EA040502B1 - INSECTICIDAL PROTEINS AND METHODS OF THEIR APPLICATION - Google Patents

Description

Cross-reference to related applications

This application claims priority from U.S. Provisional Application No. 62/331,708, filed May 4, 2016, which is incorporated herein by reference in its entirety.

Link to the sequence listing provided electronically

An official copy of this sequence listing is provided electronically via EFS-Web as a sequence listing in an ASCII file named 6441WOPCT_Sequence_Listing, created on May 1, 2017, with a size of 1094 kilobytes, and submitted at the same time as the description. The sequence listing contained herein in ASCII format is part of the specification and is incorporated herein by reference in its entirety.

Field of invention

The present invention relates to the field of molecular biology. New genes are provided that encode pesticidal proteins. These pesticidal proteins and the nucleic acid sequences that encode them are useful in the production of pesticide formulations and in the production of pest resistant transgenic plants.

Background of the invention

Biological control of agriculturally important insects using a microbial agent such as fungi, bacteria or other insect species provides an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. In general, it can be said that the use of biopesticides poses a lower risk of pollution and adverse environmental impacts, and biopesticides provide specificity for more targets than traditional broad-spectrum chemical insecticides. In addition, biopesticides are often cheaper to produce, and as a result, cost-effective yields are improved for a wide range of crops.

As is known, certain types of microorganisms of the genus Bacillus have pesticidal activity against a number of insect pests, including Lepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biological control agents developed to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus. Microbial insecticides, in particular those derived from Bacillus strains, have played an important role in agriculture as an alternative to chemical pest control.

Crop plants with increased resistance to insects have been developed by genetically engineering crop plants to produce Bacillus pesticidal proteins. For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bacillus thuringiensis. These genetically engineered crops are now widely used in agriculture and provide the farmer with an environmentally friendly alternative to traditional insect control methods. Although they have been commercially highly successful, these genetically engineered insect-resistant crop plants provide resistance to only a narrow range of economically important insect pests. In some cases, insects can develop resistance to various insecticidal compounds, raising the need to identify alternative biological controls for pest control.

Accordingly, there remains a need for new pesticidal proteins with different spectra of insecticidal activity against insect pests, for example, insecticidal proteins that are active against a number of insects from the order Lepidoptera and the order Coleoptera, including, but not limited to insect pests that have developed resistance. to existing insecticides.

Brief description of the invention

In one aspect, compositions and methods are provided for providing pesticidal activity to bacteria, plants, plant cells, tissues, and seeds. The compositions include nucleic acid molecules encoding pesticidal and insecticidal polypeptide sequences, vectors containing such nucleic acid molecules, and host cells containing the vectors. The compositions also include pesticidal polypeptide sequences and antibodies to such polypeptides. The compositions also contain transformed bacteria, plants, plant cells, tissues and seeds.

In another aspect, isolated or recombinant nucleic acid molecules encoding IPD090 polypeptides, including amino acid substitutions, deletions, insertions, and fragments thereof, are provided. Provided isolated or recombinant nucleic acid molecules capable of encoding IPD090 polypeptides with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384, as well as amino acid substitutions, deletions, inserts, their fragments and their combinations. Also included are nucleic acid sequences that are complementary to the nucleic acid sequence of the embodiments or that hybridize to the sequence of the embodiments. Nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. Nucleotide or amino acid sequences can be synthetic sequences that have been engineered for expression in an organism, including but not limited to a microorganism or plant.

In another aspect, IPD090 polypeptides are encompassed. Also contemplated are isolated or recombinant IPD090 polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, and SEQ ID NO: 384, as well as amino acid substitutions, deletions, insertions, fragments thereof, and their combinations.

In another aspect, methods are provided for preparing polypeptides and using the polypeptides to control or kill Lepidoptera, Coleoptera, Nematodes, Fungi and/or Diptera pests. Transgenic plants according to embodiments express one or more pesticidal sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional insect resistance genes, such as one or more additional genes for controlling pests from the group of beetles, lepidoptera, hemipterans, or nematodes. One skilled in the art will appreciate that the transgenic plant may contain any gene that provides the agronomic trait of interest.

In another aspect, methods are also provided for detecting nucleic acids and polypeptides of the embodiments in a sample. A kit is provided to detect the presence of an IPD090 polypeptide or to detect the presence of a polynucleotide encoding an IPD090 polypeptide in a sample. The kit may be provided along with all reagents and controls needed to carry out the method for detecting the intended agent, as well as instructions for use.

In another aspect, the compositions and methods of the embodiments are useful for producing organisms with improved pest resistance or pest tolerance. These organisms and compositions containing the organisms are desirable for agricultural purposes. The compositions of the embodiments are also useful for producing altered or improved proteins that have pesticidal activity or for detecting the presence of IPD090 polypeptides.

