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WO2009149787A1 - Gestion de la résistance aux vers de la capsule dans des plantes transgéniques - Google Patents

Gestion de la résistance aux vers de la capsule dans des plantes transgéniques Download PDF

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Publication number
WO2009149787A1
WO2009149787A1 PCT/EP2009/002788 EP2009002788W WO2009149787A1 WO 2009149787 A1 WO2009149787 A1 WO 2009149787A1 EP 2009002788 W EP2009002788 W EP 2009002788W WO 2009149787 A1 WO2009149787 A1 WO 2009149787A1
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WO
WIPO (PCT)
Prior art keywords
protein
plants
event
cotton
insecticidal
Prior art date
Application number
PCT/EP2009/002788
Other languages
English (en)
Inventor
Carmen Sara Hernandez
Adri Van Vliet
Jeroen Van Rie
Juan Ferré MANZANERO
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Bayer Bioscience N.V.
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Publication date
Priority claimed from US12/214,022 external-priority patent/US20090313717A1/en
Application filed by Bayer Bioscience N.V. filed Critical Bayer Bioscience N.V.
Priority to CN2009801221653A priority Critical patent/CN102066566A/zh
Priority to BRPI0914615A priority patent/BRPI0914615A2/pt
Priority to CA2727637A priority patent/CA2727637A1/fr
Priority to US12/997,133 priority patent/US20110088129A1/en
Priority to EP09761350A priority patent/EP2300618A1/fr
Publication of WO2009149787A1 publication Critical patent/WO2009149787A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the field of plant pest control, particularly insect control.
  • This invention relates to the use of transgenic plant cells and plants in an insect resistance management process, wherein the genomes of said cells and plants (or more typically, predecessor plant cells or plants) have been provided with at least two genes, each encoding a different protein insecticidal to Helicoverpa zea or Helicoverpa armigera, wherein such proteins bind saturably to the brush border membrane of such insect species, which proteins are: a) a Cry2A protein and b) a Cry1A, Cry1F or VIP3A protein, such as a VIP3A, a CryiAc, a CrylAb or a Cry1A.1O5 protein.
  • such plants are used to delay or prevent the development of resistance to crop plants in populations of the cotton bollworm.
  • the simultaneous or sequential use of a Cry2A protein and a VIP3A, Cry1A or Cry1 F protein or plants expressing such Cry2A protein and a VIP3A, Cry1A or Cry1 F protein, to delay or prevent resistance development in cotton bollworms, particularly Helicoverpa zea or Helicoverpa armigera, is provided.
  • Such transformed plants have advantages over plants transformed with a single insecticidal protein gene, or plants transformed with a Cry1 F- and a Cry1A- encoding gene, especially with respect to the delay or prevention of resistance development in populations of cotton bollworms, against the insecticidal proteins expressed in such plants.
  • This invention also relates to a process for the production of transgenic plants, particularly corn, cotton, rice, soybean, sorghum, tomato, sunflower and sugarcane, comprising at least two different insecticidal Cry proteins that show no competition for binding to the binding sites in the midgut brush border of Helicoverpa zea or Helicoverpa armigera larvae.
  • Simultaneous expression in plants of chimeric genes encoding a Cry2A protein and a VIP3A, Cry1 F or Cry1A protein, particularly a VIP3Aa, CrylAb or CryiAc protein is particularly useful to prevent or delay resistance development of populations of cotton bollworms against the insecticidal proteins expressed in such plants.
  • This invention further relates to a process for preventing or delaying the development of resistance in populations of Helicoverpa zea or Helicoverpa armigera to transgenic plants expressing a VIP3 or a Cry1A and/or a Cry1 F protein, comprising providing such plants also with a gene expressing a Cry2A protein. Since such Cry2A protein and such Cry1 A or VIP3 or Cry1 F protein do not compete for specific binding sites in the midgut brush border of Helicoverpa zea or Helicoverpa armigera larvae, these combinations are useful for securing long-lasting protection against said larvae.
  • This invention also relates to a method to control Helicoverpa zea or Helicoverpa armigera insects in a region where populations of said insect species have become resistant to plants comprising a VIP3, Cry1F and/or a Cry1A protein, comprising the step of sowing, planting or growing in said region, seeds or plants containing at least a gene encoding a Cry2A protein.
  • said plants can also comprise (besides the gene encoding a Cry2A protein) a gene encoding another insecticidal protein which does not share binding sites with such Cry2A, Vl P3, Cry1 F or Cry1A protein in Helicoverpa zea or Helicoverpa armigera.
  • Helicoverpa zea and Helicoverpa armigera are amongst the most significant polyphagous lepidopteran pest species in the New and Old World, respectively. These insects have a history of rather rapid resistance development to insecticides, and they are typically less sensitive to many Bt-de ⁇ ved insecticidal proteins compared to important other lepidopteran insect pests. Hence these insect species are amongst the most likely candidates to develop resistance to Bt-plants, such as Bt cotton or Bt corn plants.
  • Cry1 A The most widely used proteins introduced in plants for control of Lepidopteran insects include the Cry1 A, Cry1 F and VIP3A proteins
  • Cry1 F protein Based on competition binding assays, it has been proposed that a Cry1 F protein competes for the same midgut binding site as CryiAc in Helicoverpa zea and Helicoverpa armigera. Moreover, no evidence was found for any unshared sites for Cry1 F in these insects species (Hernandez and Ferre, 2005).
  • EPA biopesticide factsheet 006487 states that the Cry2Ab protein, and Cry2 proteins in general, produce highly potent ion channels to compensate for binding either to themselves or to a large collection of non-specific binding sites.
  • www.epa.gov/opp00001/biopesticides/ingredients/factsheets/factsheet_006487.htm Also, English et al. (1994) and Karim et al. (2000b) reported at least partial competition or the sharing of a common binding site for a Cry1A and Cry2A protein in Helicoverpa zea.
  • USDA-APHIS petition for non-regulated status 06-298-01 p (2006) states that a Cry1A and Cry2A protein share many common binding sites (www.aphis.usda.gov/brs/aphisdocs/06 29801p.pdf).
  • a method of controlling Helicoverpa zea or Helicoverpa armigera infestation in transgenic plants while securing a slower buildup of Helicoverpa zea or Helicoverpa armigera insect resistance development to said plants comprising expressing a combination of a) a Cry2Ae protein insecticidal to said insect species and b) a Cry1A, Cry1 F or VIP3A protein insecticidal to said insect species, in said plants.
  • Also provided herein is a method for preventing or delaying insect resistance development in populations of the insect species Helicoverpa zea or Helicoverpa armigera to transgenic plants expressing insecticidal proteins to control said insect pest, comprising expressing a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera in combination with a Cry1A, Cry1 F of VIP3A protein insecticidal to Helicoverpa zea or Helicoverpa armigera in said plants.
  • a method is provided to control Helicoverpa zea or Helicoverpa armigera in a region where populations of said insect species have become resistant to plants expressing a VIP3A, Cry1A or a Cry1 F protein, comprising the step of sowing or planting in said region, plants expressing at least a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • a method to control Helicoverpa zea or Helicoverpa armigera in a region where populations of said insect have become resistant to plants expressing a Cry2Ae protein comprising the step of sowing or planting in said region, plants expressing a Cry1 F, VIP3, or Cry1A protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • Also provided in accordance with this invention is a method for obtaining plants comprising chimeric genes encoding at least two different insecticidal proteins, wherein said proteins do not share binding sites in larvae of the species Helicoverpa zea or Helicoverpa armigera as determined in competition binding experiments using brush border membrane vesicles of said insect larvae, comprising the step of obtaining plants comprising a plant-expressible chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and a plant- expressible chimeric gene encoding a Cry1A, VIP3 or Cry1F protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • plants are obtained by transformation of a plant with plant-expressible chimeric genes encoding said Cry2Ae and Cry1A, VIP3 of Cry1 F proteins, and by obtaining progeny plants and seeds of said plants comprising said chimeric genes; or by the crossing of a parent plant comprising said Cry2Ae-encoding chimeric gene with a parent plant comprising said Cry1A-, VIP3- or Cry1F-encoding chimeric gene, and obtaining progeny plants and seeds comprising said chimeric genes; or by transformation of a plant comprising a plant-expressible chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera with a second plant-expressible chimeric gene encoding a Cry1A, VIP3 or Cry1 F protein insecticidal to Helicoverpa zea or Helicoverpa armigera, and obtaining progeny plants
  • a method for obtaining plants expressing at least two different insecticidal proteins wherein said proteins do not share midgut binding sites in larvae of the species Helicoverpa zea or Helicoverpa armigera as can be determined in competition binding experiments using brush border membrane vesicles of said larvae, and wherein said proteins are: a) Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and b) a Cry1 A, VIP3 or Cry1 F protein insecticidal to Helicoverpa zea or Helicoverpa armigera, particularly a VIP3 or Cry1 A protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • Also provided here is a method of sowing, planting, or growing plants protected against cotton bollworms, comprising chimeric genes expressing at least two different insecticidal proteins, wherein said proteins do not share binding sites in larvae of the species Helicoverpa zea or Helicoverpa armigera as determined in competition binding experiments using brush border membrane vesicles of said larvae, comprising the step of: sowing, planting, or growing plants comprising a chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and a chimeric gene encoding a Cry1A, VIP3 or Cry1 F protein insecticidal to Helicoverpa zea or Helicoverpa armigera, preferably a VIP3 or Cry1A protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera in combination with a Cry1A, VIP3 or Cry1 F protein insecticidal to insects of said species, to prevent or delay resistance development of insects of said species to transgenic plants expressing heterologous insecticidal toxins, particularly when said use is by expression of said protein combination in plants.
  • a chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and a chimeric gene encoding a Cry1A, VIP3 or Cry1 F protein insecticidal to Helicoverpa zea or Helicoverpa armigera particularly a chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and a chimeric gene encoding a Cry1A or VIP3 protein insecticidal to Helicoverpa zea or Helicoverpa armigera, in a method to obtain plants capable of expressing at least two different insecticidal proteins, wherein said proteins do not share binding sites in larvae of the species Helicoverpa zea or Helicoverpa armigera as can be determined in competition binding experiments, such as by using brush border membrane vesicles of said insect larvae.
  • a chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera is provided to obtain plants comprising at least two different insecticidal proteins, wherein said proteins do not share midgut binding sites in larvae of the species Helicoverpa zea or Helicoverpa armigera, as can be determined in competition binding experiments, such as by using brush border membrane vesicles of said insect larvae, wherein said Cry2Ae chimeric gene is present in plants also comprising a chimeric gene encoding a Cry1A, VIP3 or Cry1 F protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • the above uses include the step of obtaining plants comprising such different insecticidal proteins by transformation of a plant with chimeric genes encoding said Cry2Ae and Cry1A, VIP3 or Cry1 F proteins, and the obtaining of plants comprising such different insecticidal proteins by crossing plants comprising a chimeric gene encoding said Cry2Ae protein with plants comprising a chimeric gene encoding said Cry1A, VIP3 or Cry1 F protein, and obtaining progeny plants and seeds of said plant comprising said chimeric genes.
  • the invention also provides for the use, the sowing, planting or growing of a refuge area with plants not comprising a Cry2, Cry1 or VIP3 protein insecticidal to Helicoverpa zea or Helicoverpa armigera, such as by sowing, planting or growing such plants in the same field or in the vicinity of the plants comprising the Cry2Ae, VIP3 and Cry1 protein described herein.
