CA2727637A1 - Bollworm insect resistance management in transgenic plants - Google Patents

Bollworm insect resistance management in transgenic plants Download PDF

Info

Publication number
CA2727637A1
CA2727637A1 CA2727637A CA2727637A CA2727637A1 CA 2727637 A1 CA2727637 A1 CA 2727637A1 CA 2727637 A CA2727637 A CA 2727637A CA 2727637 A CA2727637 A CA 2727637A CA 2727637 A1 CA2727637 A1 CA 2727637A1
Authority
CA
Canada
Prior art keywords
protein
plants
event
cotton
insecticidal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA2727637A
Other languages
French (fr)
Inventor
Carmen Sara Hernandez
Adri Van Vliet
Jeroen Van Rie
Juan Ferre Manzanero
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer BioScience NV
Original Assignee
Bayer BioScience NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to EP08010791.5 priority Critical
Priority to EP08010791 priority
Priority to US12/214,022 priority patent/US20090313717A1/en
Priority to US12/214,022 priority
Priority to NZ57218908 priority
Priority to NZ572189 priority
Application filed by Bayer BioScience NV filed Critical Bayer BioScience NV
Priority to PCT/EP2009/002788 priority patent/WO2009149787A1/en
Publication of CA2727637A1 publication Critical patent/CA2727637A1/en
Application status is Abandoned legal-status Critical

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279Phenotypically 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
    • C12N15/8286Phenotypically 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
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/11Specially adapted for crops
    • Y02A40/16Pest or insect control
    • Y02A40/162Genetically modified [GMO] plants resistant to insects

Abstract

This invention relates to the use of a combination of different proteins insecticidal to Helicoverpa zeaor Helicoverpa armigeran an insect resistance management process, wherein such proteins are:
a) a Cry2A protein such as Cry2Aa, Cry2Ab, or Cry2Ae and b) a Cry1A, Cry1F or VIP3A protein, particularly wherein such proteins binds saturably to the insect midgut membrane of Helicoverpa zeaor Helicoverpa annigera, as well as plants and seeds expressing such combination of proteins, which are used to delay or prevent the development of resistance in populations of such insect species.

Description

Bollworm insect resistance management in transgenic plants Description Field of the invention 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 CrylAc, a CrylAb or a Cry1A.105 protein. In one embodiment, such plants are used to delay or prevent the development of resistance to crop plants in populations of the cotton bollworm.

Also, in the present invention the simultaneous or sequential use of a Cry2A
protein and a VIP3A, CrylA or Cry1F protein or plants expressing such Cry2A protein and a VIP3A, CrylA or Cryl 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 Cryl 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, Cry1Ab or CrylAc protein, is particularly useful to prevent or delay resistance development of populations of cotton bollworms against the insecticidal proteins expressed in such plants.

CONFIRMATION COPY

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 CrylA and/or a CrylF protein, comprising providing such plants also with a gene expressing a Cry2A protein. Since such Cry2A
protein and such Cry1A or VIP3 or Cryl 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. In one embodiment of the invention, 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, VIP3, Cry1 F or CrylA protein in Helicoverpa zea or Helicoverpa armigera.

Background of the invention Insect pests cause huge economic losses worldwide in crop production, and farmers face every year the threat of yield losses due to insect infestation. Genetic engineering of insect resistance in agricultural crops has been an attractive approach to reduce costs associated with crop-management and chemical control practices.
The first generation of insect resistant crops have been introduced into the market since 1996, based on the expression in plants of insecticidal proteins derived from the gram-positive soil bacterium Bacillus thuringiensis (abbreviated herein as "Bt").

In contrast to the rapid development of insect resistance to some synthetic insecticides, so far insect resistance to plant-incorporated insecticidal proteins such as B. thuringiensis proteins has not been reported despite many years of use.
This may be because of the insect resistance management programs which are being used for such transgenic plants, such as the expression of a high dose level of protein for the main target insect(s), and the use of refuge areas (either naturally present or structured refuges) containing plants without such insecticidal proteins.

Procedures for expressing B. thuringiensis or other insecticidal protein genes in plants in order to render the plants insect-resistant are well known in the art and provide a new approach to insect control in agriculture which is at the same time safe, environmentally attractive and cost-effective. An important determinant for the continued success of this approach will be whether (or when) insects will be able to develop resistance to insecticidal proteins expressed in transgenic plants. In contrast to a foliar application, after which insecticidal proteins are typically rapidly degraded, the transgenic plants will exert a continuous selection pressure on the insects. It is clear from laboratory selection experiments that a continuous selection pressure can lead to adaptation to insecticidal proteins, such as the B. thuringiensis Cry proteins, in insects.