Brief description of graphic materials

In FIG. 1A-1B show the alignment using the Vector NTI® ALIGNX® module of the amino acid sequences of the IPD090Aa polypeptide (SEQ ID NO: 2) and the IPD090Ca polypeptide (SEQ ID NO: 6). The difference in amino acid sequences between amino acid sequences is highlighted. Conservative amino acids that differ are indicated by shading (A), and non-conservative amino acids that differ are indicated by shading (A). The N-terminal amino acids deleted from the truncated IPD090Aa (SEQ ID NO: 2) polypeptide, the IPD090Aa (TR1) (SEQ ID NO: 10) polypeptide of Example 6, are underlined in the IPD090Aa (SEQ ID NO: 2) sequence. The corresponding breakpoints of the IPD090Aa/IPD090Ca chimeric proteins from Example 11 are indicated below the sequence of IPD090Aa (SEQ ID NO: 2) by a, above the sequence of IPD090Ca (SEQ ID NO: 6) by ▼.

In FIG. 2 is a bar graph showing the values of the gel fluorescence absorbance measurement, in the SDS-PAGE gel of Example 14, for homologous competition of 6.3 nM IPD090Aa ^Alexa binding to BBMV WCRW normalized to the binding signal in the absence of unlabeled protein (6 nM Alexa-IPD090) and in the presence of a saturating concentration of unlabeled protein (+ 13 μM IPD0 90). The difference in magnitude between columns reflects specific binding.

In FIG. 3 shows the corn rootworm nodule infestation score (CRWNIS) on selected T0 maize transgenic events from constructs PHP73234 and PHP73237 expressing the IPD090 polypeptide of SEQ ID NO: 377 and PHP77372 expressing the IPD090 polypeptide of SEQ ID NO: 10.

In FIG. 4 shows the structure of IPD090Aa (SEQ ID NO: 2) determined by X-ray diffraction crystallography, indicating the MACPF domain of the N-terminal region and the β-prismatic domain of the C-terminal region. The Mg+ atom is shown as a sphere at the bottom of the β-prismatic domain. The two helix clusters are labeled CH1 and CH2.

In FIG. 5 shows a 90° rotation of the IPD090Aa polypeptide (SEQ ID NO: 2) around the vertical axis showing the N-terminal β strand for the 5th member of the central β layer.

In FIG. 6 is a close-up view of the C-terminal β-prismatic domain of structure IPD090Aa (SEQ ID NO: 2) illustrating the 3D axis and Mg+2 interactions.

In FIG. 7 shows the Ramachandran map for the improved structure IPD090Aa (SEQ ID NO: 2) 2.1 A.

Detailed description of the invention

It should be understood that the present invention is not limited to the specific methods, protocols, cell lines, genera, and reagents described, and as such may vary.

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It should also be understood that the terminology used herein is only intended to describe specific embodiments and is not intended to limit the scope of the present invention.

As used herein, the singular form includes references to the plural unless the context clearly indicates otherwise. Thus, for example, a reference to a cell includes a plurality of such cells, and a reference to a protein includes a reference to one or more proteins or their equivalents known to those skilled in the art, and so on. All technical and scientific terms used in this document have the same meaning as generally understood by a person skilled in the art to which the present invention belongs, unless expressly stated otherwise.

The present invention relates to compositions and methods for pest control. The methods involve transformation of organisms with nucleic acid sequences encoding IPD090 polypeptides. In particular, the nucleic acid sequences of the embodiments are useful for producing plants and microorganisms that have pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are contemplated. The compositions include pesticidal nucleic acids and proteins from bacterial species. Nucleic acid sequences find use in constructing expression vectors for subsequent transformation of organisms of interest, as probes for isolating other homologous (or partially homologous) genes, and for generating altered IPD090 polypeptides using methods known in the art, such as site-directed mutagenesis, domain replacement or DNA shuffling. IPD090 polypeptides find use in the control or eradication of Lepidoptera, Coleoptera, Diptera, fungi, Hemiptera and nematode pest populations and in the preparation of compositions with pesticidal activity. Insect pests of interest include, but are not limited to, Lepidoptera species, including but not limited to corn cutworm (CEW) (Helicoverpa zea), corn moth (ECB) (Ostrinia nubilalis), cabbage moth, eg Helicoverpa zea Boddie; soy scoop, such as Pseudoplusia includens Walker; and velvet bean cutworm, such as Anticarsia gemmatalis Hubner, as well as species from the order Coleoptera, including, but not limited to, the western corn beetle (Diabrotica virgifera) - WCRW, the southern corn beetle (Diabrotica undecimpunctata howardi) - SCRW, and the northern corn beetle (Diabrotica barberi) - NCRW.