  • a process for growing, sowing or planting plants expressing a Cry protein or VIP3 protein for control of Helicoverpa armigera or Helicoverpa zea insects comprising the step of planting, sowing or growing an insecticide sprayed structured refuge area of less than 20 %, less than 15 %, or less than 10 % or an non-insecticide sprayed structured refuge area of less than 5 %, of the planted field or in the vicinity of the planted field, or without planting, sowing or growing a structured refuge area in a field, wherein such structured refuge area is a location in the same field or is within 2 miles, within 1 mile or within 0.5 miles of a field, and which contains plants not comprising such Cry or VIP3 protein, wherein such plants expressing a Cry or VIP3 protein express a combination of a Cry2Ae protein insecticidal to said insect species, and a Cry1A, Cry1 F or VIP3A protein, particularly a Cry2Ae and a Cry1 Ab
  • a field of plants comprising a structured refuge of less than 20 %, of less than 15 %, of less than 10 %, or of less than 5 %, or comprising no structured refuge, wherein said field is planted with plants expressing a combination of a Cry2Ae of Cry2Ab protein insecticidal to Helicoverpa armigera or Helicoverpa zea insects, and a Cry1A, Cry1F or VIP3A protein, particularly a Cry2Ae and a CrylAb, CryiAc or VIP3A protein, preferably a Cry2Ae and CrylAb and VIP3 protein, insecticidal to one of said insect species.
  • Also provided in one embodiment of this invention is the use of at least 2 insecticidal proteins binding specifically and saturably to binding sites in the midgut of Helicoverpa zea larvae, for delaying or preventing resistance development of such insect species to plants expressing insecticidal proteins, wherein one of said proteins in said plants is a Cry2A protein, such as a Cry2Ab protein, insecticidal to such insect species, and the other protein is a Cry1A, Cry1 F or VIP3 protein insecticidal to such insect species, wherein such saturable binding is determined in a saturability assay using a fixed concentration of binding sites (i.e., BBMVs) to which increasing concentrations of labeled protein are added.
  • BBMVs fixed concentration of binding sites
  • the Cry1A protein is selected from the group of: a CryiAc, CrylAb, Cry1A.1O5, or a CryiAc or CrylAb hybrid protein, such as a protein encoded by any one of the cry1A coding regions referred to herein.
  • Such Cry2Ab and Cry1 A proteins do not compete for their (saturable and specific) binding sites in the midgut of such H. zea insect larvae, as can be measured in BBMV competition binding assays.
  • a Cry2Ae protein refers to an insecticidal Cry2Ae protein such as a full length Cry2Ae protein of SEQ ID No. 2 of WO 2002/057664 (Cry2Ae1, SEQ ID No. 1 ), a Cry2Ae toxic fragment or a protein comprising a Cry2Ae toxic fragment as described in of WO 2002/057664, such as a fusion protein of a Cry2Ae protein fragment with a chloroplast transit peptide or another peptide sequence insecticidal to H. zea or H. armigera, or is a protein insecticidal to H. zea or H.
  • armigera comprising an amino acid sequence with at least 95, 97 or 99 % sequence identity to the amino acid sequence of SEQ ID No. 1 herein or to SEQ ID No. 2 of WO 2002/057664, particularly in the part corresponding to the smallest toxic fragment, or is a protein encoded by the Cry2Ae coding region part of the Cry2Ae chimeric gene contained in cotton event EE-GH6 as described in the PCT patent application claiming priority to European patent application number 07075460 or 07075485 (unpublished), particularly any protein comprising the smallest toxic fragment of any one of such Cry2Ae proteins, or a variant of any one of such Cry2Ae proteins differing in 1-5 amino acids retaining toxicity to Helicoverpa zea or Helicoverpa armigera.
  • a Cry2Ab protein refers to any one of the Cry2Ab proteins of Crickmore et al. (1998), or www.lifesci.susx.ac.uk/home/Neil_Crickmore/Bt/ insecticidal to H. zea or H. armigera, such as a full length Cry2Ab protein, a Cry2Ab toxic fragment, a Cry2Ab2 protein (SEQ ID No.
  • a protein comprising a Cry2Ab toxic fragment such as a fusion protein of a Cry2Ab2 protein fragment with a chloroplast transit peptide or another peptide sequence retaining toxicity to Helicoverpa zea or Helicoverpa armigera, or is a protein insecticidal to Helicoverpa zea or Helicoverpa armigera comprising an amino acid sequence with at least 95, 97 or 99 % sequence identity to SEQ ID No.
  • a Cry1 F protein includes any protein comprising the smallest toxic fragment of the amino acid sequence of a Cry1F protein retaining toxicity to Helicoverpa zea or Helicoverpa armigera, such as the protein of NCBI accession AAA22347 (SEQ ID No. 10 of US 2005049410), or a Cry1Fa1 protein (SEQ ID No. 3). Also included in this definition are variants of the amino acid sequence in NCBI accession AAA22347 of SEQ ID No. 3, such as amino acid sequences having a sequence identity of at least 90% to SEQ ID No.
  • a Cry1 F protein includes the protein encoded by the Cry1 F gene in Cry1F Cotton Event 281-24-236 (WO 2005/103266, see USDA APHIS petition for non-regulated status 03-036-01 p), or in corn events TC1507 or TC-2675 (US 7,288,643, WO 2004/099447, USDA APHIS petitions for non-regulated status 00-136-01 p and 03-181 -01 p), particularly any protein comprising the smallest toxic fragment of any one of such Cry1 F proteins, or a variant of any one of such Cry1 F proteins differing in 1-5 amino acids with toxicity to Helicoverpa zea or Helicoverpa armigera.
  • the VIP3 protein is a protein insecticidal ' to Helicoverpa zea or Helicoverpa armigera larvae, and which is any one of the VIP3 proteins listed in Crickmore et al. (2008), or any protein comprising the smallest toxic fragment of any one of these proteins
  • the VIP3 protein used is a VIP3A protein insecticidal to Helicoverpa zea or Helicoverpa armigera, such as the VIP3Aa1 protein of SEQ ID No. 4, the VIP3Af1 protein of SEQ ID No. 5, VIP3Aa19 (NCBI accession ABG20428, EPA experimental use permit factsheet 006499 (2007), SEQ ID No.
  • VIP3Aa20 protein (SEQ ID No. 7 herein) described herein, but also any protein comprising an insecticidal fragment or functional domain thereof, as well as any protein insecticidal to Helicoverpa zea or Helicoverpa armigera with a sequence identity of at least 70 % with the VIP3Aa1 protein of SEQ ID No. 4, or NCBI accession AAC37036 (Estruch et al., 1996), particularly with its smallest toxic fragment, or with the VIP3Af1 protein of NCBI accession CAI43275 (SEQ ID No. 5 herein, SEQ ID No.
  • VIP3A protein insecticidal to Helicoverpa zea or Helicoverpa armigera selected from the group of: VIP3Ab, VIP3Ac, VIP3Ad, VIP3Ae, VIP3Af, VIP3Ag, or VIP3Ah, particularly the VIP3Af1, VIP3Ad1 or VIP3Ae1 proteins (NCBI accessions CAI43275 (ISP3a, SEQ ID No.4 of WO 03/080656), CAI43276 (ISP3b, SEQ ID No.6 in WO 03/080656), and CAI43277 (ISP3C, SEQ ID No.
  • the VIP3 protein is the VIP3Aa19 protein (NCBI accession ABG20428, SEQ ID No. 6) introduced in cotton plants (e.g., in plants containing event COT102 described in WO 2004/039986, or in USDA APHIS petition for non- regulated status 03-155-01 p) or the VIP3Aa20 protein (NCBI accession ABG20429, SEQ ID NO: 2 in WO 2007/142840, SEQ ID No.
  • a Cry1A protein refers to a Cry1Ac1 (SEQ ID No. 8), Cry1A.1O5 (SEQ ID No. 9 ) or a Cry1Ab1 (SEQ ID No.
  • protein includes any protein comprising the smallest toxic fragment of the amino acid sequence of a CryiAc, Cry1A.1O5 or CryiAb protein retaining toxicity to Helicoverpa zea or Helicoverpa armigera, such as any protein comprising the smallest toxic fragment of the protein in SEQ ID No. 8 or in NCBI accession AAA22331 (CryiAc; Adang et al., 1985), of the protein in SEQ ID No. 10 or in NCBI accession AAA22330 (Wabiko et al., 1986 (CrylAb)), or of the Cry1A.1O5 protein in SEQ ID No.
  • variants of the amino acid sequence in NCBI accession AAA22331 (Cry1Ac1), NCBI accession AAA22330 (CrylAb, Wabiko et al., 1986), or the amino acid sequence of the Cry1A.1O5 protein described in USDA APHIS petition for non-regulated status 06-298-01 p, such as proteins having an amino acid sequence identity of at least 90% with such a CryiAc, Cry1A.1O5 or CrylAb protein, particularly of SEQ ID Nos.
  • Cry1A proteins include the CrylAb protein encoded by SEQ ID NO:3 of US 6,114,608, particularly the CrylAb protein encoded by the crylAb coding region in corn event MON810 (US 6,713,259), USDA APHIS petition for non- deregulated status 96-017-01 p and extensions thereof), the CrylAb protein encoded by the crylAb coding region in corn event Bt11 (USDA APHIS petition for non- deregulated status 95-195-01 p, US patent 6,114,608), the CryiAc protein encoded by the transgene in cotton event 3006-210-23 (US 7,179,965, WO 2005/103266, USDA APHIS petition for non-deregulated status 03-036-02p), the CrylAb protein encoded by the crylAb coding region in cotton event COT67B (USDA APHIS petition for non-deregulated status 07-108-01 p, WO 2006/128573), the CrylAb coding region contained in cotton event EE-GH5 described in PC
  • plants or seeds comprising at least 2 transgenes each encoding a different protein insecticidal to H. zea or armigera which proteins bind saturably and specifically to binding sites in the midgut of such insects, wherein said proteins do not compete for the same binding sites in such insects, and wherein said proteins are i) a Cry2A protein and ii) a Cry1A, Cry1 F or VIP3 protein.
  • said plants comprise transgenes encoding the proteins: i) Cry2Aa, Cry2Ab or Cry2Ae, and ii) CrylAb, CryiAc, Cryl Fa, or VIP3A, particularly a Cry2Ae protein and a CrylAb and/or VIP3A protein.
  • said plants or seeds are corn or cotton plants or seeds containing a chimeric gene encoding a Cry1A, Cry1F or Vl P3 protein and a chimeric gene encoding a Cry2A protein, particularly a Cry2Ae protein, wherein said plants or seeds contain a transformation event selected from the group consisting of: corn event MON89034, corn event MIR162, a corn event comprising a transgene encoding a Cry2Ae protein, corn event TC1507, corn event Bt11 , corn event MON810, cotton event EE-GH6, cotton event COT102, cotton event COT202, cotton event COT203, cotton event T342-142, cotton event 1143-14A, cotton event 1143-51 B, cotton event CE44-69D, cotton event CE46- 02A, cotton event COT67B, cotton event 15985, cotton event 3006-210-23, cotton event 531 , cotton event EE-GH5, cotton Event 281-24-236, all as defined further
  • plants comprising at least 3 transgenes each encoding a different protein insecticidal to H.zea or H.armigera which proteins bind saturably and specifically to binding sites in the midgut of such insects, wherein said proteins do not compete for the same binding sites in such insects, and wherein said plants contain a chimeric gene encoding a Cry1A or Cry1 F protein, a chimeric gene encoding a Cry2A protein, and a chimeric gene encoding a VIP3A protein, and wherein the events are selected from the group as set forth in the above paragraphs.
  • the Cry2Ae, Cry2Ab, VIP3, Cry1 F or Cry1A chimeric genes are the chimeric genes contained in any one of the above corn or cotton events.
  • armigera particularly when saturable binding is determined in a direct saturability binding assay; preferably such uses, processes, plants or seeds wherein there is no biologically significant competition between the specific binding of any of said Cry2A protein and a Cry1A, Cry1 F or VIP3 protein, in standard competition binding assays as described herein, in H. armigera or H. zea.
