed by multiple seed companies. These include combinations of IR traits conferred by B.t.
  • insecticidal proteins and HT traits such as tolerance to Acetolactate Synthase (ALS) inhibitors such as sulfonylureas, imidazolinones, triazolopyrimidine, sulfonanilides, and the like, Glutamine Synthetase (GS) inhibitors such as bialaphos, glufosinate, and the like, 4-HydroxyPhenylPyruvate Dioxygenase (HPPD) inhibitors such as mesotrione, isoxaflutole, and the like, 5-EnolPyruvylShikimate-3-Phosphate Synthase (EPSPS) inhibitors such as glyphosate and the like, and Acetyl-Coenzyme A Carboxylase (ACCase) inhibitors such as haloxyfop, quizalofop, diclofop, and the like.
  • ALS Acetolactate Synthase
  • transgenically provided proteins provide plant tolerance to herbicide chemical classes such as phenoxy acids herbicides and pyridyloxyacetates auxin herbicides (see WO 2007/053482 A2), or phenoxy acids herbicides and aryloxyphenoxypropionates herbicides (see WO 2005107437 A2, A3).
  • herbicide chemical classes such as phenoxy acids herbicides and pyridyloxyacetates auxin herbicides (see WO 2007/053482 A2), or phenoxy acids herbicides and aryloxyphenoxypropionates herbicides (see WO 2005107437 A2, A3).
  • IR traits a valuable commercial product concept, and the convenience of this product concept is enhanced if insect control traits and weed control traits are combined in the same plant. Further, improved value may be obtained via single plant combinations of IR traits conferred by a B.t.
  • insecticidal protein such as that of the subject invention, with one or more additional HT traits such as those mentioned above, plus one or more additional input traits (e.g. other insect resistance conferred by B.t.-derived or other insecticidal proteins, insect resistance conferred by mechanisms such as RNAi and the like, nematode resistance, disease resistance, stress tolerance, improved nitrogen utilization, and the like), or output traits (e.g. high oils content, healthy oil composition, nutritional improvement, and the like).
  • additional input traits e.g. other insect resistance conferred by B.t.-derived or other insecticidal proteins, insect resistance conferred by mechanisms such as RNAi and the like, nematode resistance, disease resistance, stress tolerance, improved nitrogen utilization, and the like
  • output traits e.g. high oils content, healthy oil composition, nutritional improvement, and the like.
  • Such combinations may be obtained either through conventional breeding (breeding stack) or jointly as a novel transformation event involving the simultaneous introduction of multiple genes (molecular stack).
  • Benefits include the ability
  • the transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants.
  • Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors.
  • the resulting hybrid individuals have the corresponding phenotypic properties.
  • plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Pat. No. 5,380,831.
  • methods for creating synthetic Bt genes for use in plants are known in the art (Stewart and Burgin, 2007).
  • a preferred transformed plant is a fertile maize plant comprising a plant expressible gene encoding a Cry3Aa protein, and further comprising a second plant expressible genes encoding a Cry34Ab/35Ab protein.
  • Transfer (or introgression) of the Cry3Aa- and Cry34Ab/35Ab-determined trait(s) into inbred maize lines can be achieved by recurrent selection breeding, for example by backcrossing.
  • a desired recurrent parent is first crossed to a donor inbred (the non-recurrent parent) that carries the appropriate gene(s) for the Cry-determined traits.
  • the progeny of this cross is then mated back to the recurrent parent followed by selection in the resultant progeny for the desired trait(s) to be transferred from the non-recurrent parent.
  • the progeny will be heterozygous for loci controlling the trait(s) being transferred, but will be like the recurrent parent for most or almost all other genes (see, for example, Poehlman & Sleper (1995) Breeding Field Crops, 4th Ed., 172-175; Fehr (1987) Principles of Cultivar Development, Vol. 1: Theory and Technique, 360-376).
  • IRM Insect Resistance Management
  • non-transgenic i.e., non-B.t.
  • refuges a block of non-Bt crops/corn
  • the above percentages, or similar refuge ratios, can be used for the subject double or triple stacks or pyramids.
  • DNA fragments were purified using the Millipore Ultrafree®-DA cartridge (Billerica, Mass.) after agarose Tris-acetate gel electrophoresis.