As used herein, the term pesticidal toxin or pesticidal protein refers to a toxin that has toxic activity against one or more pests, including but not limited to members of the orders Lepidoptera, Diptera, Hemiptera, and Coleoptera, or the Nematoda phylum, or a protein that is homologous to such a protein. Pesticide proteins have been isolated from organisms including, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae. Pesticidal proteins include, but are not limited to, insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens 7:1-13); from strain CHAO and Pf-5 Pseudomonas protegens (formerly fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386; GenBank accession no. EU400157); from Pseudomonas taiwanensis (Liu, et al. (2010) J. Agric. Food Chem., 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li , et al., (2007) Plant Cell Tiss Organ Cult 89:159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal 3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069); US patent No. 6048838 and US patent No. 6379946; PIP-1 polypeptide from US Patent Application Publication US20140007292; AfIP-1A and/or AfIP-1B polypeptide from US9475847; PHI-4 polypeptide from US Patent Application Publications US20140274885 and US20160040184; PIP-47 polypeptide from US Patent Application Publication No. US20160186204; PIP-72 polypeptide from US Patent Application Publication No. US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide from PCT Publication No. WO2015/120270; the PtIP-83 polypeptide from PCT Publication No. WO2015/120276; the PtIP-96 polypeptide from PCT Publication Serial No. PCT/US15/55502; polypeptide IPD079 from PCT Publication No. WO2017/23486; the IPD082 polypeptide from US Patent Document Serial No. PCT/US16/65531, the IPD093 polypeptide from US Patent Document Serial No. 62/434020; the IPD080 polypeptide from US Patent Document Serial No. US62/411318; and δ-endotoxins, including, but not limited to, δ-endotoxin genes of the classes Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18 , Cry19, Cry20, Cry21, Cry22, Cry23, Cry24,

Cry25, Cry26, Cry27, Cry28, Cry29, Cry30, Cry31, Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39,

Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry54, Cry55,

Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70,

Cry71 and Cry72 and the genes of the cytolytic toxins cyt1 and cyt2 of B. thuringiensis. Representatives of these classes of insecticidal proteins from B. thuringiensis are well known to those skilled in the art (see Crickmore, et al., Bacillus thuringiensis toxin nomenclature (2011) at lifesci.sussex.ac.uk/home/Neil_Crickmore /Bt/, which can be accessed on the World Wide Web using the prefix www).

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Examples of δ-endotoxins also include, without limitation, the CrylA proteins of US Pat. Nos. 5,880,275 and 7,858,849; mutant Cry1Ac from US9512187; DIG-3 toxin or DIG-11 toxin (variants of cry proteins with N-terminal deletion of α-helix 1 and/or α-helix 2, such as Cry1A, Cry3A) of US Pat. Cry1B from US Patent Application Publication Serial No. 10/525318, US Patent Application Publication No. US20160194364 and US Patent Nos. 9404121 and 8772577; Cry1B variants from PCT Publication No. WO2016/61197 and Serial No. PCT/US17/27160; Cry1C from US Pat. No. 6,033,874; Cry1F from US Pat. Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras from US Pat. No. 7,070,982; 6962705 and 6713063); a Cry2 protein such as the Cry2Ab protein of US Pat. No. 7,064,249); a Cry3a protein, including but not limited to a genetically engineered hybrid insecticidal protein (eHIP) obtained by fusing unique combinations of variable regions and conserved units of at least two different Cry proteins (U.S. Patent Application Publication No. 2010/0017914); Cry4 protein; protein Cry5; Cry6 protein; Cry8 proteins from US Pat. Cry9 protein, such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families, including the Cry9 protein of US Pat. No. 9,000,261 and 8,802,933 and US Pat. Cry15 protein from Naimov, et al., (2008) Applied and Environmental Microbiology, 74:7145-7151; protein Cry14 from US patent No. US8933299; Cry22, Cry34Ab1 protein from US Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; truncated Cry34 protein from US Pat. No. US8816157; CryET33 and CryET34 protein from US Pat. CryET33 and CryET34 homologues from US Patent Application Publication No. 2006/0191034, 2012/0278954 and PCT Publication No. WO 2012/139004; protein Cry35Ab1 from US patent No. 6083499, 6548291 and 6340593; Cry46 protein from US Pat. No. 9,403,881, Cry 51 protein, Cry binary toxin; TIC901 or related toxin; TIC807 from US Patent Application Publication No. 2008/0295207; TIC853 from US Patent US8513493; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 from PCT US 2006/033867; Hemiptera engineered toxic proteins from US Patent Application Publication No. US20160150795, AXMI-027, AXMI-036 and AXMI-038 from US Patent No. 8236757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 from US Patent No. 7923602; AXMI-018, AXMI-020 and AXMI-021 from WO 2006/083891; AXMI-010 from WO 2005/038032; AXMI-003 from WO 2005/021585; AXMI-008 from US Patent Application Publication No. 2004/0250311; AXMI-006 from US Patent Application Publication No. 2004/0216186; AXMI-007 from US Patent Application Publication No. 2004/0210965; AXMI-009 from US Patent Application No. 2004/0210964; AXMI-014 from US Patent Application Publication No. 2004/0197917; AXMI-004 from US Patent Application Publication No. 2004/0197916; AXMI-028 and AXMI-029 from WO 2006/119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 from WO 2004/074462; AXMI-150 from US Pat. No. 8,084,416; AXMI-205 from US Patent Application Publication No. 2011/0023184; AXMI-011, AXMI012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI022, AXMI-023, AXMI-041, AXMI- 063 and AXMI-064 from US Patent Application Publication No. 2011/0263488;