  • preferred plants such as for stacking or combining different chimeric genes in the same plants by crossing, are plants comprising any one of the above corn events or any one of the above cotton events, as well as their progeny or descendants comprising said Cry2A, and said VIP3 and/or Cry1 protein-encoding chimeric genes.
  • Plants or seeds as used herein include plants or seeds of any plant species significantly damaged by cotton bollworms, but particularly include corn, cotton, rice, soybean, sorghum, tomato, sunflower and sugarcane.
  • a method for deregulating or for obtaining regulatory approval for planting or commercialization of plants expressing proteins insecticidal to H. zea or H. armigera, or for obtaining a reduction in structured refuge area containing plants not producing any protein insecticidal to H. zea or H. armigera, or for planting fields without a structured refuge area comprising the step of referring to, submitting or relying on insect assay binding data showing that Cry2A proteins bind specifically and saturably to the insect midgut membrane of such insects, and that said Cry2A proteins do not compete with binding sites for Cry1A, Cry1 F or VIP3 proteins in such insects, such as the data disclosed herein or similar data reported in another document.
  • such Cry2A protein is a Cry2Aa, Cry2Ab or Cry2Ae protein and such Cry1A protein is a CryiAc, CryiAb, or Cry1A.1O5 protein, and said VIP3 protein is a VIP3Aa protein.
  • a field planted with plants containing insecticidal proteins to protect said plants from Helicoverpa armigera or Helicoverpa zea insects wherein said field has a structured refuge of less than 20 %, or a structured refuge of less than 5 %, or has no structured refuge in said field, and wherein said plants express a combination of a) a Cry2Ae protein insecticidal to said insect species and b) a Cry1 A, Cry1 F or VIP3A protein insecticidal to said insect species, in said plants.
  • Said plants are preferably corn or cotton plants.
  • Bt toxin enhancer protein is expressed in said plants, wherein said Bt toxin enhancer protein is a protein or a fragments thereof which is a part, preferably a part comprising or corresponding to the binding domain, of a Bt toxin receptor in an insect, such as a fragment of a cadherin-like protein.
  • Bt toxin enhancer proteins are fed to target insects together with one or more Bt insecticidal toxins such as Cry proteins.
  • Bt toxin enhancer proteins can enhance the toxin activity of the Bt insecticidal protein against the insect species that was the source of the receptor but also against other insect species.
  • said Bt toxin enhancer protein is a part of a midgut cell Bt toxin receptor of a H. zea or H. armigera insect.
  • the current invention relates to Cry2A proteins that do not show competition for the Cry1 F, Vl P3 or Cry1 A receptor in Helicoverpa zea or Helicoverpa armigera, making it most interesting to combine in the same plant at least a Cry2Ae, Cry2Aa or Cry2Ab protein with a VIP3, Cry1 F or Cry1 A protein, preferably at least a Cry2Ae protein and a CrylAb, CryiAc, Cry1 A.105 or VIP3A protein, to prevent or delay the development of insect resistance to Helicoverpa zea or Helicoverpa armigera.
  • This approach should ideally be part of a global approach for insect resistance management including, where desired or required, structured refuge areas and the expression of the proteins at a high dose for the target insect.
  • binding sites which are referred to herein only refer to the specific binding sites for proteins insecticidal to H. zea or H. armigera, such as the Cry2Ae, Cry2Ab, VIP3A, CryiAc or CrylAb proteins. These are the binding sites to which a protein binds specifically, i.e., for which the binding of a labeled ligand (such as a Cry2Ae or VIP3A protein), to its binding site, can be displaced (or competed for) by an excess of non-labeled homologous ligand (a Cry2Ae or VIP3A protein, respectively).
  • the terms binding site or receptor are used interchangeably herein and are equivalent.
  • the binding to such specific binding sites is saturable as measured in a direct saturability assay.
  • a "direct saturability assay” is an assay in which a fixed amount of receptor (in this case BBMV) is incubated with increasing amounts of labeled ligand.
  • BBMV receptor-in this case
  • a plateau -or at least a deviation from linearity- will be evident when the binding data are plotted (% binding on the Y-axis, concentration of labeled ligand on the X-axis)
  • no plateau - or deviation from linearity - will be evident, but % binding keeps increasing linearly with increasing concentrations of labeled ligand.
  • the plateau is the maximum binding that can be obtained in the experimental conditions because all the available specific binding sites have been occupied by the labeled ligand.
  • competition of one protein for the binding site of another protein is not considered biologically significant (or, in other words, is considered biologically insignificant competition) if the competition takes place only at very high concentrations of the heterologous competitor (e.g., if 100 nM (or more) of the unlabeled heterologous competitor displaces only a minimal amount of bound labeled ligand (e.g., about 25 % or less of the specific binding of the labeled ligand) as determined when the binding data are plotted (% binding vs. concentration of unlabeled ligand)).
  • a protein X binds only with low affinity (e.g., if 100 nM (or more) of the unlabeled heterologous competitor displaces only a minimal amount of bound labeled ligand (e.g., about 25 % or less of the specific binding of the labeled ligand) as determined when the binding data are plotted (% binding vs. concentration of unlabeled ligand)) to the binding sites of a labeled protein Y, but there is no evidence of any different binding site in reciprocal binding assays using labeled protein X, both proteins effectively bind to the same binding site and hence are not suitable to be combined for resistance management purposes.
  • measuring Cry or VIP3 protein binding by ligand blotting using denatured BBMV proteins is not deemed to be a reliable measure of the actual specific binding sites present in the midgut or in BBMV preparations (which can be measured in BBMV binding assays using radiolabeled, or biotinylated proteins, since binding is to non-denatured BBMV proteins in such assays), as (binding) characteristics of denatured proteins may be different from non-denatured proteins.
  • BBMV BBMV specific binding of purified labeled protein (such as a Cry2Ae, Cry2Ab, VIP3 or Cry1 protein) to such BBMV is analyzed.
  • Homologous competition assays are done to determine if the binding is specific (herein an excess of the same unlabeled protein is used as competitor for the labeled ligand), and heterologous competition assays are done to determine if another protein competes for the same binding site in these BBMV (herein an excess of a different, unlabeled protein is used as competitor for the labeled ligand).
  • heterologous competition assays when no competition is found using labeled protein X and unlabeled protein Y as competitor, also the reciprocal experiment is done to confirm absence of competition, using the labeled protein Y and the unlabeled protein X as competitor.
  • homologous competition assays the binding is specific if a significant part of the binding of labeled protein is competed for (or displaced by) the unlabeled protein (i.e., the homologous competitor) - the part of the binding which is not displaced or competed for by homologous ligand is considered non-specific binding.
  • a "nucleic acid sequence” refers to a DNA or RNA molecule in single or double stranded form, preferably a DNA or RNA, particularly a DNA, encoding any of the proteins used in this invention.
  • isolated nucleic acid sequence refers to a nucleic acid sequence which is no longer in the natural environment where it was isolated from, e.g., the nucleic acid sequence in another bacterial host or in a plant nuclear genome.
  • heterologous proteins such as when referring to the use of heterologous insecticidal proteins in plants, refers to proteins not present in such organism (such as a plant) in nature, particularly to proteins encoded by transgenes introduced into the genome of plants, wherein such proteins are derived from bacterial proteins.
  • protein or “polypeptide” are used interchangeably to refer to a molecule consisting of a chain of amino acids, without reference to any specific mode of action, size, three-dimensional structures or origin. Hence, a fragment or portion of a protein used in the invention is still referred to herein as a "protein”.
  • the natural environment of the protein refers to the environment in which the protein could be found when the nucleotide sequence encoding it was expressed and translated in its natural environment, i.e., in the environment from which the nucleotide sequence was isolated.
  • an isolated protein can be present in vitro, or in another bacterial host or in a plant cell or it can be secreted from another bacterial host or from a plant cell.
  • insecticidal protein should be understood as an intact protein or a part thereof which has insecticidal activity, particularly insecticidal to Helicoverpa zea or Helicoverpa armigera larvae.
  • This can be a naturally-occurring protein or a chimeric or hybrid protein comprising parts of different insecticidal proteins (such as mixing domains from different proteins, or mixing parts of different proteins by using gene shuffling), or can be a variant having substantially the amino acid sequence of a bacterial protein but modified in some amino acids.
  • such an insecticidal protein can be a VIP or a Cry protein derived from Bt or other bacterial strains, or proteins encoded by mutant or recombinant genes as can be obtained by gene shuffling, mutagenesis, and the like from genes encoding Bt insecticidal proteins, such as Cry or VIP proteins.
  • protoxin should be understood as the primary translation product of a full-length gene encoding an insecticidal protein, before any cleavage has occurred in the midgut.
  • a VIP3 protoxin has a molecular weight of about 88 kD
  • a Cry1 F or Cry1A protoxin has a molecular weight of about 130-140 kD
  • a Cry2A protoxin has a molecular weight of about 60-70 kD.
  • toxin or "smallest toxic fragment” should be understood as that part of an insecticidal protein, such as a Cry2A, VIP3 or Cry1 F or Cry1A protein, which can be obtained by trypsin digestion or by proteolysis in (target insect, e.g., Helicoverpa zea or Helicoverpa armigera) midgut juice, and which still has insecticidal activity.
  • a VIP3 or Cry1 toxin has a molecular weight of about 60-65 kD
  • a Cry2A toxin has a molecular weight of about 50-58 kD on SDS- PAGE gel.
  • VIP3 protein refers to a protein insecticidal to Helicoverpa zea or Helicoverpa armigera larvae, and which is any one of the VIP3 proteins listed in Crickmore et al. (2008, see Table 3) on the VIP nomenclature website at: www.lifesci.susx.ac.uk/home/Neil Crickmore/Bt/VIP.html. or any protein comprising the smallest toxic fragment of any one of these proteins.
  • this is a VIP3A protein insecticidal to Helicoverpa zea or Helicoverpa armigera, such as a VIP3Aa1 (NCBI accession AAC37036), VIP3Af1 (NCBI accession CAI43275), VIP3Aa19 (NCBI accession ABG20428) or VIP3Aa20 protein (NCBI accession ABG20429), but also any insecticidal fragments thereof, or proteins with a sequence identity of at least 70 %, particularly at least 75 %, 80 %, 85 %, 90%, 95 %, 96 %, 97 %, 98 % or 99 % at the amino acid sequence level with the VIP3Aa1 protein of NCBI accession AAC37036, or the VIP3Af1 protein of NCBI accession CAI43275 (ISP3A, SEQ ID No.
  • VIP3Aa1 NCBI accession AAC37036
  • VIP3Af1 NCBI accession CAI43275
  • VIP3Aa19 NCBI accession ABG
  • a VIP3 protein as used herein is a VIP3A protein such as the VIP3Aa1 protein described in Estruch et al.
  • VIP3A protein insecticidal to Helicoverpa zea or Helicoverpa armigera selected from the group of: VIP3Ab, VIP3Ac, VIP3Ad, VIP3Ae, VIP3Af, VIP3Ag, or VIP3Ah, particularly the VIP3Af1 , VIP3Ad1 or VIP3Ae1 proteins (NCBI accessions CAI43275 (ISP3a, SEQ ID No.4 of WO 03/080656), CAI43276 (ISP3b, SEQ ID No.6 in WO 03/080656), and CAI43277 (ISP3C, SEQ ID No.