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-derived 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.

The most widely used proteins introduced in plants for control of Lepidopteran insects include the CrylA, Cryl F and VIP3A proteins. Based on competition binding assays, it has been proposed that a Cryl F protein competes for the same midgut binding site as CrylAc in Helicoverpa zea and Helicoverpa armigera. Moreover, no evidence was found for any unshared sites for Cryl F in these insects species (Hernandez and Ferre, 2005). Hence a combination of these two proteins in the same plant is not a suitable approach for resistance management of Helicoverpa zea or Helicoverpa armigera insects. Only a low affinity of Cry1 Fa for the Cryl Ac binding site was found, this low affinity likely reflects the low toxicity observed for the Cryl F
protein in these insect species (Liao et al., 2002).

There appears to be a generally accepted proposition that the mode of action of Cry2 toxins is unique, and different from other three-domain Cry toxins, due to their non-specific and/or non-saturable binding to an unlimited number of binding sites (English et al., 1994; Lee et al., 2006). Since the publication by English et al.
(1994), the binding characteristics of the Cry2A protein described therein have apparently been reiterated, and several authors still refer to the method described therein for the preparation of Cry2A protein for binding assays (e.g., Luo et al., 2007).
Also, EPA
biopesticide factsheet 006487 (2002) 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. Also, USDA-APHIS petition for non-regulated status 06-298-01p (2006) states that a Cry1A and Cry2A protein share many common binding sites (www.aphis.usda.gov/brs/aphisdocs/06 29801p.pdf).

There is no report available that demonstrates saturable binding of a Cry2A
protein based on a direct saturability assay, wherein a fixed concentration of binding sites (i.e., BBMVs) are used to which increasing concentrations of labeled protein are added. In contrast to the reports and findings in the art, the inventors conclusively show herein that a Cry2A toxin can bind in a specific and saturable manner to receptors in susceptible insects, and that a Cry2A toxin does not share (or compete for) binding sites with a Cry1A toxin in the cotton bollworms, H.zea and H.armigera.
The current document contains the first report showing that Cry2A proteins bind saturably to the midgut brush border membrane of susceptible insects in a direct saturability assay, and also contains the first report analyzing binding competition between different Cry2A proteins.

SUMMARY OF THE INVENTION

Provided herein is 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 Cryl A, Cryl 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, Cry1F of VIP3A protein insecticidal to Helicoverpa zea or Helicoverpa armigera in said plants.

In one embodiment of this invention, 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 Cry1F 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.

Further provided herein is 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 Cryl 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. Further provided herein is such method wherein said plants are obtained by transformation of a plant with plant-expressible chimeric genes encoding said Cry2Ae and Cry1A, VIP3 of Cry1F 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 CrylA-, VIP3- or CrylF-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 Cryl F protein insecticidal to Helicoverpa zea or Helicoverpa armigera, and obtaining progeny plants and seed comprising such at least two chimeric genes.

In another embodiment of this invention a method is provided 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 Cryl A, VIP3 or Cryl F protein insecticidal to Helicoverpa zea or Helicoverpa armigera, particularly a VIP3 or CrylA 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.

Also provided herein is the use of at least two different insecticidal proteins in transgenic plants to prevent or delay insect resistance development in populations of Helicoverpa zea or Helicoverpa armigera, wherein said proteins do not share binding sites in the midgut of insects of said insect species, as can be determined by competition binding experiments, comprising expressing a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and a Cryl F, VIP3 or Cry1A
protein insecticidal to Helicoverpa zea or Helicoverpa armigera in said transgenic plants, as well as the use of a chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and a chimeric gene encoding a Cryl F, VIP3 or Cry1A 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 VIP3 or Cry1A
protein insecticidal to Helicoverpa zea or Helicoverpa armigera, 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.

In one embodiment herein is provided the use of a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera in combination with a Cry1A, VIP3 or Cryl 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.