AXMI046, AXMI048, AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMI055, AXMI056, AXMI057, AXMI058, AXMI059, AXMI060, AXMI061, AXMI067, AXMI069, AXMI071, AXMI072, AXMI073, AXMI074, AXMI075, AXMI087, AXMI088, AXMI093, AXMI070, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125, AXMI126, AXMI127, AXMI129, AXMI151, AXMI161, AXMI164, AXMI183,

AXMI132, AXMI137, AXMI138 from US Patent US8461421 and US8461422; AXMI-R1 and related proteins from US Patent Application Publication No. 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z from WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 from WO 2011/103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163, and AXMI-184 from US Pat. No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 from US Patent Application Publication No. 2010/0298211; AXMI-066 and AXMI-076 from US Patent Application Publication No. 2009/0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156,

AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170,

AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179,

AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 from US Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, dsAXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI1376 of US Patent42 US84; AXMI192 from US Patent US8461415; AXMI281 from US Patent Application Publication No. US20160177332; AXMI422 from US Patent No. US8252872; cry proteins such as Cry1A and Oy3A with modified

- 4 040502 cited proteolytic cleavage sites from US patent No. 8319019; CrylAc, Cry2Aa and Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 from US Patent Application Publication No. 2011/0064710. Cry proteins MP237S, MP242S, MP243, MP248, MP249S, MP251M, MP252S, MP253, MP259S, MP287S, MP288S, MP295S, MP296S, MP297S, MP300S, MP304S, MP306S, MP310S, MP312S,