  • VIP3 protein variants having some, preferably 5-10, particularly less than 5, amino acids added, replaced or deleted, preferably in the part corresponding to the smallest toxic fragment, without significantly changing the Helicoverpa zea or Helicoverpa armigera insecticidal activity of the protein, such as the VIP3Aa19 protein (NCBI accession ABG20428, EPA experimental use permit factsheet 006499 (2007)) introduced in cotton plants (e.g., in plants containing event COT102 described in WO 2004/039986, or in USDA APHIS petition for non- regulated status 03-155-01 p) or the VIP3Aa20 protein (NCBI accession ABG20429, SEQ ID NO: 2 in WO 2007/142840) introduced in corn plants (e.g., event MIR162, USDA APHIS petition for non-regulated status 07-253-01 p), or the VIP3A protein produced in cotton event COT202 or COT203 (WO 2005/054479 and WO 2005/05
  • any putative native (bacterial) secretion signal peptide can be deleted or can be replaced by a Met amino acid or Met-Ala dipeptide, or by an appropriate signal peptide, such as a chloroplast transit peptide.
  • Putative signal peptides can be detected using computer based analysis, using programs such as the program Signal Peptide search (SignalP V1.1 or 2.0), using a matrix for prokaryotic gram-positive bacteria and a threshold score of less than 0.5, especially a threshold score of 0.25 or less (Von Heijne, Gunnar, 1986 and Nielsen et al.,1996).
  • a “Cry1 F protein” or “Cry1 F”, as used herein, includes any protein comprising the smallest toxic fragment of the amino acid sequence of a Cry1 F protein retaining toxicity to Helicoverpa zea or Helicoverpa armigera, such as the protein of NCBI accession AAA22347 (SEQ ID No. 10 of US 2005049410), or a Cryi Fa protein.
  • This includes hybrid or chimeric proteins comprising this smallest toxic fragment, or at least one of the structural domains, preferably at least 2 of the 3 structural domains, of a Cry1 F protein, such as the hybrid proteins derived from Cry1 F in WO 1999/024581.
  • variants of the amino acid sequence in NCBI accession AAA22347 such as amino acid sequences having a sequence identity of at least 90%, 95 %, 96 %, 97 %, 98 % or 99 % to the Cry1 F protein of NCBI accession AAA22347 (SEQ ID No. 10 of US 2005049410), as determined using pairwise alignments using the GAP program of the Wisconsin package of GCG (Madison, Wisconsin, USA, version 10.2), particularly such identity is with the part corresponding to the smallest toxic fragment.
  • the GAP program is used with the following parameters for the amino acid sequence comparisons: the 'blosum62' scoring matrix, a 'gap creation penalty' (or 'gap weight 1 ) of 8 and a 'gap extension penalty' (or 'length weight 1 ) of 2.
  • proteins having some, preferably 5-10, particularly less than 5, amino acids added, replaced or deleted without significantly decreasing the Helicoverpa zea or Helicoverpa armigera insecticidal activity of the protein such as a Cry1 F protein with one or more conservative amino acid substitutions for cloning purposes, are included in this definition.
  • a Cry1 F protein includes the protein encoded by the Cry1 F genes in Cry1 F Cotton Event 281-24-236 (WO 2005/103266, see USDA APHIS petition for non-regulated status 03-036-01 p), or in corn events TC1507 or TC-2675 (US 7,288,643, WO 2004/099447, USDA APHIS petitions for non-regulated status 00-136-01 p and 03- 181-01p), particularly a protein comprising the smallest toxic fragment of any one of such Cry1 F proteins, or a variant of any one of such Cry1 F proteins differing in 1-5 amino acids retaining toxicity to Helicoverpa zea or Helicoverpa armigera.
  • a "Cry2Ae” protein refers to an insecticidal Cry2Ae protein such as a full length Cry2Ae protein of SEQ ID No. 2 of WO 2002/057664, a Cry2Ae toxic fragment or a protein comprising a Cry2Ae toxic fragment as described in of WO 2002/057664, such as a fusion protein of a Cry2Ae protein fragment with a chloroplast transit peptide or another peptide sequence insecticidal to H. zea or H. armigera, or is a protein insecticidal to H. zea or H.
  • armigera comprising an amino acid sequence with at least 95, 97 or 99 % sequence identity to the amino acid sequence of SEQ ID No. 2 of WO 2002/057664, particularly in the part corresponding to the smallest toxic fragment, or is a protein encoded by the Cry2Ae gene contained in cotton event EE-GH6 as described in the PCT patent application claiming priority to European patent application number 07075460 or 07075485 (unpublished), or a protein comprising the smallest toxic fragment of any one of such Cry2Ae proteins, or a variant of any one of such Cry2Ae proteins differing in 1-5 amino acids retaining toxicity to Helicoverpa zea or Helicoverpa armigera.
  • Cry2Ab protein refers to any one of the Cry2Ab proteins of Crickmore et al. (1998, 2008) or www.lifesci.susx.ac.uk/home/NeiLCrickmore/Bt/ insecticidal to H. zea or H.
  • armigera such as a full length Cry2Ab protein, a Cry2Ab toxic fragment, or a protein comprising a Cry2Ab toxic fragment, such as a fusion protein of a Cry2Ab2 protein fragment with a chloroplast transit peptide or another peptide sequence retaining toxicity to Helicoverpa zea or Helicoverpa armigera, or is a protein insecticidal to Helicoverpa zea or Helicoverpa armigera comprising an amino acid sequence with at least 95, 97 or 99 % sequence identity to the coding region of NCBI accession CAA39075 (Dankocsik et al., 1990), particularly in the part corresponding to the smallest toxic fragment, or is the protein encoded by the Cry2Ab2 gene contained in cotton event 15985 as described in USDA-APHIS petition for non-regulated status 00-342-01 p, the protein encoded by the Cry2Ab2 gene contained in corn event MON89034 as described in USDA-APHIS petition
  • a “Cry1A” protein refers to a CryiAc, Cry1A.1O5 or a CrylAb protein, and includes any protein comprising the smallest toxic fragment of the amino acid sequence of a CryiAc, Cry1A.1O5 or CrylAb protein retaining toxicity to Helicoverpa zea or Helicoverpa armigera, such as the smallest toxic fragment of the protein in NCBI accession AAA22331 (CryiAc; Adang et al., 1985), NCBI accession AAA22330 (Wabiko et al., 1986 (CrylAb)), or the Cry1 A.105 protein encoded by the Cry1A transgene in corn event MON89034 (USDA APHIS petition for non-regulated status 06-298-01 p, WO 2007/140256, SEQ ID NO: 2 or 4 in WO 2007/027777), or the CrylAb protein encoded by the crylAb coding region in cotton event COT67B
  • a Cry1A protein such as CrylAb or CryiAc
  • variants of the amino acid sequence in NCBI accession AAA22331 (Cry1Ac1 ), NCBI accession AAA22330 (CrylAb, Wabiko et al., 1986), or the amino acid sequence of the Cry1A.1O5 protein described in USDA APHIS petition for non-regulated status 06-298-01 p, such as proteins having an amino acid sequence identity of at least 90%, 95 %, 96 %, 97 %, 98 % or 99 % at the amino acid sequence level with such a CryiAc, Cry1A.1O5 or CrylAb protein, particularly in the part corresponding to the smallest toxic fragment, as determined using pairwise alignments using the GAP program of the Wisconsin package of GCG (Madison, Wisconsin, USA, version 10.2), with the smallest toxic fragment of a Cry1 A protein.
  • the GAP program is used with the following parameters for the amino acid sequence comparisons: the 'blosum62' scoring matrix, a 'gap creation penalty' (or 'gap weight') of 8 and a 'gap extension penalty' (or 'length weight') of 2.
  • proteins having some, preferably 5-10, particularly less than 5, amino acids added, replaced or deleted without significantly changing the Helicoverpa zea or Helicoverpa armigera insecticidal activity of the protein such as a Cry1 A protein with one or more conservative amino acid substitutions (e.g., for gene cloning purposes), are included in this definition.
  • Cry1A proteins for use in this invention include the CrylAb protein encoded by SEQ ID NO:3 of US 6,114,608, particularly the CrylAb protein encoded by the crylAb coding region in corn event MON810 (US 6,713,259), USDA APHIS petition for non-deregulated status 96-017-01p and extensions thereof), the CrylAb protein encoded by the crylAb coding region in corn event Bt11 (USDA APHIS petition for non-deregulated status 95-195-01p, US patent 6,114,608), the CryiAc protein encoded by the transgene in cotton event 3006-210-23 (US 7,179,965, WO 2005/103266, USDA APHIS petition for non-deregulated status 03-036-02p), the CrylAb protein encoded by the crylAb coding region in cotton event COT67B (USDA APHIS petition for non-deregulated status 07-108-01 p, WO 2006/128573), the CrylAb coding region contained in cotton event EE-GH5 described in PC
  • a Cry1 Ab or a Cry1A.1O5 protein, or a protein comprising the smallest toxic fragment thereof, from this above list is used, or a protein comprising the smallest toxic fragment of any one of such Cry1A proteins, or a variant of any one of such Cry1A proteins differing in 1-5 amino acids retaining toxicity to Helicoverpa zea or Helicoverpa armigera.
  • Cry2Ae and Cry2Aa proteins compete for the same binding site as the Cry2Ab protein in H. armigera, and that this binding site is different from (i.e. not shared with) the binding site of CryiAc in Helicoverpa zea and Helicoverpa armigera. Also, CryiAc did not compete for the binding of Cry2Ab in these insect species. Also, it has already been reported that Cry1 F and CryiAc, and CryiAc and CrylAb share binding sites in Helicoverpa zea or Helicoverpa armigera (e.g., Hernandez and Ferre, 2005, Karim et al., 2000b; Estela et al., 2004).
  • CrylAb, Cry1 F and CryiAc bind to a binding site that is different from the binding site of Cry2Ae or Cry2Aa in H. zea or H. armigera.
  • the VIP3A protein binds to a different binding site than Cry2Ab (Lee et al., 2006). Since Cry2Aa and Cry2Ae share a common binding site with Cry2Ab, Cry2Ae or Cry2Aa proteins bind to a different binding site than the VIP3A protein in H. zea and H. armigera.
  • Cry1F proteins generally have a lower activity to these insect species compared to the Cry1A, VIP3A or Cry2A proteins tested, they are amongst the most widely used Cry1 proteins in plants, and since they do not share binding sites with Cry2A proteins, they can also be used for insect resistance management, certainly if the plants can provide for sufficiently high levels of expression of the Cry1 F protein.
  • Some Cry1 F-derived proteins have a higher intrinsic activity to H. zea or H. armigera, and these are a more preferred Cry1 F protein in this invention.
  • a Cry1A protein such as a CrylAb or Cry1A.1O5 protein, will be the better choice to delay or prevent resistance development to Helicoverpa zea or Helicoverpa armigera, given their higher intrinsic toxicity to these insect species.
  • Bt Cry proteins such as Cry1 F, Cry2A and Cry1A proteins are expressed as protoxins in their native host cells (Bacillus thuringiensis), which are converted into the toxin form by proteolysis in the insect gut.
  • a Cry1 F, Cry2A or Cry1A protein, as used herein, refers to either the full protoxin or the toxin, or any intermediate form with insecticidal activity.
  • a Cry1 F protein includes a protein comprising the amino acid sequence of NCBI accession AAA22347 from amino acid position 29 to amino acid position 604, and a Cry1A protein includes a protein comprising the amino acid sequence of NCBI accession AAA22331 (Cry1Ac1 ; Adang et al., 1985) from amino acid position 29 to 607, comprising the amino acid sequence of NCBI accession AAA22330 (CrylAb, Wabiko et al., 1986) from amino acid position 29 to amino acid position 607, or comprising the amino acid sequence of Fig. IV-1 in USDA APHIS petition for non-regulated status 06-298-01 p from amino acid position 29 to amino acid position 607.