Also provided herein is the use of plants comprising a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera in a region where populations of said insect species have become resistant to plants comprising a Cry1 F, VIP3 and/or Cry1A
protein, wherein said use can comprise the sowing, planting or growing of plants comprising a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera in said region, as well as the use of plants comprising a Cry1F, VIP3 and/or Cry1A
protein insecticidal to Helicoverpa zea or Helicoverpa armigera in a region where populations of said insect species have become resistant to plants comprising a Cry2Ae protein, wherein said use can comprise the sowing, planting or growing of plants comprising a Cryl F, VIP3 and/or CrylA protein insecticidal to Helicoverpa zea or Helicoverpa armigera in said region.

Also provided herein is the use of a chimeric gene encoding a Cry2Ae protein insecticidal to Helicoverpa zea or Helicoverpa armigera and a chimeric gene encoding a Cry1A, VIP3 or Cryl 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.

In one embodiment of this invention, the use of 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 CrylF protein insecticidal to Helicoverpa zea or Helicoverpa armigera.

In one embodiment, 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 CrylF 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 CrylA, VIP3 or Cryl 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, Cryl 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 Cryl protein described herein.

Also provided herein are the above uses or processes wherein the plants express the Cry2Ae, VIP3, CrylF or CrylA proteins at a high dose for Helicoverpa zea or Helicoverpa armigera.

Further provided herein is 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, Cryl F or VIP3A
protein, particularly a Cry2Ae and a CrylAb or CrylAc or VIP3A protein, preferably a Cry2Ae and CrylAb and VIP3 protein, insecticidal to said insect species. Also provided herein is a field of plants, particularly corn or cotton 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 Cryl A, Cryl F or VIP3A protein, particularly a Cry2Ae and a CrylAb, CrylAc 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. Particularly, in such use the Cry1A
protein is selected from the group of: a CrylAc, CrylAb, CrylA.105, or a CrylAc or CrylAb hybrid protein, such as a protein encoded by any one of the cry1A
coding regions referred to herein. Such Cry2Ab and Cry1A 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, as used herein, refers to an insecticidal Cry2Ae protein such as a full length Cry2Ae protein of SEQ ID No. 2 of WO 2002/057664 (Cry2Ael, 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, as used herein, 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. 2 herein), or a protein comprising a Cry2Ab toxic fragment, such as a fusion protein of a Cry2Ab2 protein fragment with a chioroplast 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. 2 herein, or to the amino acid sequence 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 coding region part of the Cry2Ab chimeric gene contained in cotton event 15985 as described in USDA-APHIS petition for non-regulated status 00-342-01p, the protein encoded by the Cry2Ab2 coding region part of the Cry2Ab chimeric gene contained in corn event MON89034 as described in USDA-APHIS petition for non-regulated status 06-298-01p, particularly any protein comprising the smallest toxic fragment of any one of such Cry2Ab proteins, or a variant of any one of such Cry2Ab proteins differing in 1-5 amino acids retaining toxicity to Helicoverpa zea or Helicoverpa armigera.

A Cryl F protein, as used herein, includes any protein comprising the smallest toxic fragment of the amino acid sequence of a Cryl 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 CrylFal 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. 3 or to the Cryl F protein of NCBI
accession AAA22347, 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. A Cryl F protein, as used herein, includes the protein encoded by the Cryl F gene in Cryl 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 TC1 507 or (US 7,288,643, WO 2004/099447, USDA APHIS petitions for non-regulated status 00-136-01p and 03-181-01p), particularly any protein comprising the smallest toxic fragment of any one of such Cryl F proteins, or a variant of any one of such Cryl F

proteins differing in 1-5 amino acids with toxicity to Helicoverpa zea or Helicoverpa armigera.

In one embodiment in the invention, the VIP3 protein is a protein insecticidal' to Helicoverpa zea or Helicoverpa armigera larvae, and which is any one of the 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. 6 herein) or 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 He/icoverpa 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 CA143275 (SEQ ID No. 5 herein, SEQ ID No. 4 in W003/080656), particularly with its smallest toxic fragment, as determined using pairwise alignments using the GAP program of the Wisconsin package of GCG, as well as a 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 CA143275 (ISP3a, SEQ ID No.4 of WO 03/080656), CA143276 (ISP3b, SEQ ID No.6 in WO 03/080656), and CA143277 (ISP3C, SEQ ID No. 2 of WO 03/080656), respectively) and insecticidal fragments, hybrids or variants thereof.
In one embodiment, 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 03-155-01p) or the VIP3Aa2O protein (NCBI accession ABG20429, SEQ ID NO: 2 in WO 2007/142840, SEQ ID No. 7 herein) introduced in corn plants (e.g., event MIR162, USDA APHIS petition for non-regulated status 07-253-01p), or the VIP3A protein produced in cotton event COT202 or COT203 (WO 2005/054479 and WO 2005/054480, respectively), or a variant of any one of the above VIP3 proteins differing in 1-5 amino acids and retaining toxicity to Helicoverpa zea or Helicoverpa armigera.