MP314S, MP319S, MP325S, MP326S, MP327S, MP328S, MP334S, MP337S, MP342S, MP349S, MP356S,

MP359S, MP360S, MP437S, MP451S, MP452S, MP466S, MP468S, MP476S, MP482S, MP522S, MP529S,

MP548S, MP552S, MP562S, MP564S, MP566S, MP567S, MP569S, MP573S, MP574S, MP575S, MP581S,

MP590, MP594S, MP596S, MP597, MP599S, MP600S, MP601S, MP602S, MP604S, MP626S, MP629S,

MP630S, MP631S, MP632S, MP633S, MP634S, MP635S, MP639S, MP640S, MP644S, MP649S, MP651S,

MP652S, MP653S, MP661S, MP666S, MP672S, MP696S, MP704S, MP724S, MP729S, MP739S, MP755S,

MP773S, MP799S, MP800S, MP801S, MP802S, MP803S, MP805S, MP809S, MP815S, MP828S, MP831S,

MP844S, МР852, MP865S, MP879S, MP887S, MP891S, MP896S, MP898S, MP935S, МР968, МР989, МР993, МР997, МР1049, МР1066, МР1067, МР1080, МР1081, МР1200, МР1206, МР1233, и МР1311 из патентного документа США с серийным No. 62/429426. The insecticidal activity of Cry proteins is well known to those skilled in the art (for a review, see van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cry proteins as traits of a transgenic plant is well known to those skilled in the art, and transgenic Cry plants, including but not limited to plants expressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Oy9c, and CBI-Bt have been approved by regulatory authorities (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and CERA. (2010) base GM plant data from the Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington DC at cera-gmc.org/index.php?action=gm_crop_database, which can be accessed on the World Wide Web using the prefix www). Plants may also express more than one pesticidal protein well known to the person skilled in the art, such as Vip3Ab and Cry1Fa (US2012/0317682); Cry1BE and Cry1F (US2012/0311746); Cry1CA and Cry1AB (US2012/0311745); Cry1F and CryCa (US2012/0317681); Cry1DA and Cry1BE (US2012/0331590); Cry1DA and Cry1Fa (US2012/0331589); Cry1AB and Cry1BE (US2012/0324606); Cry1Fa and Cry2Aa and Cry1I and Cry1E (US2012/0324605); Cry34Ab/35Ab and Cry6Aa (US20130167269); Cry34Ab/VCry35Ab and Cry3Aa (US20130167268); as well as StuZA and Cry1Ab or Vip3Aa (US9045766). Pesticide proteins also include insecticidal lipases, including lipid acyl hydrolases of US Pat. No. 7,491,869 and cholesterol oxidases such as those from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticide proteins also include VIP toxins (Vegetative Insecticide Proteins) of US Pat. Other VIP proteins are well known to those skilled in the art (see website at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html, which can be accessed via the World Wide Web using the prefix www) . Pesticide proteins also include toxin complex (TC) proteins, which can be obtained from organisms such as Xenorhabdus, Photorhabdus, and Paenibacillus (see US Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have independent insecticidal activity, while other TC proteins increase the activity of independent toxins produced by the same specified organism. The toxicity of a standalone TC protein (eg, from Photorhabdus, Xenorhabdus, or Paenibacillus) can be increased by one or more TC proteins, enhancers derived from a source organism from a different genus. There are three main types of TS proteins. As set forth herein, Class A proteins (Protein A) are toxins in their own right. Class B proteins (protein B) and class C proteins (protein C) increase the toxicity of class A proteins. Examples of class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi. Examples of class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticide proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include, but are not limited to, lycotoxin-1 peptides and mutant forms thereof (US Pat. No. 8,334,366).

In some embodiments, the IPD090 polypeptides include amino acid sequences derived from the full length nucleic acid sequences disclosed herein and amino acid sequences that are shorter than the full length sequences, either derived from an alternative downstream origin or from processing, resulting in a shorter protein with pesticidal activity. Processing can take place in the organism in which the protein is expressed, or in the pest after the protein has been ingested.

Thus, this document provides new isolated or recombinant nucleic acid sequences that provide pesticidal activity. Also provided are the amino acid sequences of the IPD090 polypeptides. The protein resulting from the translation of these IPD090 genes in cells provides the ability to control or kill pests that ingest them.

IPD090 proteins, as well as their variants and fragments.

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The IPD090 polypeptides are within the scope of the present invention. The terms IPD090 polypeptide and IPD090 protein, used interchangeably herein, refer to a polypeptide with insecticidal activity, including but not limited to insecticidal activity against one or more insect pests from the orders Lepidoptera and/or Coleoptera, and a sufficiently homologous IPD090Aa polypeptide with SEQ ID NO: 2. Suggested range of IPD090 polypeptides. Sources of IPD090 polypeptides or related proteins include bacterial species selected without limitation from species of the genus Pseudomonas and Woodsholea. Aligning the amino acid sequences of the IPD090 polypeptide homologues (eg, in FIG. 1) allows the identification of residues that are highly conserved among natural homologues in a given family.

The term sufficiently homologous is used herein to refer to an amino acid sequence that is characterized by at least about 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or greater sequence homology when compared to a reference sequence using one of the alignment programs described herein using standard parameters. In some embodiments, sequence homology is determined relative to the full length sequence of the IPD090 polypeptide. In some embodiments, the IPD090 polypeptide sequence is at least about 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity when compared to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384. The term approximately when used herein in the context of percent sequence identity means +/- 0.5%. One skilled in the art will appreciate that these values can be adjusted accordingly to determine appropriate protein homology, taking into account amino acid similarity and the like. In some embodiments, sequence identity is calculated using the ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all standard parameters. In some embodiments, sequence identity is calculated over the entire length of the polypeptide using the ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all standard parameters.

As used herein, the terms protein, peptide molecule, or polypeptide include any molecule that contains five or more amino acids. It is well known in the art that protein, peptide, or polypeptide molecules may undergo modification, including post-translational modifications such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation, or oligomerization. Thus, as used herein, the terms protein, peptide molecule, or polypeptide include any protein that has been modified by any biological or non-biological process. The terms amino acid and amino acids refer to all naturally occurring L-amino acids.

The term recombinant protein is used herein to refer to a protein that is no longer found in its natural environment, eg in vitro, or in a recombinant bacterial or plant host cell. An IPD090 polypeptide that contains virtually no cellular material includes protein preparations having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-pesticide protein (also referred to herein as contaminant protein).