  • a Cry2A protein includes a protein comprising the amino acid sequence of SEQ ID No. 2 of US Patent 7,265,269 from amino acid position 50 to 625, a protein comprising the amino acid sequence of Fig. IV-2 in USDA APHIS petition for non-regulated status 06-298-01 p from amino acid position 81 to 746, or a protein comprising the amino acid sequence of NCBI accession CAA39075 from amino acid position 50 to amino acid position 626.
  • a “Cry1” protein refers to a Cry1 F or Cry1A protein as defined above.
  • a "Cry2A” protein, as used herein, refers to a Cry2Ae or Cry2Ab protein as defined herein, but can also refer to any Cry2A protein in Crickmore et al. (2008), such as a Cry2Aa protein, insecticidal to H. zea or H. armigera.
  • a gene can be naturally occurring, artificial (modified) or synthetic in whole or in part.
  • event refers to a specific integration of one or more transgenes at a specific location in the plant genome, which can be considered as a part of DNA containing the inserted sequences and the flanking plant sequences. Such an event can be crossed into many other plants of the same species or in plants of a different species allowing intercrossing with the plants containing the event by breeding techniques, including techniques such as embryo rescue.
  • DNA/protein comprising the sequence or region X refers to a DNA or protein including or containing at least the sequence or region X, so that other nucleotide or amino acid sequences can be included at the 5' (or N-terminal) and/or 3' (or C-terminal) end, e.g. (the nucleotide sequence of) a transit peptide, and/or a 5 1 or 3" leader sequence.
  • a VIP3 or Cry protein-encoding "chimeric gene”, as used herein, refers to a VIP3 or Cry-encoding DNA (or coding region) having 5' and/or 3 1 regulatory sequences, at least a 5' regulatory sequence or promoter, different from the naturally-occurring bacterial 5' and/or 3' regulatory sequences which drive the expression of the Vl P3 or Cry protein in its native host cell, e.g., a VIP3 or cry DNA operably-linked to a plant- expressible promoter such that said chimeric gene can be expressed in the plants containing it.
  • the chimeric gene need not be expressed the entire time or in every cell of the plant, e.g., expression can be induced by insect feeding or wounding using a wound-induced promoter, or expression can be localized in those plant parts mostly attacked by insects such as Helicoverpa zea or Helicoverpa armigera larvae, particularly those most valuable for the grower or farmer, e.g., the leaves and ears of a com plant, or the leaves and bolls of cotton plants, or the leaves and pods of soybean plants.
  • a plant expressing a VIP3, Cry2A, Cry1F or Cry1A protein as used herein refers to a plant containing the necessary plant-expressible chimeric gene encoding such a protein, so that the protein is expressed in the relevant tissues or at the relevant time periods, which need not be in all plant tissues or need not be all the time.
  • sequence identity of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (x100) divided by the number of positions compared.
  • a gap i.e. a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues.
  • GAP program which uses the Needleman and Wunsch algorithm (1970) and which is provided by the Wisconsin Package, Version 10.2, Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin 53711 , USA, is used.
  • GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps.
  • gap creation penalty 50 (nucleotides) / 8 (proteins)
  • gap extension penalty 3 (nucleotides) / 2 (proteins).
  • the default scoring matrix used is "nwsgapdna" and for proteins the default scoring matrix is "blosum62" (Henikoff & Henikoff, 1992).
  • DNAs included herein as a VIP3 or Cry DNA are those DNAs that encode a VIP3 or Cry protein, or a variant or hybrid thereof, insecticidal to H. zea or H. armigera, and that hybridizes under stringent hybridization conditions to a DNA that can encode a VIP3 or Cry protein.
  • “Stringent hybridization conditions”, as used herein, refers particularly to the following conditions: immobilizing the relevant DNA on a filter, and prehybridizing the filters for either 1 to 2 hours in 50 % formamide, 5 % SSPE, 2x Denhardt's reagent and 0.1 % SDS at 42 ° C or 1 to 2 hours in 6x SSC, 2xDenhardt's reagent and 0.1 % SDS at 68 0 C.
  • the denatured (Digoxigenin- or radio-) labeled probe is then added directly to the prehybridization fluid and incubation is carried out for 16 to 24 hours at the appropriate temperature mentioned above.
  • the filters are then washed for 30 minutes at room temperature in 2x SSC, 0.1 % SDS, followed by 2 washes of 30 minutes each at 68 0 C in 0.5 x SSC and 0.1 % SDS.
  • An autoradiograph is established by exposing the filters for 24 to 48 hours to X-ray film (Kodak XAR-2 or equivalent) at -70 0 C with an intensifying screen.
  • equivalent conditions and parameters can be used in this process while still retaining the desired stringent hybridization conditions.
  • insecticidal activity of a protein means the capacity of a protein to kill insects when such protein is fed to insects, preferably by expression in a recombinant host such as a plant. It is understood that a protein has insecticidal activity if it has the capacity to kill the insect during at least one of its developmental stages, preferably the larval stage.
  • a population of insect species that "has developed resistance” or “has become resistant” to plants expressing an insecticidal protein refers to the detection of repeated, significant unacceptable yield damage in such plants, caused by such insect population as compared to the level of yield damage of such plants by the same insect species when such plants were first introduced. This has to be confirmed to check that the plants are indeed producing the insecticidal protein (i.e., they are not non-transgenic plants), and that members of this insect population indeed need a higher amount of insecticidal protein to be controlled or killed.
  • insect resistance development leads to an increased plant damage that is detected.
  • insect resistance of an insect species population is readily observed if insects from such population can complete their life cycle on such plants, and continue to damage the plants instead of being arrested in their growth and feeding habits because of the insecticidal proteins produced in such plants - in an extreme form of insect resistance such plant can be as damaged as conventional non- transgenic plants with the same genetic background by an insect attack.
  • the binding to Cry or VIP3 proteins to such resistant insects can be analyzed in (standard) competition binding assays using BBMV of H. zea or H. armigera, to confirm that resistance is due to binding site modification.
  • Bt toxin enhancer protein is expressed in said plants, wherein said Bt toxin enhancer protein is a protein or a fragment thereof which is a part, preferably a part comprising or corresponding to the binding domain, of a Bt toxin receptor in an insect, such as a fragment of a cadherin-like protein.
  • Bt toxin enhancer proteins are shown in published US patent application 20090018075. These Bt toxin enhancer proteins are fed to target insects together with one or more Bt insecticidal toxins such as Cry proteins.
  • Bt toxin enhancer proteins can enhance the toxic activity of the Bt insecticidal protein against the insect species that was the source of the receptor but also against other insect species.
  • said Bt toxin enhancer protein is a part of a midgut cell Bt toxin receptor of a H. zea or H. armigera insect.
  • /-/. zea refers to Helicoverpa zea (Boddie), an important Lepidopteran pest insect, also known as the (American) cotton bollworm, the corn earworm or the tomato fruitworm, sorghum headworm, or vetchworm.
  • This insect is an important pest in corn, cotton and tomato, but also attacks plants like artichoke, asparagus, cabbage, cantaloupe, collard, cowpea, cucumber, eggplant, lettuce, lima bean, melon, okra, pea, pepper, potato, pumpkin, snap bean, spinach, squash, sweet potato, watermelon, alfalfa, clover, flax, oat, millet, rice, sorghum, soybean, sugarcane, sunflower, tobacco, vetch, and wheat.
  • plants like artichoke, asparagus, cabbage, cantaloupe, collard, cowpea, cucumber, eggplant, lettuce, lima bean, melon, okra, pea, pepper, potato, pumpkin, snap bean, spinach, squash, sweet potato, watermelon, alfalfa, clover, flax, oat, millet, rice, sorghum, soybean, sugarcane, sunflower, tobacco, vetch, and wheat.
  • H ⁇ bner Helicoverpa armigera
  • This insect is an important pest in corn and cotton, but it also attacks plants like tobacco, sunflower, linseed, soybean, Lucerne, peas such as pigeonpea or chickpea, chili, okra, besides carnations, geraniums and other ornamental or flower crops, fruits, and vegetables such as cabbage, aubergines, peppers, tomato, and cucumber.
  • Cotton bollworm(s) or “bollworm(s)", as used herein in a general sense, refers to H. zea and/or H. armigera.
  • insects-controlling amounts of a protein, as used herein, refers to an amount of protein which is sufficient to limit damage on a plant, caused by insects (e.g. insect larvae) feeding on such plant, to commercially acceptable levels, e.g. by killing the insects or by inhibiting the insect development, fertility or growth in such a manner that they provide less damage to a plant and plant yield is not significantly adversely affected.
  • the VIP3 and/or Cry protein of the invention are expressed at a high dose in the plants used in the invention.
  • 'High dose' expression refers to a concentration of the insecticidal protein in a plant (measured by ELISA as a percentage of the total soluble protein, which total soluble protein is measured after extraction of soluble proteins in a standard extraction buffer using Bradford analysis (Bio-Rad, Richmond, CA; Bradford, 1976)) which kills at least 95% of insects in a developmental stage of the target insect which is significantly less susceptible, preferably at least 25 times less susceptible to the insecticidal protein than the first larval stage of the insect (as can be analyzed in standard insecticidal protein bio- assays), and can thus can be expected to ensure full control of the target insect species.
  • a "structured refuge” as used herein, refers to a part of the field or land of a grower or farmer that is otherwise planted with Bt-plants, but which is planted with plants not containing a Bt transgene (as compared to using weeds or other non-Bt plants around a farmer's fields as a natural refuge).
  • General procedures for the evaluation and exploitation of at least two insecticidal genes for prevention of the development, in a target insect, of resistance to transgenic plants expressing those genes can be found in published European patent application EP408403. Definitions used in the field of receptor binding analysis can be found at: www.unmc.edu/Pharmacology/receptortuto ⁇ al/definitions/definitions.htm
  • the binding of Cry proteins to the brush border membrane of the midgut cells of Helicoverpa zea or Helicoverpa armigera insect larvae has been investigated.
  • the brush border membrane is the primary target of the VIP3 or Cry proteins, and membrane vesicles, preferentially derived from the insect midgut brush border membrane (named BBMV herein, for brush border membrane vesicles), can be obtained according to procedures known in the art, e.g., Wolfersberger et al. (1987).
  • This invention involves the combined expression of at least two insecticidal protein genes in transgenic plants to delay or prevent resistance development in populations of the target insect Helicoverpa zea or Helicoverpa armigera.
  • the genes are inserted in a plant cell genome, preferably in its nuclear genome, so that the inserted genes are downstream of, and operably linked to, a promoter which can direct the expression of the genes in plant cells
  • a plant with a lasting resistance to Helicoverpa zea or Helicoverpa armigera comprising a chimeric gene encoding a Cry2A protein, such as a Cry2Ab or Cry2Ae protein, insecticidal to Helicoverpa zea or Helicoverpa armigera, and a chimeric gene encoding a Cry1A, VIP3 and/or Cry1F protein, preferably a CrylAb, VIP3A or a Cry1A.1O5 protein as defined above, insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • a Cry2A protein such as a Cry2Ab or Cry2Ae protein
  • insecticidal to Helicoverpa zea or Helicoverpa armigera preferably a CrylAb, VIP3A or a Cry1A.1O5 protein as defined above, insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • a method of controlling Helicoverpa zea or Helicoverpa armigera infestation in transgenic plants while securing a slower buildup of Helicoverpa zea or Helicoverpa armigera insect resistance development to said plants comprising expressing a combination of a) a Cry2Ae protein insecticidal to said insect species and b) a Cry1A, Cry1 F or VIP3A protein insecticidal to said insect species, in said plants, as well as a method for preventing or delaying insect resistance development in populations of the insect species Helicoverpa zea or Helicoverpa armigera to transgenic plants expressing insecticidal proteins to control said insect pest, comprising expressing a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera in combination with a Cry1A, Cry1 F of VIP3A protein insecticidal to Helicoverpa zea or Helicoverpa armigera in said plants.