A Cry1A protein, as used herein, refers to a CrylAcl (SEQ ID No. 8), CrylA.105 (SEQ ID No. 9 ) or a CrylAb1 (SEQ ID No. 10) protein, and includes any protein comprising the smallest toxic fragment of the amino acid sequence of a CrylAc, CrylA.105 or CrylAb 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 (CrylAc; 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 CrylA.105 protein in SEQ ID No. 9 herein, encoded by the Cry1A transgene in corn event MON89034 (USDA APHIS petition for non-regulated status 06-298-01p, WO 2007/140256, SEQ ID NO: 2 or 4 in WO 2007/027777), or of the CrylAb protein encoded by the crylAb coding region in cotton event COT67B
(USDA APHIS petition for non-deregulated status 07-108-01p, WO 2006/128573).
Also included in this definition are variants of the amino acid sequence in NCBI
accession AAA22331 (CrylAc1), NCBI accession AAA22330 (CrylAb, Wabiko et al., 1986), or the amino acid sequence of the CrylA.105 protein described in USDA
APHIS petition for non-regulated status 06-298-01p, such as proteins having an amino acid sequence identity of at least 90% with such a CrylAc, CrylA.105 or CrylAb protein, particularly of SEQ ID Nos. 8, 9 or 10, more, 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 Cry1A
protein.
Included herein as Cry1A proteins are 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 CrylAc 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 Cry1Ab protein encoded by the crylAb coding region in cotton event COT67B (USDA APHIS
petition for non-deregulated status 07-108-01p, WO 2006/128573), the CrylAb coding region contained in cotton event EE-GH5 described in PCT patent application PCT/EP2008/002667 (unpublished), the Cry1Ab coding region of SEQ ID No. 2 of US patent 7,049,491, the CrylA.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), the CrylAc-like protein encoded by the hybrid crylAc coding region in cotton event 15985 or cotton event 531, 757, or 1076 (USDA APHIS petition for non-regulated status 94-308-01p, the chimeric CrylAc protein encoded by the cry1A cotton event of WO 2002/100163), the crylAb protein encoded by the crylAb coding region in cotton events T342-142, 1143-14A, 1143-51B,CE44-69D, or CE46-02A of WO 2006/128568, WO
2006/128569, WO 20061128570, WO 2006/128571, or WO 2006/128572 respectively (i.e., the protein encoded by the DNA of SEQ ID No. 7 in WO 2006/128568, WO
2006/128569, WO 2006/128571, or WO 2006/128572, or by the DNA of SEQ ID No.
in WO 2006/128570), or a protein comprising the smallest toxic fragment of any one of such Cry1A proteins, or a variant of any one of the above Cry1A
proteins differing in 1-5 amino acids but retaining toxicity to H.zea or H.armigera.

Also provided herein are 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. In one embodiment said plants comprise transgenes encoding the proteins: i) Cry2Aa, Cry2Ab or Cry2Ae, and ii) CrylAb, Cry1Ac, CrylFa, or VIP3A, particularly a Cry2Ae protein and a CrylAb and/or VIP3A protein. In another embodiment said plants or seeds are corn or cotton plants or seeds containing a chimeric gene encoding a Cry1A, Cry1F 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 11 43-14A, cotton event 1143-51 B, cotton event CE44-69D, cotton event 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 herein.

Also provided herein are 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 Cry1F 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.

In one embodiment of this invention, in the uses, methods or plant of the invention the Cry2Ae, Cry2Ab, VIP3, Cry1F or CrylA chimeric genes are the chimeric genes contained in any one of the above corn or cotton events. In accordance with the invention is also included any one of the herein described uses, methods, plants or seeds wherein the term Cry2Ae is replaced by the term Cry2Aa or Cry2Ab, as well as any of the above uses, methods, plants or seeds involving a Cry2Ae, Cry2Aa or Cry2Ab protein wherein the binding of such Cry2A protein is specific and saturable to the midgut BBMVs of H. zea or H. 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 CrylA, Cry1F or VIP3 protein, in standard competition binding assays as described herein, in H.armigera or H.
zea.