The terms fragments or biologically active parts include polypeptide fragments containing amino acid sequences sufficiently identical to the IPD090 polypeptide and which exhibit insecticidal activity. The terms fragments or biologically active parts of IPD090 polypeptides include fragments containing amino acid sequences sufficiently identical to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384, where the IPD090 polypeptide is characterized by insecticidal activity. Such biologically active parts can be obtained using recombinant techniques and evaluated for insecticidal activity. In some embodiments, an IPD090 polypeptide fragment is an N-terminal and/or C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more amino acids from the N-terminus and/or C-terminus relative to SEQ ID NO : 2, SEQ ID NO: 4 or SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384, e.g. -codon and/or insertion of a stop codon. In some embodiments, an IPD090 polypeptide fragment is an N-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 amino acids from the N-terminus of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or

- 6 040502

SEQ ID NO: 384. In some embodiments, the IPD090 polypeptide fragment is an N-terminal and/or C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 amino acids with N -terminus and/or C-terminus relative to SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384. In some embodiments, the IPD090 polypeptide fragment contains amino acids 1- 315, amino acids 1-330, amino acids 1-349, amino acids 1-4 50, amino acids 25-315, amino acids 25-330, amino acids 25-349, amino acids 25-450, or amino acids 25-483 from any of SEQ ID NO: 2 , SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384. In some embodiments, the truncated variant is the polypeptide of SEQ ID NO: 10.

The term variants as used herein refers to proteins or polypeptides with an amino acid sequence that is at least about 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87 , 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the original amino acid sequence.

In some embodiments, the IPD090 polypeptide contains an amino acid sequence that is at least about 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384, wherein the IPD090 polypeptide has insecticidal activity.

In some embodiments, the IPD090 polypeptide contains an amino acid sequence that is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97%, 98%, 99% or more identical throughout the amino acid sequence to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384.

In some embodiments, sequence identity is calculated over the entire length of the polypeptide using the ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all standard parameters.

In some embodiments, the IPD090 polypeptide contains an amino acid sequence that is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97%, 98%, 99% or more identical throughout the amino acid sequence to SEQ ID NO: 2.

In some embodiments, the IPD090 polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 , 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70.71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or more amino acid substitutions compared to the native amino acid at the corresponding position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384.

In some embodiments, a variant IPD090 polypeptide contains any one or more amino acid substitutions corresponding to positions 3, 4, 8, 12, 15, 16, 21, 23, 24, 26, 28, 30, 38, 46, 47, 50, 52 , 55, 62, 63, 67, 68, 70, 73, 74, 75, 76, 80, 90, 91, 94, 99, 100, 108, 115, 127, 129, 161, 169, 175, 177, 178 , 180, 185, 207, 213, 223, 240, 241, 247, 255, 266, 273, 275, 277, 278, 287, 288, 302, 306, 309, 310,

311, 312, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,

336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,

358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379,

380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 391, 392, 395, 397, 400, 401, 402, 405, 407, 410, 423, 425,

426, 431, 433, 434, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454,

455, 457, 458, 459, 460, 468, and 471 SEQ ID NO: 2 in any combination.

In some embodiments, a variant IPD090 polypeptide contains any one or more active amino acid substitutions from Table 1. 10 and/or 12.

Methods for performing such manipulations are generally known in the art. For example, variants of the amino acid sequence of the IPD090 polypeptide can be generated by mutations in the DNA. This can also be done by one of several forms of mutagenesis and/or directed evolution. In some aspects, the changes encoded in the amino acid sequence will not significantly affect the function of the protein. Such variants will have the desired pesticidal activity. However, it is understood that the ability of an IPD090 polypeptide to provide pesticidal activity can be enhanced by the use of such techniques in the compositions of the present invention.

For example, conservative amino acid substitutions can be made at one or more predicted non-essential amino acid residues. A non-essential amino acid residue is a residue that can be changed relative to the sequence of the polypeptide

- 7 040502

Wild-type IPD090 with no change in biological activity. Non-essential amino acid residues can be identified by aligning related IPD090 homologues such as those shown in FIG. 1. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (eg, lysine, arginine, histidine); acidic side chains (eg aspartic acid, glutamic acid); polar negatively charged residues and their amides (eg aspartic acid, asparagine, glutamic acid, glutamine); uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); small aliphatic non-polar or weakly polar residues (eg, alanine, serine, threonine, proline, glycine); non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); large aliphatic non-polar residues (eg methionine, leucine, isoleucine, valine, cystine); beta-branched side chains (eg threonine, valine, isoleucine); aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine); large aromatic side chains (eg tyrosine, phenylalanine, tryptophan).

Amino acid substitutions can be made at non-conserved sites that retain function. In general, such substitutions should not be made for conservative amino acid residues or for amino acid residues in a conservative motif where such residues are essential for the activity of the protein. Examples of residues that are conserved and that may be essential to protein activity include, for example, residues that are identical across all proteins contained in an alignment of similar or related toxins with sequences according to embodiments (e.g., residues that are identical when aligned with homologous proteins ). Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions on all proteins contained in an alignment of similar or related toxins with sequences according to embodiments (e.g., residues that are characterized only by conservative substitutions for all proteins contained in an alignment of homologous proteins). However, one of skill in the art will appreciate that functional variants may have minor conservative or non-conservative changes in conservative residues. Guidance on suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C. ), incorporated herein by reference.