  • a method is provided to control Helicoverpa zea or Helicoverpa armigera in a region where populations of said insect species have become resistant to plants expressing a VIP3A, Cry1A or a Cry1 F protein, comprising the step of sowing or planting in said region, plants expressing at least a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • a method to control Helicoverpa zea or Helicoverpa armigera in a region where populations of said insect have become resistant to plants expressing a Cry2Ae protein comprising the step of sowing or planting in said region, plants expressing a Cry1 F, VIP3, or Cry1A protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • Also provided in accordance with this invention is a method for obtaining plants comprising chimeric genes encoding at least two different insecticidal proteins, wherein said proteins do not share binding sites in larvae of the species Helicoverpa zea or Helicoverpa armigera as determined in competition binding experiments using brush border membrane vesicles of said insect larvae, comprising the step of obtaining plants comprising a plant-expressible chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and a plant- expressible chimeric gene encoding a Cry1A, VIP3 or Cry1 F protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • Also provided here is a method of sowing, planting, or growing plants protected against cotton bollworms, comprising chimeric genes expressing at least two different insecticidal proteins, wherein said proteins do not share binding sites in larvae of the species Helicoverpa zea or Helicoverpa armigera as determined in competition binding experiments using brush border membrane vesicles of said larvae, comprising the step of: sowing, planting, or growing plants comprising a chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and a chimeric gene encoding a Cry1A, VIP3 or Cry1 F protein insecticidal to Helicoverpa zea or Helicoverpa armigera, preferably a Vl P3 or Cry1A protein insecticidal to Helicoverpa zea or Helicoverpa armigera.
  • a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera in combination with a Cry1 A, VIP3 or Cry1 F protein insecticidal to insects of said species, to prevent or delay resistance development of insects of said species to transgenic plants expressing heterologous insecticidal toxins, particularly when said use is by expression of said proteins in plants.
  • the invention also provides for the use, the sowing, planting or growing of a refuge area with plants not comprising a Cry2, Cry1 or VIP3 protein insecticidal to Helicoverpa zea or Helicoverpa armigera, such as by sowing, planting or growing such plants in the same field or in the vicinity of the plants comprising the Cry2Ae, VIP3 and Cry1 protein described herein.
  • a process for growing, sowing or planting plants expressing a Cry protein or Vl P3 protein for control of Helicoverpa armigera or Helicoverpa zea insects comprising the step of planting, sowing or growing an insecticide sprayed structured refuge area of less than 20 %, or an non-insecticide sprayed structured refuge area of less than 5 %, of the planted field or in the vicinity of the planted field, or without planting, sowing or growing a structured refuge area in a field, wherein such structured refuge area is a location in the same field or is within 2 miles, within 1 mile or within 0.5 miles of a field, and which contains plants not comprising such Cry or VIP3 protein, wherein such plants expressing a Cry or VIP3 protein express a combination of a Cry2Ae protein insecticidal to said insect species, and a Cry1A, Cry1F or VIP3A protein, particularly a Cry2Ae and a CrylAb or CryiAc or VIP3A protein
  • Also provided in one embodiment of this invention is the use of at least 2 insecticidal proteins binding specifically and saturably to binding sites in the midgut of Helicoverpa zea larvae, for delaying or preventing resistance development of such insect species to plants expressing insecticidal proteins, wherein one of said proteins in said plants is a Cry2A protein, such as a Cry2Ab protein, insecticidal to such insect species, and the other protein is a Cry1A, Cry1F or VIP3 protein insecticidal to such insect species, wherein such saturable binding is determined in a saturability assay using a fixed concentration of binding sites (i.e., BBMVs) to which increasing concentrations of labeled protein are added.
  • BBMVs fixed concentration of binding sites
  • the Cry1A protein is selected from the group of: a CryiAc, CrylAb, Cry1A.1O5, or a CryiAc or CrylAb hybrid protein, such as a protein encoded by any one of the cry1A coding regions referred to herein.
  • Such Cry2Ab and Cry1 A proteins do not compete for their (saturable and specific) binding sites in the midgut of such H. zea insect larvae, as can be measured in BBMV competition binding assays.
  • plants or seeds comprising at least 2 transgenes each encoding a different protein insecticidal to H.
  • said plants comprise transgenes encoding the proteins: i) Cry2Aa, Cry2Ab or Cry2Ae, and ii) CrylAb, CryiAc, CrylFa, or VIP3A, particularly a Cry2Ae protein and a CrylAb and/or VIP3A protein.
  • said plants or seeds are corn or cotton plants or seeds containing a chimeric gene encoding a Cry1A, Cry1 F or VIP3 protein and a chimeric gene encoding a Cry2A protein, particularly a Cry2Ae protein, wherein said plants or seeds contain a transformation event selected from the group consisting of: corn event MON89034, corn event MIR162, a corn event comprising a transgene encoding a Cry2Ae protein, corn event TC1507, corn event Bt11 , corn event MON810, cotton event EE-GH6, cotton event COT102, cotton event COT202, cotton event COT203, cotton event T342- 142, cotton event 1143-14A, cotton event 1143-51 B, cotton event CE44-69D, cotton event CE46- 02A, cotton event COT67B, cotton event 15985, cotton event 3006-210-23, cotton event 531 , cotton event EE-GH5, cotton Event 281-24-236, all as defined further here
  • plants comprising at least 3 transgenes each encoding a different protein insecticidal to H. zea or H. armigera which proteins bind saturably and specifically to binding sites in the midgut of such insects, wherein said proteins do not compete for the same binding sites in such insects, and wherein said plants contain a chimeric gene encoding a Cry1A or Cry1 F protein, a chimeric gene encoding a Cry2A protein, and a chimeric gene encoding a VIP3A protein, and wherein the events are selected from the group as set forth in the above paragraphs.
  • the Cry2Ae, Cry2Ab, VIP3, Cry1 F or Cry1A chimeric genes are the chimeric genes contained in any one of the above specific corn or cotton events.
  • armigera particularly when saturable binding is determined in a direct saturability binding assay; preferably such uses, processes, plants or seeds wherein there is no biologically significant-competition between the specific binding of any of said Cry2A protein and a Cry1A, Cry1 F or VIP3 protein, in standard competition binding assays as described herein, in H.armigera or H. zea.
  • preferred plants such as for stacking or combining different chimeric genes in the same plants by crossing, are plants comprising any one of the above corn events or any one of the above cotton events, as well as their progeny or descendants comprising said Cry2A, and said VIP3 and/or Cry1 protein-encoding chimeric genes.
  • Plants or seeds as used herein include plants or seeds of any plant species significantly damaged by cotton bollworms, but particularly include corn, cotton, rice, soybean, sorghum, tomato, sunflower and sugarcane.
  • a method for deregulating or for obtaining regulatory approval for planting or commercialization of plants expressing proteins insecticidal to H. zea or H. armigera, or for obtaining a reduction in structured refuge area containing plants not producing any protein insecticidal to H. zea or H. armigera, or for planting fields without a structured refuge area comprising the step of referring to, submitting or relying on insect assay binding data showing that Cry2A proteins bind specifically and saturably to the insect midgut membrane of such insects, and that said Cry2A proteins do not compete with binding sites for Cry1A, Cry1 F or VIP3 proteins in such insects, such as the data disclosed herein or similar data reported in another document.
  • such Cry2A protein is a Cry2Aa, Cry2Ab or Cry2Ae protein and such Cry1A protein is a Cry 1Ac, CrylAb, or Cry1A.1O5 protein, and said VIP3 protein is a VIP3Aa protein.
  • restriction sites can be introduced, flanking the DNA sequence. This can be done by site-directed mutagenesis, using well-known procedures (Stanssens et al., 1989; White et al., 1989).
  • the codon usage of the genes or insecticidally effective gene part of this invention can be modified to form an equivalent, modified or artificial gene or gene part in accordance with PCT publications WO 91/16432 and WO 93/09218 and publications EP 0 385 962, EP 0 359 472 and US 5,689,052, or the genes or gene parts can be inserted in the plastid, mitochondrial or chloroplast genome and expressed there using a suitable promoter (e.g., Mc Bride et al., 1995; US patent 5,693,507, WO 2004/053133).
  • a suitable promoter e.g., Mc Bride et al., 1995; US patent 5,693,507, WO 2004/053133.
  • amino acid codons can be replaced by others without changing the amino acid sequence of the protein.
  • amino acids can be substituted by other equivalent amino acids without significantly changing, preferably without changing, the insecticidal activity of the protein, at least without changing the insecticidal activity of the protein in a negative way.
  • conservative amino acid substitutions within the categories basic e.g. Arg, His, Lys
  • acidic e.g. Asp, GIu
  • nonpolar e.g. Ala, VaI, GIy, Leu, lie, Met
  • polar e.g.
  • variants of the DNA sequences of the invention include DNA sequences having a different codon usage compared to the native genes of the Cry2A, VIP3, Cry1 F or Cry1A proteins used in this invention but which encode a protein with the same insecticidal activity and with substantially the same, preferably the same, amino acid sequence.
  • the DNA sequences can be codon-optimized by adapting the codon usage to that most preferred in plant genes, particularly to genes native to the plant genus or species of interest (Bennetzen & Hall, 1982; ltakura et al., 1977) using available codon usage tables (e.g. more adapted towards expression in corn, cotton, rice, soybean, sorghum, tomato, sunflower or sugarcane). Codon usage tables for various plant species are published for example by lkemura (1993) and Nakamura et al. (2000).
  • an intron preferably a monocot intron
  • a monocot intron can also be added to the chimeric gene.
  • the insertion of the intron of the maize Adh1 gene into the 5" regulatory region has been shown to enhance expression in maize (Callis et. al., 1987).
  • the HSP70 intron as described in US 5,859,347, may be used to enhance expression.
  • the DNA sequence of the insecticidal protein gene or its insecticidal part can be further changed in a translationally neutral manner, to modify possibly inhibiting DNA sequences present in the gene part by means of site-directed intron insertion and/or by introducing changes to the codon usage, e.g., adapting the codon usage to that most preferred by plants, preferably the specific relevant target plant species/genus (Murray et al., 1989), without changing significantly, preferably without changing, the encoded amino acid sequence.
  • cotton bollworms (Helicoverpa zea or Helicoverpa armigera) susceptible to a VIP3, Cry2A , a Cry1 F or Cry1A protein are contacted with a combination of these proteins in insect-controlling amounts, preferably insecticidal amounts, e.g., by expressing these proteins in plants targeted by these armyworms or by transforming plants so that these plants and their descendants contain chimeric genes encoding such proteins.
  • target plants for these armyworms are corn, cotton, rice, soybean, sorghum, tomato, sunflower or sugarcane plants, particularly in Northern, Central and Southern American countries.
  • the term plant encompasses whole plants as well as parts of plants, such as leaves, stems, flowers or seeds.
  • at least 3 different proteins of the invention binding to different binding sites in H. zea or H. armigera are combined in such target plants by providing them with the necessary chimeric genes encoding such proteins, such as any one of the following combinations of Cry or VIP3 proteins: a Cry2Ab, a CrylAb and a VIP3A protein; a Cry2Ab, a CryiAc and a VIP3A protein; a Cry2Ab, a Cry1 F and a VIP3A protein; a Cry2Ab, a Cry1A.1O5 and a VIP3A protein; a Cry2Ae, a CrylAb and a VIP3A protein; a Cry2Ae, a Cry1A.1O5 and a VIP3A protein; a Cry2Ae, a CryiAc and a VIP3A protein;
  • the insecticidally effective gene preferably the chimeric gene, encoding an insecticidally effective portion of the Cry2A, VIP3, Cry1 F or Cry1A protein, can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so-transformed plant cell can be used in a conventional manner to produce a transformed plant that is insect-resistant.
