(19) TZZ¥Z¥Z¥_T (11) EP 3 030 663 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) C07K 14/435 (2006.01) 04.09.2019 Bulletin 2019/36 (86) International application number: (21) Application number: 14747277.3 PCT/US2014/047204 (22) Date of filing: 18.07.2014 (87) International publication number: WO 2015/010026 (22.01.2015 Gazette 2015/03) (54) COMPOSITIONS AND METHODS FOR CONTROLLING LEPTINOTARSA ZUSAMMENSETZUNGEN UND VERFAHREN ZUR BEKÄMPFUNG VON LEPTINOTARSA COMPOSITIONS ET MÉTHODES DE LUTTE CONTRE LEPTINOTARSA (84) Designated Contracting States: (74) Representative: Uexküll & Stolberg AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Partnerschaft von GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Patent- und Rechtsanwälten mbB PL PT RO RS SE SI SK SM TR Beselerstraße 4 22607 Hamburg (DE) (30) Priority: 19.07.2013 US 201361856137 P 01.11.2013 US 201361899000 P (56) References cited: 17.04.2014 US 201461980800 P WO-A1-2013/010691 WO-A2-2007/035650 WO-A2-2007/074405 (43) Date of publication of application: 15.06.2016 Bulletin 2016/24 • J. K. COLBOURNE ET AL: "The Ecoresponsive Genome of Daphnia pulex", SCIENCE, vol. 331, (60) Divisional application: no. 6017, 3 February 2011 (2011-02-03), pages 19185431.4 555-561, XP055151455, ISSN: 0036-8075, DOI: 10.1126/science.1197761 -& DATABASE EMBL (73) Proprietor: Monsanto Technology LLC [Online] 27 February 2008 (2008-02-27), St. Louis, MO 63167 (US) "CBIB7954.fwd CBIB_Daphnia_pulex_Chosen_One_Library_2 (72) Inventors: Daphnia pulex cDNA clone CBIB7954 5’, mRNA • BEATTIE, Jodi Lynn sequence.", XP002732239, retrieved from EBI St. Louis, Missouri 63167 (US) accession no. EM_EST:FE348695 Database • CRAWFORD, Michael John accession no. FE348695 St. Louis, Missouri 63167 (US) • James A. Baum ET AL: "Progress Towards • EADS, Brian Donovan RNAi-Mediated Insect Pest Management" In: St. Louis, Missouri 63167 (US) "Advances in Insect Physiology", 1 January 2014 • FLAGEL, Lex Evan (2014-01-01), Academic Press, XP055392568, St. Louis, Missouri 63167 (US) ISSN: 0065-2806 vol. 47, pages 249-295, DOI: • KAPOOR, Mahak 10.1016/B978-0-12-800197-4.00005-1, St. Louis, Missouri 63167 (US) • Jesús Gómez-Zurita ET AL: "Recalibrated Tree of • TAYLOR, Christina Marie Leaf Beetles (Chrysomelidae) Indicates St. Louis, Missouri 63167 (US) Independent Diversification of Angiosperms and Their Insect Herbivores", PLoS ONE, vol. 2, no. 4, 11 April 2007 (2007-04-11), page e360, XP055480916, DOI: 10.1371/journal.pone.0000360 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 3 030 663 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 3 030 663 B1 Remarks: The complete document including Reference Tables and the Sequence Listing can be downloaded from the EPO website 2 EP 3 030 663 B1 Description FIELD 5 [0001] Methods for controlling invertebrate pest infestations, particularly in plants, as well as compositions, polynu- cleotides, and recombinant DNA constructs useful in such methods are disclosed. More specifically, this invention is related to polynucleotides and methods of use thereof for modifying the expression of genes in an insect pest, particularly through RNA interference. Pest species of interest include Leptinotarsa species, especially those that infest crop plants. 10 BACKGROUND [0002] Commercial crops are often the targets of attack by invertebrate pests such as insects. Compositions for controlling insect infestations in plants have typically been in the form of chemical insecticides. However, there are several disadvantages to using chemical insecticides. For example, chemical insecticides are generally not selective, 15 and applications of chemical insecticides intended to control insect pests in crop plants can exert their effects on non- target insects and other invertebrates as well. Chemical insecticides often persist in the environment and can be slow to degrade, thus potentially accumulating in the food chain. Furthermore the use of persistent chemical insecticides can result in the development of resistance in the target insect species. Thus there has been a long felt need for more environmentally friendly methods for controlling or eradicating insect infestation on or in plants, i.e., methods which are 20 species-selective, environmentally inert, non-persistent, and biodegradable, and that fit well into pest resistance man- agement schemes. [0003] RNA interference (RNAi, RNA-mediated gene suppression) is another approach used for pest control. In in- vertebrates, RNAi-based gene suppression was first demonstrated in nematodes (Fire et al., (1998) Nature, 391:806-811; Timmons & Fire (1998) Nature, 395:854). Subsequently, RNAi-based suppression of invertebrate genes using recom- 25 binant nucleic acid techniqueshas been reported in anumber ofspecies, including agriculturally or economically important pests from various insect and nematode taxa. [0004] Leptinotarsa spp. form a genus including a number of species that infest commercially important plants, including many solanaceous plants (e. g., potato, tomato, eggplant, peppers, tobacco, and petunia). For example, Leptinotarsa decemlineata (Colorado potato beetle, CPB) is an early- to mid-season pest affecting solanaceous plants such as potato. 