When making such changes, the hydropathic index of amino acids can be taken into account. The importance of the amino acid hydropathicity index to ensure consistent biological function of a protein is generally understood in the art (Kyte and Doolittle, (1982) J Mol Biol. 157(1):105-32). It is recognized that the relative hydropathicity of an amino acid contributes to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

It is known in the art that certain amino acids can be substituted with other amino acids having a similar hydropathic index or index and still result in a protein with similar biological activity, i.e. still receive an equivalent protein with biological function. Each amino acid was assigned a hydropathic index based on its hydrophobicity and charging characteristics (Kyte and Doolittle, ibid). They are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); series (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5). When making such changes, it is preferable to replace amino acids whose hydropathic indices are in the range up to +2, especially those with indices in the range up to +1, and even more preferred those with indices in the range up to +0.5.

It is also clear from the prior art that substitution of such amino acids can be efficiently carried out on the basis of hydrophilicity. US Pat. No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as determined by the hydrophilicity of adjacent amino acids, correlates with the biological property of the protein.

As detailed in US Pat. No. 4,554,101, the amino acid residues were assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); series (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

Alternatively, changes can be made to the protein sequence of many proteins at the amino or carboxy terminus without significantly affecting activity. They may include insertion

- 8 040502 ci, deletions or changes introduced by modern molecular methods such as PCR, including PCR amplifications, which alter or extend the protein coding sequence by incorporating amino acid coding sequences into the oligonucleotides used in the amplification through PCR. Alternatively, the added protein sequences may include sequences encoding the entire protein, such as those commonly used in the art to obtain protein fusions. Such fusion proteins are often used to (1) increase the expression of a protein of interest, (2) introduce a binding domain, enzymatic activity, or epitope to facilitate or purify the protein, detect the protein, or other experimental applications known in the art, (3) target secretion or translation of the protein into an intracellular organelle such as the periplasmic space of Gram-negative bacteria, the mitochondria or chloroplasts of plants, or the endoplasmic reticulum of eukaryotic cells, the latter often resulting in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the present invention also encompass sequences resulting from mutation and recombination procedures such as DNA shuffling. Using this procedure, one or more different coding regions of an IPD090 polypeptide can be used to generate a new IPD090 polypeptide having the desired properties. Thus, libraries of recombinant polynucleotides are generated from a population of sequence-related polynucleotides containing regions of sequences that share significant sequence identity and can undergo homologous recombination in vitro or in vivo. For example, using this approach, motifs with domain-of-interest-coding sequences can be shuffled between a pesticide gene and other known pesticide genes to produce a new gene encoding a protein with an improved property of interest, such as increased insecticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. sci. USA 91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. sci. USA 94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and US Pat. Nos. 5,605,793 and 5,837,458.

Domain replacement or shuffling is another mechanism for generating altered IPD090 polypeptides. Domain substitutions can be made between IPD090 polypeptides, resulting in hybrid or chimeric toxins with increased pesticidal activity or a broader target spectrum. Methods for obtaining recombinant proteins and testing them for pesticidal activity are well known in the art (see, for example, Naimov, et al., (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd, et al., (1996) Appl Environ Microbiol 62:1537-1543 Ge, et al., (1991) J Biol Chem 266:17954-17958 Schnepf, et al., (1990) J Biol Chem 265:20923-20930; Rang, et al., 91999) Appl. Environ. microbiol. 65:2918-2925).

Phylogenetic analysis, sequence motif analysis, and structural analysis of insecticidal protein families. A sequence and structure analysis method can be applied, which consists of four components: building a phylogenetic tree, detecting protein sequence motifs, predicting secondary structure, and aligning protein sequences and secondary structures. Detailed descriptions of each component are illustrated below.

1) Construction of a phylogenetic tree.

Phylogenetic analysis can be performed using MEGA5 software. Protein sequences can be analyzed using ClustalW version 2 (Larkin M.A et al (2007) Bioinformatics 23(21):2947-2948) for multiple sequence alignment. The evolutionary history is then determined using a maximum likelihood method based on the JTT matrix model. The tree with the highest log-likelihood is obtained, exported to Newick format, and further processed to extract the sequence IDs in the same order as they appear on the tree. Some clades representing subfamilies can be manually identified for each family of insecticidal proteins.

2) Detection of protein sequence motifs.