  • a T-DNA vector containing the insecticidally effective gene, in Agrobacterium tumefaciens can be used to transform the plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0 116 718, EP 0 270 822, PCT publication WO 84/02913 and published European Patent application EPO 242 246 and in Gould et al. (1991 ).
  • the construction of a T-DNA vector for Agrobacterium mediated plant transformation is well known in the art.
  • the T-DNA vector may be either a binary vector as described in EP 0 120 561 and EP 0 120 515 or a co-integrate vector which can integrate into the Agrobacterium Ti-plasmid by homologous recombination, as described in EP 0 116 718.
  • Preferred T-DNA vectors each contain a promoter operably linked to the insecticidally effective gene between T-DNA border sequences, or at least located to the left of the right border sequence. Border sequences are described in Gielen et al. (1984).
  • vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 223 247), pollen mediated transformation (as described, for example in EP 0 270 356 and WO 85/01856), protoplast transformation as, for example, described in US 4,684,611, plant RNA virus-mediated transformation (as described, for example in EP 0 067 553 and US 4,407,956), liposome-mediated transformation (as described, for example in US 4,536,475), and other methods such as the recently described methods for transforming certain lines of corn (e.g., US 6,140,553; Fromm et al., 1990; Gordon-Kamm et al., 1990) and rice (Shimamoto et al., 1989; Datta et al.
  • direct gene transfer as described, for example in EP 0 223 247)
  • pollen mediated transformation as described, for example in EP 0 270 356 and WO 85/01856
  • Cry2A and a VIP3, Cry1 F or Cry1A protein are most useful in plants targeted by (or damaged by) the cotton bollworm, including corn (field and sweet corn), cotton, tomato, artichoke, asparagus, aubergines, cabbage, cantaloupe, collard, cowpea, cucumber, eggplant, lettuce, lima bean, melon, okra, pepper, potato, pumpkin, snap bean, spinach, squash, sweet potato, watermelon, alfalfa, clover, flax, oat, millet, rice, sorghum, soybean, sugarcane, sunflower, tobacco, vetch, wheat, tobacco, linseed, peas such as pigeonpea or chickpea, chili, okra, besides carnations, geraniums and other ornamental or flower crops, or fruit crops; preferably in corn, cotton, rice, soybean, sunflower, tomato, or sugarcane plants.
  • a Cry2A protein and a VIP3, Cry1 F or Cry1A protein in accordance with the invention for delaying or preventing resistance development of cotton bollworm, is preferably in any one of these plants.
  • the term "corn” is used herein to refer to Zea mays.
  • Cotton as used herein refers to Gossypium spp., particularly G. hirsutum and G. barbadense.
  • the term “rice” refers to Oryza spp., particularly O. sativa.
  • Soybean refers to Glycine spp, particularly G. max.
  • Sugarcane is used herein to refer to plants of the genus Saccharum, a tall perennial grass of the family Poaceae, native to warm temperate to tropical regions that can be used for sugar extraction.
  • Sunflower as used herein refers to Helianthus annuus.
  • Transformed plants can be used in a conventional plant breeding scheme to produce more transformed plants with the same characteristics or to introduce the insecticidally effective gene part into other varieties of the same or related plant species. Seeds, which are obtained from the transformed plants, contain the insecticidally effective gene as a stable genomic insert.
  • Cells of the transformed plant can be cultured in a conventional manner to produce the insecticidally effective portion of the Cry2A, VIP3 or Cry1 toxin or protein, which can be recovered for use in conventional insecticide compositions against Lepidoptera.
  • the insecticidally effective gene is inserted in a plant cell genome so that the inserted gene is downstream (i.e., 3') of, and under the control of, a promoter which can direct the expression of the gene part in the plant cell (a plant-expressible promoter).
  • a promoter which can direct the expression of the gene part in the plant cell (a plant-expressible promoter). This is preferably accomplished by inserting the chimeric gene in the plant cell genome, particularly in the nuclear or plastid (e.g., chloroplast) genome.
  • Plant-expressible promoters that can be used in the invention include but are not limited to : the strong constitutive 35S promoters (the "35S promoters") of the cauliflower mosaic virus (CaMV) of isolates CM 1841 (Gardner et al., 1981), CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); the 35S promoter described by Odell et al.
  • the 35S promoters the strong constitutive 35S promoters of the cauliflower mosaic virus (CaMV) of isolates CM 1841 (Gardner et al., 1981), CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); the 35S promoter described by Odell et al.
  • promoters from the ubiquitin family e.g., the maize ubiquitin promoter of Christensen et al., 1992, EP 0 342 926, see also Cornejo et al., 1993
  • the gos2 promoter de Pater et al., 1992
  • the emu promoter Last et al., 1990
  • Arabidopsis actin promoters such as the promoter described by An et al. (1996)
  • rice actin promoters such as the promoter described by Zhang et al. (1991 ) and the promoter described in US 5,641 ,876
  • promoters of the Cassava vein mosaic virus WO 97/48819, Verdaguer et al.
  • a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (e.g., leaves and/or roots) whereby the inserted gene part is expressed only in cells of the specific tissue(s) or organ(s).
  • the insecticidally effective gene could be selectively expressed in the leaves of a plant (e.g., corn, cotton, rice, soybean) by placing the insecticidally effective gene part under the control of a light-inducible promoter such as the promoter of the ribulose-1 ,5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant, such as pea, as disclosed in US 5,254,799.
  • the promoter can, for example, be chosen so that the gene of the invention is only expressed in those tissues or cells on which the target insect pest feeds so that feeding by the susceptible target insect will result in reduced insect damage to the host plant, compared to plants which do not express the gene.
  • a promoter whose expression is inducible, e.g., the MPI promoter described by Cordera et al. (1994), which is induced by wounding (such as caused by insect feeding), or a promoter inducible by a chemical, such as dexamethasone as described by Aoyama and Chua (1997) or a promoter inducible by temperature, such as the heat shock promoter described in US 5,447,858, or a promoter inducible by other external stimuli.
  • the insecticidally effective gene is inserted into the plant genome so that the inserted gene is upstream (i.e., 5') of suitable 3' end transcription regulation signals (i.e., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the chimeric gene in the plant cell genome.
  • suitable 3' end transcription regulation signals i.e., transcript formation and polyadenylation signals.
  • the type of polyadenylation and transcript formation signals is not critical, and can include those of the CaMV 35S gene, the nopaline synthase gene (Depicker et al., 1982), the octopine synthase gene (Gielen et al., 1984) or the T-DNA gene 7 (Velten and Schell, 1985), which act as 3'-untranslated DNA sequences in transformed plant cells.
  • marker genes for the chimaeric genes of this invention also is not critical, and any conventional DNA sequence can be used which encodes a protein or polypeptide which renders plant cells, expressing the DNA sequence, readily distinguishable from plant cells not expressing the DNA sequence (EP 0344029).
  • the marker gene can be under the control of its own promoter and have its own 3' non-translated DNA sequence as disclosed above, provided the marker gene is in a genetic locus in the vicinity of the locus of the gene(s) which it identifies.
  • the marker gene can be, for example: a herbicide resistance gene, such as the sfr or sfrv genes (EPA 87400141 ); a gene encoding a modified target enzyme for a herbicide having a lower affinity for the herbicide than the natural (non-modified) target enzyme, such as a modified 5-EPSP as a target for glyphosate (U.S. Pat. No. 4,535,060; EP 0218571 ) or a modified glutamine synthetase as a target for a glutamine synthetase inhibitor (EP 0240972); or an antibiotic resistance gene, such as a neo gene (PCT publication WO 84/02913; EP 0193259).
  • a herbicide resistance gene such as the sfr or sfrv genes (EPA 87400141 )
  • a gene encoding a modified target enzyme for a herbicide having a lower affinity for the herbicide than the natural (non-modified) target enzyme
  • the transgenic plant obtained can be used in further plant breeding schemes.
  • the transformed plant can be selfed to obtain a plant which is homozygous for the inserted genes. If the plant is an inbred line, this homozygous plant can be used to produce seeds directly or as a parental line for a hybrid variety.
  • the gene can also be crossed into open pollinated populations or other inbred lines of the same plant using conventional plant breeding approaches.
  • Cry2A proteins bind specifically and saturably to the insect midgut of susceptible target insect pests
  • Cry2A proteins include methods known in the art.
  • included in this invention is also a process for isolating Cry2A protein receptors, particularly the proteins functioning as Cry2Ab receptor, as well as the many uses that can be had of such an isolated receptor protein.
  • Such isolated receptor molecules can be used in screening assays to find proteins binding to a different receptor, to find proteins with improved binding to the receptor, and the like.
  • the DNA sequence of such Cry2A receptor may provide a useful screening tool for screening for alterations in such DNA, which can be an indication of alteration of the protein resulting in resistance development.
  • screening can be done quickly using standard DNA detection tools, such as PCR, from insects collected in the field.
  • SEQ ID No. 1 Cry2Ae1 protein
  • SEQ ID No. 2 Cry2Ab2 protein
  • SEQ ID No. 3 Cry1 Fa1 protein
  • SEQ ID No. 4 VIP3Aa1 protein
  • SEQ ID No. 5 VIP3Af1 protein
  • SEQ ID No. 6 VIP3Aa19 protein
  • SEQ ID No. 7 VIP3Aa20 protein
  • SEQ ID No. 8 Cry1Ac1 protein
  • SEQ ID No. 9 Cry1A.1O5 protein
  • SEQ ID No. 10 Cry1Ab1 protein
  • B. thuringiensis strain HD73 from the Bacillus Genetic Stock Collection (Columbus, OH) expressing CryiAc was grown in CCY medium (Stewart et al., 1981) at 28.5 0 C with continuous shaking and air supplement for 48 hours. The pelleted insoluble fraction was washed twice with 1 M NaCI, 10 mM EDTA, and once with 10 mM KCI. CryiAc crystals were solubilized in freshly prepared carbonate buffer (50 mM Na2CO3/NaHCO3, 10 mM DTT; pH 10.5) and incubated at room temperature with shaking at 150 rpm for 2.5 h. Insoluble debris was discarded by centrifugation at 25000 x g for 10 min at 4 0 C.
  • the solubilised CryiAc protoxin was activated by incubation with trypsin (Sigma T-8642) with a trypsin:protein ratio of 1:10 (w:w) at 37 0 C for 2 h. After centrifugation at 25000 x g for 10 min at 4 0 C, the supernatant was dialysed in buffer A (20 mM Tris-HCI, pH 8.65) and filtered prior to anion exchange purification in a MonoQ 5/5 column using an AKTA chromatography system (GE Healthcare, UK). A continuous gradient of buffer B (20 mM Tris-HCI, 1 M NaCI, pH 8.65) up to 60% was used to elute the Cry1 Ac activated toxin.
  • Recombinant B. thuringiensis strain BtlPS78/11 expressing Cry2Ab2 was grown in C2 medium (Donovan et al., 1988) containing 6 ⁇ g/ml choramphenicol at 28°C with shaking at 80 rpm for 47 hours. Following two wash steps in phosphate-buffered saline (PBS) (8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCI; pH 7.4) to which 250 mM NaCI was added, the cell pellet was solubilized in NEE buffer.
  • PBS phosphate-buffered saline
  • Trypsin was added to a final concentration of 0.3 mg/ml out of a freshly prepared 25 mg trypsin/ml and the mixture was incubated at 37°C for 75 min and centrifuged.
  • the Cry2Ab toxin solution was precipitated with ammonium sulfate and the resulting pellet was dissolved in TEE buffer (50 mM Tris-HCI, 5 mM EDTA, 10 mM EGTA; pH 8.6).