30 Colorado potato beetles primarily feed on above-ground portions of the plant, and defoliation leads to lower tuber yields.Methods and compositions for controlling insect pests, in particular Leptinotarsa spp. which infest crop plants are desired. SUMMARY 35 [0005] The present embodiments are related to control of Leptinotarsa species, especially those that are economically or agriculturally important pests. In various embodiments, theLeptinotarsa species is at least one selected from the group consisting of Leptinotarsa behrensi, Leptinotarsa collinsi, Leptinotarsa decemlineata (Colorado potato beetle), Leptinotarsa defecta, Leptinotarsa haldemani (Haldeman’s green potato beetle), Leptinotarsa heydeni, Leptinotarsa 40 juncta (false potato beetle), Leptinotarsa lineolata (burrobrush leaf beetle), Leptinotarsa peninsularis, Leptinotarsa ru- biginosa, Leptinotarsa texana, Leptinotarsa tlascalana, Leptinotarsa tumamoca, and Leptinotarsa typographica. In spe- cific embodiments, the Leptinotarsa species is at least one selected from the group consisting of Leptinotarsa decem- lineata (Colorado potato beetle), Leptinotarsa juncta (false potato beetle), Leptinotarsa haldemani (Haldeman’s green potato beetle), and Leptinotarsa lineolata (burrobrush leaf beetle). 45 [0006] The present invention provides a method for controlling a Leptinotarsa species infestation of a plant comprising causing mortality or stunting in larvae of said Leptinotarsa species by providing in the diet of said larvae at least one double-stranded RNA polynucleotide comprising at least one silencing element comprising 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence of SEQ ID NO:730, or an RNA transcribed from said target gene. 50 [0007] The invention further provides an insecticidal composition for controlling a Leptinotarsa species, comprising: (a) an insecticidal double-stranded RNA molecule that causes mortality or stunting of growth inLeptinotarsa a species when ingested or contacted by said Leptinotarsa species, wherein at least one strand of said insecticidal double-stranded RNA molecule comprises 21 contiguous nucleotides that are complementary to a target gene or 55 an RNA transcribed from said target gene, wherein said target gene has a sequence of SEQ ID NO:730; or (b) an insecticidally effective amount of at least one double-stranded RNA comprising a sequence selected from the group consisting of: SEQ ID NOs:989 and 988. 3 EP 3 030 663 B1 [0008] In a related embodiment the invention provides a recombinant DNA construct comprising a heterologous pro- moter operably linked to: (a) DNA encoding an insecticidal double-stranded RNA molecule that causes mortality or stunting of growth in a 5 Leptinotarsa species when ingested or contacted by said Leptinotarsa species, wherein at least one strand of said insecticidal double-stranded RNA molecule comprises 21 contiguous nucleotides that are complementary to a target gene or an RNA transcribed from said target gene, wherein said target gene has a sequence of SEQ ID NO:730; or (b) DNA encoding an insecticidally effective amount of at least one double-stranded RNA comprising a sequence selected from the group consisting of: SEQ ID NOs:989 and 988. 10 [0009] In a further alternative the invention provides a plant chromosome or a plastid or a recombinant plant virus vector or a recombinant baculovirus vector comprising the recombinant DNA construct as described above. [0010] The invention also provides a transgenic solanaceous plant cell having in its genome the recombinant DNA construct described above. 15 [0011] The transgenic solanaceous plant or a fruit, seed, or propagatable part of said transgenic solanaceous plant comprising the transgenic solanaceous plant cell described above represents a further embodiment of the invention. DETAILED DESCRIPTION 20 [0012] Unless defined otherwise, all technical