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Insecticidal Proteins and Methods for Their Use Insektizidproteine Und Verfahren Zu Deren Verwendung Protéines Insecticides Et Leurs Procédés D’Utilisation

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Insecticidal Proteins and Methods for Their Use Insektizidproteine Und Verfahren Zu Deren Verwendung Protéines Insecticides Et Leurs Procédés D’Utilisation (19) *EP003102592B1* (11) EP 3 102 592 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 14/415 (2006.01) C12N 15/29 (2006.01) (2006.01) (2006.01) 20.05.2020 Bulletin 2020/21 C12N 15/82 C12N 15/62 A01N 65/04 (2009.01) (21) Application number: 15705872.8 (86) International application number: (22) Date of filing: 06.02.2015 PCT/US2015/014824 (87) International publication number: WO 2015/120276 (13.08.2015 Gazette 2015/32) (54) INSECTICIDAL PROTEINS AND METHODS FOR THEIR USE INSEKTIZIDPROTEINE UND VERFAHREN ZU DEREN VERWENDUNG PROTÉINES INSECTICIDES ET LEURS PROCÉDÉS D’UTILISATION (84) Designated Contracting States: •LIU, Lu AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Palo Alto, California 94301 (US) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO •LUM, Amy PL PT RO RS SE SI SK SM TR Hayward, California 94545 (US) • SCHEPERS, Eric (30) Priority: 07.02.2014 US 201461937295 P Port Deposit, Maryland 21904 (US) 17.09.2014 US 201462051720 P • YALPANI, Nasser Johnston, Iowa 50131 (US) (43) Date of publication of application: • ZHU, Genhai 14.12.2016 Bulletin 2016/50 San Jose, California 95131 (US) • GERBER, Ryan Michael (60) Divisional application: Apex, North Carolina 27539 (US) 20169137.5 (74) Representative: J A Kemp LLP (73) Proprietors: 14 South Square • Pioneer Hi-Bred International, Inc. Gray’s Inn Johnston, Iowa 50131-1014 (US) London WC1R 5JJ (GB) • E. I. du Pont de Nemours and Company Wilmington, DE 19805 (US) (56) References cited: WO-A2-2013/098858 US-A1- 2012 233 726 (72) Inventors: • BARRY, Jennifer Ames, Iowa 50014 (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 3 102 592 B1 Printed by Jouve, 75001 PARIS (FR) EP 3 102 592 B1 Description FIELD 5 [0001] This disclosure relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These pesticidal proteins and the nuchcleic acid sequences that encode them are useful in preparing pesticidal formu- lations and in the production of transgenic pest-resistant plants. BACKGROUND 10 [0002] Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally speaking, the use of biopesticides presents a lower risk of pollution and environmental hazards and biopesticides provide greater target specificity than is characteristic of traditional broad-spectrum chemical insecti- 15 cides. In addition, biopesticides often cost less to produce and thus improve economic yield for a wide variety of crops. [0003] Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus. Microbial insecticides, particularly 20 those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pest control. [0004] Crop plants have been developed with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from Bacillus. For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bt. These genetically engineered crops are now widely used in agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect-control methods. 25 While they have proven to be very successful commercially, these genetically engineered, insect-resistant crop plants provide resistance to only a narrow range of the economically important insect pests. In some cases, insects can develop resistance to different insecticidal compounds, which raises the need to identify alternative biological control agents for pest control. [0005] Accordingly, there remains a need for new pesticidal proteins with different ranges of insecticidal activity against 30 insect pests, e.g., insecticidal proteins which are active against a variety of insects in the order Lepidoptera and/or the order Coleoptera including but not limited to insect pests that have developed resistance to existing insecticides. [0006] US 2012/233726 discloses a novel Bacillus thuringiensis gene with lepidopteran activity. [0007] US 2013/098858 discloses a novel insecticidal chitinase protein, its encoding nucleotide and application thereof. 35 SUMMARY [0008] Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are disclosed. Compositions include nucleic acid molecules encoding sequences for pesticidal and insecticidal polypep- tides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. Compositions also include 40 the pesticidal polypeptide sequences and antibodies to those polypeptides. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant. Compositions also comprise transformed bacteria, plants, plant cells, tissues and seeds. 45 [0009] In particular, the invention provides a purified PtIP-83 polypeptide having at least 80% sequence identity across the full-length of the amino acid sequence PtIP-83Aa as shown in Figures 2a-2j, wherein the PtIP-83 polypeptide has insecticidal activity against Corn Earworm (Helicoverpa zea). [0010] The invention further provides a recombinant polynucleotide encoding the PtIP-83 as defined above. [0011] Yet further provided by the invention is a transgenic monocot or dicot plant comprising the polynucleotide as 50 defined above, a DNA construct comprising said polynucleotide and a transgenic monocot or dicot plant or plant cell comprising said DNA construct. [0012] The invention further provides an agricultural composition comprising an insecticidally effective dose of the PtIP-83 polypeptide as defined above. [0013] Yet further provided by the invention is a fusion protein comprising the PtIP-83 polypeptide as defined above. 55 [0014] The invention further provides a method: (a) for controlling an insect pest population, comprising contacting the insect pest population with the PtIP-83 polypep- tide as defined above; 2 EP 3 102 592 B1 (b) of inhibiting growth or killing an insect pest, comprising contacting the insect pest with a composition comprising the PtIP-83 polypeptide as defined above; and (c) of controlling Lepidoptera and/or Coleoptera insect infestation in a transgenic plant and providing insect resistance management, comprising expressing in the plant the PtIP-83 polypeptide as defined above. 5 [0015] Yet further provided by the invention is a method: (a) for controlling an insect pest population, comprising contacting the insect pest population with the transgenic plant or plant cell as defined above; and 10 (b) of inhibiting growth or killing an insect pest, comprising contacting the insect pest with the transgenic plant or plant cell as defined above. [0016] The invention further provides the use of the PtIP-83 polypeptide as defined above to inhibit growth of or kill an insect or insect population. 15 [0017] Isolated or recombinant nucleic acid molecules are disclosed encoding Pteridophyta Insecticidal Protein-83 (PtIP-83) polypeptides including amino acid substitutions, deletions, insertions, fragments thereof. Additionally, amino acid sequences corresponding to the PtIP-83 polypeptides are disclosed. Disclosed are isolated or recombinant nucleic acid molecules capable of encoding PtIP-83 polypeptides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, as well as amino 20 acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Nucleic acid sequences that are complementary to a nucleic acid sequence of the embodiments or that hybridize to a sequence of the embodiments are also disclosed. Also disclosed are isolated or recombinant PtIP-83 polypeptides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19, as well as amino acid substitutions, deletions, insertions, fragments thereof and combinations thereof. 25 [0018] Methods are disclosed for producing the polypeptides and for using those polypeptides for controlling or killing a Lepidopteran, Coleopteran, nematode, fungi, and/or Dipteran pests. The transgenic plants of the embodiments express one or more of the pesticidal sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional genes for insect resistance, for example, one or more additional genes for controlling Coleopteran, Lepidopteran, Hemipteran or nematode pests. It will be understood by one of skill in the art that the transgenic plant 30 may comprise any gene imparting an agronomic trait of interest. [0019] Methods for detecting the nucleic acids and polypeptides of the embodiments in a sample are also disclosed. A kit for detecting the presence of a PtIP-83 polypeptide or detecting the presence of a polynucleotide encoding a PtIP- 83 polypeptide in a sample is disclosed. The kit may be provided along with all reagents and control samples necessary for carrying out a method for detecting the intended agent, as well as instructions for use.
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