  • the basic cloning strategy entailed subcloning the coding sequences (CDS) of full-length Cry34Ab1, Cry35Ab1, and Cry3Aa1 Cry proteins into pMYC1803 at, for example, SpeI and XhoI (or XbaI, or HindIII) restriction sites, respectively, whereby they were placed under the expression control of the Ptac promoter and the rrnBT1T2 terminator from plasmid pKK223-3 (PL Pharmacia, Milwaukee, Wis.), respectively.
  • pMYC1803 is a medium copy plasmid with the RSF1010 origin of replication, a tetracycline resistance gene, and a ribosome binding site preceding the restriction enzyme recognition sites into which DNA fragments containing protein coding regions may be introduced (US Patent Application No. 20080193974).
  • the expression plasmid was transformed by electroporation into a P. fluorescens strain MB214, recovered in SOC-Soy hydrolysate medium, and plated on Luria broth (LB) medium containing 20 ⁇ g/ml tetracycline. Details of the microbiological manipulations are available US Patent Application No. 20060008877, US Patent Application No. 20080193974, and US Patent Application No.
  • Colonies were screened by restriction digestion of miniprep plasmid DNA. Plasmid DNA of selected clones containing inserts was sequenced by contract with a commercial sequencing vendor such as MWG Biotech (Huntsville, Ala.). Sequence data were assembled and analyzed using the SequencherTM software (Gene Codes Corp., Ann Arbor, Mich.).
  • Cry34Ab1, Cry35Ab1, and Cry3Aa1 toxins via the Ptac promoter were induced by addition of isopropyl- ⁇ -D-1-thiogalactopyranoside (IPTG) after an initial incubation of 24 hours at 30° C. with shaking Cultures were sampled at the time of induction and at various times post-induction. Cell density was measured by optical density at 600 nm (OD 600 ).
  • Cry protein inclusion body (IB) preparations were performed on cells from P. fluorescens fermentations that produced insoluble B.t. insecticidal protein, as demonstrated by SDS-PAGE and MALDI-MS (Matrix Assisted Laser Desorption/Ionization Mass Spectrometry). P. fluorescens fermentation pellets are thawed in a 37° C. water bath.
  • the cells were resuspended to 25% w/v in lysis buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 20 mM EDTA disodium salt (Ethylenediaminetetraacetic acid), 1% Triton X-100, and 5 mM Dithiothreitol (DTT); 5 mL/L of bacterial protease inhibitor cocktail (P8465 Sigma-Aldrich, St. Louis, Mo.) were added just prior to use.
  • the cells were suspended using a homogenizer at lowest setting (Tissue Tearor, BioSpec Products, Inc., Bartlesville, Okla.).
  • Lysozyme 25 mg of Sigma L7651, from chicken egg white was added to the cell suspension by mixing with a metal spatula, and the suspension was incubated at room temperature for one hour. The suspension was cooled on ice for 15 minutes, then sonicated using a Branson Sonifier 250 (two 1-minute sessions, at 50% duty cycle, 30% output). Cell lysis was checked by microscopy. An additional 25 mg of lysozyme was added if necessary, and the incubation and sonication were repeated. When cell lysis was confirmed via microscopy, the lysate was centrifuged at 11,500 ⁇ g for 25 minutes (4° C.) to form the IB pellet, and the supernatant was discarded.
  • the IB pellet was resuspended with 100 mL lysis buffer, homogenized with the hand-held mixer and centrifuged as above. The IB pellet was repeatedly washed by resuspension (in 50 mL lysis buffer), homogenization, sonication, and centrifugation until the supernatant became colorless and the IB pellet became firm and off-white in color. For the final wash, the IB pellet was resuspended in sterile-filtered (0.22 ⁇ m) distilled water containing 2 mM EDTA, and centrifuged. The final pellet was resuspended in sterile-filtered distilled water containing 2 mM EDTA, and stored in 1 mL aliquots at ⁇ 80° C.
  • SDS-PAGE analysis and quantification SDS-PAGE analysis and quantification of protein in IB preparations were done by thawing a 1 mL aliquot of IB pellet and diluting 1:20 with sterile-filtered distilled water. The diluted sample was then boiled with 4 ⁇ reducing sample buffer [250 mM Tris, pH6.8, 40% glycerol (v/v), 0.4% Bromophenol Blue (w/v), 8% SDS (w/v) and 8% ⁇ -Mercapto-ethanol (v/v)] and loaded onto a Novex® 4-20% Tris-Glycine, 12+2 well gel (Invitrogen) run with 1 ⁇ Tris/Glycine/SDS buffer (Invitrogen).