In the above plants, seeds, uses or methods of the current invention, 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 Cryl 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.

Further provided herein is 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, such method 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 CrylA, Cry1 F or VIP3 proteins in such insects, such as .the data disclosed herein or similar data reported in another document. In one embodiment such Cry2A protein is a Cry2Aa, Cry2Ab or Cry2Ae protein and such Cry1A protein is a CrylAc, CrylAb, or CrylA.105 protein, and said VIP3 protein is a VIP3Aa protein.

Further provided herein is 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 Cry1A, Cryl F or VIP3A protein insecticidal to said insect species, in said plants.
Said plants are preferably corn or cotton plants.

Also included herein are the above methods or plants, wherein besides the Cry or VIP3 proteins, also a 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. These Bt toxin enhancer proteins are fed to target insects together with one or more Bt insecticidal toxins such as Cry proteins. These 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. In one embodiment, said Bt toxin enhancer protein is a part of a midgut cell Bt toxin receptor of a H. zea or H. armigera insect.

DETAILED DESCRIPTION OF THE INVENTION

Because of the success and the increasing number of plants comprising introduced insecticidal proteins such as Bt Cry or VIP3 proteins, resistance management is even more important now than in the past.

While the insecticidal spectrum of different insecticidal proteins derived from Bt or other bacteria, such as the Cry or VIP proteins, can be different, the major pathway of their toxic action is common. All Bt-derived insecticidal proteins used in transgenic plants, for which the mechanism of action has been studied in at least one target insect (e.g., Cryl and VIP3 toxins), are proteolytically activated in the insect gut and interact with the midgut epithelium of sensitive species and cause lysis of the epithelial cells. In the pathway of toxic action of Cry proteins and VIP
proteins, the specific binding of the toxin to receptor sites on the brush border membrane of these cells is a crucial feature (Hofmann et al., 1988; Lee et al., 2003). The binding sites are typically referred to as receptors, since the binding is saturable and with high affinity.

When two different insecticidal proteins share receptor binding sites in insects, they do not provide a good combination for insect resistance management purposes.
Indeed, the most likely mechanism of resistance to insecticidal proteins such as Bt Cry proteins - and the only major mechanism found in field-developed insect resistance to Bt sprays so far - is receptor binding modification. Proteins that are highly similar in amino acid sequence often share receptor sites (e.g., the CrylAb and CrylAc proteins). But, even two different proteins having quite a different amino acid sequence may bind with high affinity to a common binding site in an insect species (such as, e.g., the CrylAb and CrylF proteins in Plutella xylostella).
Also, it has been found that two proteins that do not share binding sites in one insect species, may share a common binding site in another insect species (e.g., the CrylAc and CrylBa proteins were found to share a binding site in Chilo suppressalis by Fiuza et al. (1996) while they were found to bind to different binding sites in Plutella xylostella (Ballester et al. 1999)).

The current invention relates to Cry2A proteins that do not show competition for the Cryl F, VIP3 or Cryl 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, Cryl F or CrylA protein, preferably at least a Cry2Ae protein and a CrylAb, CrylAc, CrylA.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.

The 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, CrylAc 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. In one embodiment, the binding to such specific binding sites is saturable as measured in a direct saturability assay. As used herein, 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. In case of saturable binding, 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), whereas in case of non-saturable binding, 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.

It is important when combining different insecticidal proteins in plants with the aim to delay or decrease insect resistance development of a target insect species, to check experimentally (i.e., by performing binding assays) in the target insect species if a proposed combination of different insecticidal proteins shares binding sites in the midgut of the target insect. In the current invention, when there is competition between two different insecticidal proteins for a single binding site (meaning when the binding data from competition binding experiments are plotted, both proteins reach the same plateau at the bottom of the competition curve), such proteins are not a useful combination in plants from an insect resistance management perspective.
As used herein, when two different insecticidal proteins bind to two different binding sites, such proteins are useful from an insect resistance management perspective.
As used herein, for proteins binding to different binding sites, 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)). If 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.

In this invention, 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.
The methods and techniques for testing sharing of binding sites to insect larvae for a pair of different insecticidal proteins are well known