  • TEE buffer 50 mM Tris-HCI, 5 mM EDTA, 10 mM EGTA; pH 8.6
  • berliner 1715 cry- strain harbouring plasmid pGA32 expressing Cry2Ae was grown in C2 medium containing erythromycin at 20 ⁇ g/ml at 28°C with shaking at 80 rpm for 144 hours. Following two wash steps in PBS plus 250 mM NaCI, the cell pellet was solubilized in alkaline buffer (0.1 M CAPS, 10 mM EGTA, 5 mM EDTA, pH 12.0) and incubated for 1 hour at 37°C Trypsin was added (at a 3:1 ratio, w/w) out of a freshly prepared 7.5 mg trypsin/ml and the mixture was incubated overnight at 37 0 C and centrifuged. The Cry2Ae toxin solution was dialyzed to PBS.
  • alkaline buffer 0.1 M CAPS, 10 mM EGTA, 5 mM EDTA, pH 12.0
  • BBMV Last instar larvae of H. armigera (ANGR strain, CSIRO Entomology, Australia) and H. zea (USDA-ARS, MS) were dissected and the midguts were preserved at -8O 0 C until required.
  • BBMV were prepared by the differential magnesium precipitation method (Wolfersberger et al., 1987), frozen in liquid nitrogen and stored at -80 0 C. The protein concentration in the BBMV preparations was determined by the method of Bradford (1976) using bovine serum albumin as standard.
  • Cry1 Ac and Cry2Ab toxins were labeled using the chloramine T method.
  • Na 1251 (0.5 mCi) (PerkinElmer, Boston, MA) was added to 25 microgram of Cry toxin in presence of 1/3 v of 18 mM of chloramine T in PBS. After incubation for 45 s, the reaction was stopped by adding 1/4 v of 23 mM potassium metabisulfite in H2O. Finally, 1/4 v of 1M NaI was added, and the mixture was loaded onto a PD10 desalting column (GE Healthcare, UK) equilibrated with buffer column (20 mM Tris-HCI, 150 mM NaCI, 0.1% BSA).
  • the purity of the labeled protein was checked by analysing the elution fractions by SDS-PAGE with further exposure of the dry gel to an X-ray film.
  • the specific activity of the labeled toxin was calculated based on the input toxin, the radioactivity eluting in the protein peak, and the percentage of radioactivity in the toxin band vs. that in minor bands as reveled by SDS-PAGE (Fig.1 ).
  • the estimated specific activities of 125l-Cry1Ac and 125l-Cry2Ab toxins were 3 mCi/mg, and 2.4 mCi/mg, respectively.
  • BBMV Prior to use, BBMV were centrifuged for 10 min at 16000 x g and resuspended in binding buffer (8 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCI; pH 7.4; 0.1% bovine serum albumin). Saturation experiments were carried out by incubating 20 microgram of BBMV from H. armigera with increasing amount of 125l-Cry2Ab in binding buffer for 1 h at 25 0 C. After incubation, samples were centrifuged at 16000 x g for 10 min and the pellet was washed twice with 500 microliter of cold binding buffer. The radioactivity retained in the pellet was measured in an LKB 1282 Compugamma CS gamma counter.
  • Non-specific binding was determined by adding an excess of unlabeled Cry2Ab (1 micromolar) to the reaction. Specific binding was calculated by subtracting the non-specific binding from the total binding.
  • increasing amounts of BBMV were incubated with 0.4 nM and 1.2 nM of labeled Cry1 Ac and Cry2Ab, respectively, in a final volume of 0.1 ml of binding buffer for 1 h at 25 0 C. An excess of unlabeled toxin was used to calculate the non-specific binding.
  • CryiAc and Cry2Ab preparations were tested. Activated CryiAc and Cry2Ab proteins were toxic to H. armigera larvae and the LC50 values are shown in Table 2. Also activated Cry2Ae protein shows significant insecticidal activity to H. armigera.
  • Binding of Cry2Ab was shown to be saturable by incubating H. armigera BBMV with increasing concentrations of labeled Cry2Ab. In the conditions used (0.2 mg BBMV protein/ml), the curve deviated from linearity starting approximately at 5 nM 1251- Cry2Ab and reached a maximum at approximately 20 nM (Fig. 2). Competition experiments with H. armigera BBMV.
  • Binding parameters dissociation constant (Kd) and concentration of binding sites (Rt), were calculated for Cry2Ab and CryiAc from homologous competition curves (Table 1 ). In both cases, homologous competition data fit a single-site model equation. As shown in Table 1 , Cry2Ab has slightly more specific binding sites than CryiAc in H. armigera, but the affinity of Cry2Ab is lower than the affinity of CryiAc for their respective binding sites.
  • Radiolabeling of Cry2Ab and CryiAc was done twice independently and all the experiments described above were repeated with a newly prepared second preparation of radiolabeled CryiAc and Cry2Ab. Results obtained with the second set of radiolabeled toxins were similar to those described above.
  • 125l-Cry2Ab was used to perform homologous and heterologous competition assays in H. zea. As in H. armigera, unlabeled Cry2Ab competed for 125l-Cry2Ab binding sites (Fig. 4B). The homologous competition indirectly demonstrated saturable binding of Cry2Ab in this insect, since the curve reflects a limited number of binding sites in the concentration range of unlabeled competitor. The heterologous competition assay showed that CryiAc did not compete with 125l-Cry2Ab.
  • the concentration range of Cry2Aa protein used in saturation experiments might not have been sufficient to saturate binding sites.
  • the range used was the same as that for Cry 1Ac toxin, but due to the lower affinity of Cry2A protein, a higher concentration of Cry2Aa protein may have been required to show saturation of the binding sites.
  • the concentration of BBMV used might have been inadequate to distinguish specific binding. High levels of nonspecific binding of Cry2A proteins (to vesicle components and/or vials or filters) might have masked specific binding if the concentration of binding sites was not high enough.
  • the BBMV binding ability assay i.e. fixed concentration of labeled toxin and increasing amounts of BBMV
  • a 125 l-Cry2A saturation assay i.e. fixed BBMV concentration and increasing amounts of labeled toxin
  • Cry2A proteins have a saturable, high affinity binding site different from that of Cry1A proteins, has important implications for insect resistance management: it offers a biochemical explanation of why cross-resistance between Cry1A and Cry2A proteins is so rare and provides a solid support for the resistance management strategy of combining cry1A and cry2A genes in the same crop to target H. armigera or H. zea.
  • a first procedure is based on sequential transformation steps in which a plant, already transformed with a first chimeric gene, is retransformed in order to introduce a second gene.
  • the sequential transformation preferably makes use of two different selectable marker genes, such as the resistance genes for kanamycin and phosphinotricin acetyl transferase (e.g., the well known pat or bar genes), which confers resistance to glufosinate herbicides.
  • the use of both these selectable markers has been described in De Block et al. (1987).
  • the second procedure is based on the cotransformation of two chimeric genes encoding different insecticidal proteins on different plasmids in a single step.
  • the integration of both genes can be selected by making use of the selectable markers, linked with the respective genes.
  • separate transfer of two insecticidal protein genes to the nuclear genome of separate plants can be done in independent transformation events, which can subsequently be combined in a single plant through crossing, and plants comprising the different genes can be selected using DNA marker technology.
  • Corn plants comprising the MIR162 event (WO 2007/142840, USDA APHIS petition for non-regulated status 07-253-01 p) are crossed with corn plants containing a chimeric gene comprising the coding region of SEQ ID No. 9 of WO 2002/057664, creating corn plants expressing a VIP3A and a Cry2Ae insect control protein.
  • corn plants containing event Bt11 (USDA APHIS petition for non- regulated status 95-195-01 p) or corn plants containing event MON810 (USDA APHIS petition 96-017-01 p) are crossed with corn plants containing a chimeric gene comprising the coding region of SEQ ID No. 9 of WO 2002/057664, creating corn plants expressing a CrylAb and a Cry2Ae insect control protein.
  • Cotton plants comprising event EE-GH6 as described in the PCT patent application claiming priority to European patent application number 07075460 or 07075485 (unpublished) are crossed with cotton plants comprising comprising event EE-GH5 described in PCT patent application PCT/EP2008/002667, to obtain cotton plants expressing a Cry2A and Cry1 A protein with built-in insect resistance management for H.zea and H.armigera.
  • cotton plants comprising the COT102 event of USDA APHIS petition 03-155- 01 p (WO 2004/039986) are crossed with the above 2-gene cotton plants, or with cotton plants containing a chimeric gene comprising the coding region of SEQ ID N. 7 of WO 2002/057664 and a chimeric gene comprising the CrylAb coding region of SEQ ID No. 2 of US patent 7,049,491 , so that cotton plants expressing the VIP3A, CrylAb and Cry2Ae protein are obtained.
  • Co-expression of at least two insecticidal protein genes in the individual transformants can be evaluated by insect toxicity tests and by biochemical means known in the art.
  • Specific probes allow for the quantitive analysis of the transcript levels; monoclonal antibodies cross-reacting with the respective gene products allow the quantitative analysis of the respective gene products in ELISA tests; and specific DNA probes allow the characterization of the genomic integrations of the transgenes in the transformants.
  • these plants can also comprise other transgenes, such as genes conferring protection to other Lepidopteran insect species or to insect species from other insect orders, such as Coleopteran or Homopteran insect species, or genes conferring tolerance to herbicides, and the like.
  • Vip3Aa19 protein and the genetic material necessary for its production (vector pCOT1 ) in Event COT102 cotton plants (006499) Experimental Use Permit Factsheet (Environmental Protection Agency, USA, www.epa.gov)
  • a K d values represent the equilibrium dissociation constant and were calculated from the homologous competition assay and are expressed in nanomolar concentrations.
  • b Rt values represent the binding site concentration and are expressed in picomoles per milligram of BBMV protein.
  • c Values are the mean of two replicates d Values are the mean of at least four replicates e Mean ⁇ SEM.
  • 125l-Cry2Ab was incubated with BBMV in the absence or presence of an excess of competitor and the pellet from centrifuging the binding reaction mixture was subjected to SDS-PAGE and exposed to an X-ray film for a week.
  • Lane 1 1251- Cry2Ab toxin
  • lane 2 125l-Cry2Ab incubated with BBMV in absence of competitor
  • lane 3 homologous competition (excess of unlabeled Cry2Ab)
  • lane 4 heterologous competition with CryiAc.
  • Figure 2 Saturation of 125l-Cry2Ab specific binding to H. armigera BBMV.
  • BBMV BBMV (20 microgram of vesicle proteins) was incubated with increasing amounts of 125l-Cry2Ab for 1h. The binding reaction was stopped by centrifugation and the radioactivity retained in the pellet was measured. Nonspecific binding was calculated by incubating with an excess of unlabeled Cry2Ab, and subtracted from total binding.

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Abstract

L’invention concerne l’utilisation d’une combinaison de différentes protéines insecticides envers Helicoverpa zea ou Helicoverpa armigeran dans un procédé de gestion de la résistance à des insectes, lesdites protéines étant : a) une protéine Cry2A telle que Cry2Aa, Cry2Ab ou Cry2Ae et b) une protéine Cry1A, Cry1F ou VIP3A, de telles protéines se liant notamment de manière saturée à la membrane vitelline de l’insecte Helicoverpa zea ou Helicoverpa armigera. L’invention concerne également des plantes et des graines qui expriment une telle combinaison de protéines, qui sont utilisées pour retarder ou inhiber le développement d’une résistance chez les populations de telles espèces d’insectes.
PCT/EP2009/002788 2008-06-13 2009-04-16 Gestion de la résistance aux vers de la capsule dans des plantes transgéniques WO2009149787A1 (fr)

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US12/997,133 US20110088129A1 (en) 2008-06-13 2009-04-16 Bollworm Insect Resistance Management in Transgenic Plants
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