  • 4 ⁇ reducing sample buffer [250 mM Tris, pH6.8, 40% glycerol (v/v), 0.4% Bromophenol Blue (w/v), 8% SDS (w/v) and 8% ⁇ -Mercapto-ethanol (v/v
  • the gel was run for approximately 60 min at 200 volts then stained and distained by following the SimplyBlueTM Safe Stain (Invitrogen) procedures. Quantification of target bands was done by comparing densitometric values for the bands against Bovine Serum Albumin (BSA) samples run on the same gel to generate a standard curve using the Bio-Rad Quantity One software.
  • BSA Bovine Serum Albumin
  • Inclusions were resuspended using a pipette and vortexed to mix thoroughly.
  • the tubes were placed on a gently rocking platform at 4° C. overnight to extract full-length Cry34Ab1, Cry35Ab1, and Cry3Aa1 proteins.
  • the extracts were centrifuged at 30,000 ⁇ g for 30 min at 4° C., and saved the resulting supernatants containing solubilized full-length Cry proteins.
  • Full-length Cry35Ab1 and Cry3Aa1 were truncated or digested with chymotrypsin or trypsin to get their chymotrypsin or trypsin core that are an active form the proteins.
  • the molecular mass of full-length Cry35Ab1 and Cry3Aa1 was ⁇ 44 and ⁇ 73 kDa, and their chymotrypsin or trypsin core was ⁇ 40 and ⁇ 55 kDa, respectively.
  • the amino acid sequences of the chymotrypsin Cry35Ab core is provided as SEQ ID NO:5.
  • the amino acid sequences of the trypsin Cry3Aa core is provided as SEQ ID NO:2. Neither chymotrypsin or trypsin core is available for Cry34Ab1; thus the full-length Cry34Ab1 was used for binding assays.
  • the amino acid sequence of the full-length Cry34Ab1 is provided as SEQ ID NO:3.
  • the chymotrypsinized Cry35Ab1 and trypsinized Cry3Aa1 were purified. Specifically, the digestion reactions were centrifuged at 30,000 ⁇ g for 30 min at 4° C. to remove lipids, and the resulting supernatant were concentrated 5-fold using an Amicon Ultra-15 regenerated cellulose centrifugal filter device (10,000 Molecular Weight Cutoff; Millipore).
  • sample buffers were then changed to 20 mM sodium acetate buffer, pH 3.5, for both Cry34Ab1 and Cry35Ab1, and to 10 mM CAPS [3-(cyclohexamino)l-propanesulfonic acid], pH 10.5, for Cry3Aa1, using disposable PD-10 columns (GE Healthcare, Piscataway, N.J.).
  • the final volumes were adjusted to 15 ml using the corresponding buffer for purification using ATKA Explorer liquid chromatography system (Amersham Biosciences).
  • buffer A was 20 mM sodium acetate buffer, pH 3.5
  • buffer B was buffer A+1 M NaCl, pH 3.5.
  • a HiTrap SP (5 ml) column (GE) was used.
  • the Cry35Ab1 solution was injected into the column at a flow rate of 5 ml/min. Elution was performed using gradient 0-100% of buffer B at 5 ml/min with 1 ml/fraction.
  • buffer A was 10 mM CAPS buffer, pH 10.5
  • buffer B was 10 mM CAPS buffer, pH 10.5+1 M NaCl.
  • a Capto Q, 5 ml (5 ml) column (GE) was used and the all other procedures were similar to that for Cry35Ab1. After SDS-PAGE analysis of the selected fractions to further select fractions containing the best quality target protein, pooled those fractions.
  • the buffer was changed for the purified Cry35Ab1 chymotrypsin core with 20 mM Bist-Tris, pH 6.0, as described above.
  • the salt was removed using disposable PD-10 columns (GE Healthcare, Piscataway, N.J.). The samples were saved at 4° C. for later binding assay after being quantified using SDS-PAGE and the Typhoon imaging system (GE) analyses with BSA as a standard.
  • BBMV preparations Brush border membrane vesicle (BBMV) preparations of insect midguts have been widely used for Cry toxin receptor binding assays.
  • the BBMV preparations used in this invention were prepared from isolated midguts of third instars of the western corn rootworm ( Diabrotica virgifera virgifera LeConte) using the method described by Wolferberger et al. (1987).
