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Use of Cry1ab in Combination with Cry1be For (19) TZZ _¥¥__T (11) EP 2 513 318 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/82 (2006.01) A01H 5/00 (2018.01) 24.01.2018 Bulletin 2018/04 A01H 5/10 (2018.01) A01N 63/02 (2006.01) A01N 65/00 (2009.01) (21) Application number: 10842620.6 (86) International application number: (22) Date of filing: 16.12.2010 PCT/US2010/060830 (87) International publication number: WO 2011/084631 (14.07.2011 Gazette 2011/28) (54) USE OF CRY1AB IN COMBINATION WITH CRY1BE FOR MANAGEMENT OF RESISTANT INSECTS VERWENDUNG VON CRY1AB IN KOMBINATION MIT CRY1BE ZUR BEKÄMPFUNG RESISTENTER INSEKTEN UTILISATION DE CRY1AB EN COMBINAISON AVEC CRY1BE POUR LA PRISE EN CHARGE D’INSECTES RÉSISTANTS (84) Designated Contracting States: • BURTON, Stephanie, L. AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Indianapolis GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO IN 46278 (US) PL PT RO RS SE SI SK SM TR Designated Extension States: (74) Representative: Zwicker, Jörk et al ME ZSP Patentanwälte PartG mbB Hansastraße 32 (30) Priority: 16.12.2009 US 284290 P 80686 München (DE) (43) Date of publication of application: (56) References cited: 24.10.2012 Bulletin 2012/43 US-A- 5 723 758 US-A- 5 990 390 US-A1- 2005 155 103 US-A1- 2008 311 096 (73) Proprietor: Dow AgroSciences, LLC Indianapolis, Indiana 46268 (US) • AHMAD ANWAAR ET AL: "Expression of synthetic cry1Ab and cry1Ac genes in Basmati (72) Inventors: rice (Oryza sativa L.) variety 370 via • MEADE, Thomas Agrobacterium-mediated transformation for the Zionsville control of the European corn borer (Ostrinia IN 46077 (US) nubilalis)", IN VITRO CELLULAR & • NARVA, Kenneth DEVELOPMENT BIOLOGY. PLANT, Zionsville GAITHERSBURG, MD, US, vol. 38, no. 2, 1 March IN 46077 (US) 2002 (2002-03-01), pages 213-220, XP002711544, • STORER, Nicholas, P. ISSN: 1054-5476 Kensington • BRAVO A ET AL: "How to cope with insect MD 20895 (US) resistance to Bt toxins?", TRENDS IN • SHEETS, Joel, J. BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, Zionsville CAMBRIDGE, GB, vol. 26, no. 10, 2 October 2008 IN 46077 (US) (2008-10-02), pages573-579, XP025406825, ISSN: • WOOSLEY, Aaron, T. 0167-7799, DOI: 10.1016/J.TIBTECH.2008.06.005 Fishers [retrieved on 2008-08-14] IN 46038 (US) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 513 318 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 513 318 B1 • BOHOROVA ET AL.: ’Novel synthetic Bacillus • GUTIERREZ ET AL.: ’Physiologically based thuringiensiscry1B gene and the cry1 B-cry1 Ab demographics of Bt cotton-pest interactions I.’ translational fusion confer resistance to PINK BOLLWORM RESISTANCE, REFUGE AND southwestern corn borer, sugarcane borer and RISK ECOLOGICAL MODELLING vol. 191, 2006, fall armyworm in transgenic tropical maize.’ pages 346 - 359, XP005239868 THEOR APPL GENET vol. 103, 2001, pages 817 - 826, XP008161572 • ’UniProt Entry Q6PYW8.’, [Online] 2007, Retrieved from the Internet: <URL:http://www.uniprot.org/uniprot/Q6PYW8. txt?version=22> [retrieved on 2011-02-18] • SCHUPHAN ET AL.: ’The European corn borer (Ostrinia nubilalis, Hbn.), its susceptibility to the Bt- toxin Cry1 F, its pheromone races and its gene flow in Europe in view of an Insect Resistance Management’’ DARWIN, [Online] October 2009, page 10, XP008161574 Retrieved from the Internet: <URL:http://darwin.bth.rwth-aachen.de/opus3 /volltexte/2010/3341/pdf/3341.pdf> [retrieved on 2011-02-18] 2 EP 2 513 318 B1 Description Background of the Invention 5 [0001] Humans grow corn for food and energy applications. Humans also grow many other crops, including soybeans and cotton. Insects eat and damage plants and thereby undermine these human efforts. Billions of dollars are spent each year to control insect pests and additional billions are lost to the damage they inflict. Synthetic organic chemical insecticides have been the primary tools used to control insect pests but biological insecticides, such as the insecticidal proteins derived from Bacillus thuringiensis (Bt), have played an important role in some areas. The ability to produce 10 insect-resistant plants through transformation with Bt insecticidal protein genes has revolutionized modem agriculture and heightened the importance and value of insecticidal proteins and their genes. [0002] Several Bt proteins have been used to create the insect-resistant transgenic plants that have been successfully registered and commercialized to date. These include Cry1Ab, Cry1Ac, Cry1F and Cry3Bb in corn, Cry1Ac and Cry2Ab in cotton, and Cry3A in potato. 