  • Leucine aminopeptidase was used as a marker of membrane proteins in the preparation and Leucine aminopeptidase activities of crude homogenate and BBMV preparation were determined as previously described (Li et al. 2004a). Protein concentration of the BBMV preparation was measured using the Bradford method (1976).
  • 125 I Labeling Purified full-length Cry34Ab1, chymotrypsinized Cry35Ab1, and trypsinized Cry3Aa were labeled using 125 I for homologous and competition binding assays. To ensure the radio-labeling does not abolish the biological activity of the Cry toxins, cold iodination was conducted using NaI followed the instructions of Pierce® Iodination Beads (Pierce Biotechnology, Thermo Scientific, Rockford, Ill.). Bioassay results indicated that iodinated Cry35Ab1 chymotrypsin core remained active against the larvae of the western corn rootworm, but iodination inactivated Cry34Ab1.
  • Radiolabeled 125 I-Cry34Ab1 did not specifically bind to the insect BBMV, and Cry34Ab1 requires another labeling method to assess membrane receptor binding. Bioassay using cold iodinated Cry3Aa trypsin core is on going and the result will be available in one or two weeks. Radiolabeled 125 I-Cry35Ab1 and 125 I-Cry3Aa1 were obtained with Pierce® Iodination Beads (Pierce) and Na 125 I. ZebaTM Desalt Spin Columns (Pierce) were used to remove unincorporated or free Na 125 I from the iodinated protein. The specific radioactivities of the iodinated Cry proteins ranged from 1-5 uCi/ug. Multiple batches of labeling and binding assays were conducted.
  • Specific binding assays were performed using 125 I-labeled Cry toxins as described previously (Li et al. 2004b). To determine specific binding and estimate the binding affinity (disassociation constant, Kd) and binding site concentration (Bmax) of Cry35Ab1 and Cry3Aa to the insect BBMV, a series of increasing concentrations of either 125 I-Cry35Ab1 or 125 I-Cry3Aa were incubated with a given concentration (0.05 mg/ml) of the insect BBMV, respectively, in 150 ul of 20 mM Bis-Tris, pH 6.0, 136.9 mM NaCl, 2.7 mM KCl, supplemented with 0.1% BSA at room temperature for 60 min with gentle shaking Toxin bound to BBMV was separated from free toxins in the suspension by centrifugation at 20,000 ⁇ g at room temperature for 8 min.
  • Kd binding affinity
  • Bmax binding site concentration
  • the pellet was washed twice with 900 ul of ice-cold the same buffer containing 0.1% BSA.
  • the radioactivity remaining in the pellet was measured with a COBRAII Auto-Gamma counter (Packard, a Canberra company) and considered total binding.
  • Another series of binding reactions were setup at side by side, and a 500-1,000-fold excess of unlabeled corresponding toxin was included in each of the binding reactions to fully occupy all specific binding sites on the BBMV, which was used to determine non-specific binding. Specific binding was estimated by subtracting the non-specific binding from the total binding.
  • the Kd and Bmax values of these toxins were estimated using the specific binding against the concentrations of the labeled toxin used by running GraphPad Prism 5.01 (GraphPad Software, San Diego, Calif.). The charts were made using either Microsoft Excel or GraphPad Prism programs. The experiments were replicated at least three times. These binding experiments demonstrated that both 125 I-Cry35Ab1 and 125 I-Cry3Aa were able to specifically bind to the BBMV ( FIGS. 1A and 1B ).
  • Competition binding assays were further conducted to determine if Cry35Ab1 and Cry3Aa share a same set of receptors.
  • Cry3Aa homologous competition binding assays increasing amounts (0-5,000 nM) of unlabeled Cry3Aa were first mixed with 5 nM labeled Cry3Aa, and then incubated with a given concentration (0.05 mg/ml) of BBMV at room temperature for 60 min, respectively. The percentages of bound 125 I-Cry3Aa with BBMV were determined for each of the reactions as compared to the initial specific binding at absence of unlabeled competitor.
  • Heterologous competition binding assays between 125 I-Cry3Aa and unlabeled Cry35Ab1 alone or unlabeled Cry35Ab1+Cry34Ab1 (1:3 molar ratio) were also performed respectively to identify if they share a same set of receptor(s). This was achieved by increasing the amount of unlabeled Cry35Ab1 alone or the Cry35Ab1+Cry34Ab1 mixture as one or two competitors included in the reactions to compete for the putative receptor(s) on the BBMV with the labeled Cry3Aa. The experiments were replicated at least three times.

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