15 [0003] The commercial products expressing these proteins express a single protein except in cases where the com- bined insecticidal spectrum of 2 proteins is desired ( e.g., Cry1Ab and Cry3Bb in corn combined to provide resistance to lepidopteran pests and rootworm, respectively) or where the independent action of the proteins makes them useful as a tool for delaying the development of resistance in susceptible insect populations (e.g., Cry1Ac and Cry2Ab in cotton combined to provide resistance management for tobacco budworm). See also U.S. Patent Application Publication No. 20 2009/0313717, which relates to a Cry2 protein plus a Vip3Aa, Cry 1F, or Cry 1A for controlHelicoverpa of zea or armigerain. WO 2009/132850 relates to Cry 1F or Cry 1A and Vip3Aa for controlling Spodoptera frugiperda. U.S. Patent Application Publication No. 2008/0311096 relates in part to Cry1Ab for controlling Cry1F-resistant European corn borer (ECB; Ostrinia nubilalis (Hübner)). [0004] That is, some of the qualities of insect-resistant transgenic plants that have led to rapid and widespread adoption 25 of this technology also give rise to the concern that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies have been suggested for preserving the utility of Bt-based insect resistance traits which include deploying proteins at a high dose in combination with a refuge, and alternation with, or co-deployment of, different toxins (McGaughey et al. (1998), "B.t. Resistance Management," Nature Biotechnol. 16:144-146). [0005] The proteins selected for use in an insect resistant management (IRM) stack need to exert their insecticidal 30 effect independently so that resistance developed to one protein does not confer resistance to the second protein ( i.e., there is not cross resistance to the proteins). If, for example, a pest population selected for resistance to "Protein A" is sensitive to "Protein B", one would conclude that there is not cross resistance and that a combination of Protein A and Protein B would be effective in delaying resistance to Protein A alone. [0006] In the absence of resistant insect populations, assessments can be made based on other characteristics pre- 35 sumed to be related to mechanism of action and cross-resistance potential. The utility of receptor-mediated binding in identifying insecticidal proteins likely to not exhibit cross resistance has been suggested (van Mellaert et al. 1999). The key predictor of lack of cross resistance inherent in this approach is that the insecticidal proteins do not compete for receptors in a sensitive insect species. [0007] In the event that two Bt toxins compete for the same receptor in an insect, then if that receptor mutates in that 40 insect so that one of the toxins no longer binds to that receptor and thus is no longer insecticidal against the insect, it might be the case that the insect will also be resistant to the second toxin (which competitively bound to the same receptor). That is, the insect is cross-resistant to both Bt toxins. However, if two toxins bind to two different receptors, this could be an indication that the insect would not be simultaneously resistant to those two toxins. [0008] For example, Cry1Fa protein is useful in controlling many lepidopteran pests species including ECB and the 45 fall armyworm (FAW; Spodoptera frugiperda), and is active against the sugarcane borer (SCB; Diatraea saccharalis). The Cry1Fa protein, as produced in transgenic corn plants containing event TC1507, is responsible for an industry- leading insect resistance trait for FAW control. Cry1Fa is further deployed in the Herculex®, SmartStax™, and Wid- eStrike™ products. [0009] The ability to conduct (competitive or homologous) receptor binding studies using Cry1Fa protein has been 50 limited because a common technique available for labeling proteins for detection in receptor binding assays tends to inactivate the insecticidal activity of the Cry1Fa protein. [0010] Additional Cry toxins are listed at the website of the officialB.t. nomenclature committee (Crickmore et al.; lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). There are currently nearly 60 main groups of "Cry" toxins (Cry1-Cry59), with additional Cyt toxins and VIP toxins and the like. Many of each numeric group have capital-letter subgroups, and 55 the capital letter subgroups have lower-cased letter sub-subgroups. (Cry 1 has A-L, and Cry1A has a-i, for example). 3 EP 2 513 318 B1 Brief Summary of the Invention [0011] The subject invention relates in part to the surprising discovery that Cry1Ab and Cry 1 Be do not compete for binding to sites in European corn borer (ECB; Ostrinia nubilalis (Hübner)) or fall armyworm (FAW; Spodoptera frugiperda) 5 gut cell membrane preparations. As one skilled in the art will recognize with the benefit of this disclosure, plants that produce both of these proteins (including insecticidal portions of the full-length proteins) can delay or prevent the devel- opment of resistance to any of these insecticidal proteins alone.
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