The protein sequences are rearranged according to the previously constructed phylogenetic tree and inserted into the MEME (Multiple EM for MOTIF Elicitation) tool (Bailey T.L., and Elkan C., Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36 , AAAI Press, Menlo Park, California, 1994.) to identify key sequence motifs. The initial MEME settings are as follows: minimum number of sites 2, minimum motif size 5, and maximum number of motifs 30. Sequence motifs unique to each subfamily were identified by visual inspection. The distribution of motifs throughout the gene family could be visualized on an HTML web page. motifs are numbered according to the location of the E-value for each motif.

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3) Prediction of the secondary structure.

PSIPRED, a leading secondary structure prediction technology (Jones DT. (1999) J. Mol.

Biol. 292: 195-202) can be used to predict the secondary structure of a protein. The tool provides accurate structure prediction using two neural feed-forward circuits based on the PSI-BLAST result. A database for PSI-BLAST was created by deleting the low complexity regions, transmembrane region and supercoil regions in Uniref100. PSIPRED results contain the predicted secondary structures (alpha helix: H, beta strand: E, and coil: C) and the corresponding confidence level for each amino acid in a given protein sequence.

4) Alignment of protein sequences and secondary structures.

A script can be developed to create gapped secondary structure alignments according to multiple protein sequence alignments from step 1 for all proteins. All aligned protein sequences and structures are combined into a single FASTA file and then imported into MEGA for visualization and identification of conserved structures.

In some embodiments, the implementation of the IPD090 polypeptide is characterized by a modified physical property.

Used in this document, the term physical property refers to any parameter that is suitable for describing the physico-chemical characteristics of the protein. As used herein, the terms physical property of interest and property of interest are used interchangeably to refer to the physical properties of proteins to be investigated and/or modified. Examples of physical properties include, without limitation, net surface charge and charge distribution on the protein surface, net hydrophobicity and distribution of hydrophobic residues on the protein surface, surface charge density, surface hydrophobicity density, total number of surface ionizable groups, surface tension, protein size and distribution in solution. , melting point, heat capacity, and the second virial coefficient. Examples of physical properties also include an IPD090 polypeptide characterized by increased expression, increased solubility, reduced phytotoxicity, and digestibility of proteolytic fragments in the insect gut. Models for digestion with artificial gastric juice are known to those skilled in the art (Fuchs, R.L. and J.D. Astwood. Food Technology 50: 83-88, 1996; Astwood, J.D., et al Nature Biotechnology 14: 1269-1273, 1996; Fu TJ et al J Agric Food Chem 50: 7154-7160, 2002).

In some embodiments, the variants include polypeptides that differ in amino acid sequence due to mutagenesis. The variant proteins encompassed by the present invention are biologically active, that is, they still have the desired biological activity (ie pesticidal activity) of the native protein. In some embodiments, the variant will have at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 80%, or more of the insecticidal activity of the native protein. In some embodiments, the implementation of the options may have enhanced activity compared to the native protein.

Bacterial genes quite often have several methionine start codons in the vicinity of the start site of the open reading frame. Often, translation initiation at one or more of these start codons will result in the formation of a functional protein. These start codons may include ATG codons. However, bacteria such as Bacillus sp. also recognize the GTG codon as a start codon, and proteins whose translation is initiated at GTG codons contain methionine as the first amino acid. In rare cases, translation in bacterial systems may be initiated at the TTG codon, although in this case TTG codes for methionine. In addition, it is often not determined a priori which of these codons is naturally used in a bacterium. Thus, it is understood that the use of one of the reciprocal methionine codons may also result in the formation of pesticidal proteins. These pesticidal proteins are within the scope of the present invention and may be used in the methods of the present invention. It will be understood that when expressed in plants, it will be necessary to change the mutual start codon to ATG for proper translation.

In some embodiments, the IPD090 polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384.

In some embodiments, the IPD090 polypeptide comprises the amino acid sequence of SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ

ID NO

ID NO

ID NO

ID NO

ID NO

ID NO

ID NO

ID NO

119 SEQ ID NO

125 SEQ ID NO

131 SEQ ID NO

137 SEQ ID NO

143 SEQ ID NO

149 SEQ ID NO

155 SEQ ID NO

161 SEQ ID NO

120 SEQ ID NO

126 SEQ ID NO

132 SEQ ID NO

138 SEQ ID NO

144 SEQ ID NO

150 SEQ ID NO

156 SEQ ID NO

162 SEQ ID NO

121 SEQ ID NO

127 SEQ ID NO

133 SEQ ID NO

139 SEQ ID NO

145 SEQ ID NO

151 SEQ ID NO

157 SEQ ID NO

163 SEQ ID NO

122 SEQ ID NO

128 SEQ ID NO

134 SEQ ID NO

140 SEQ ID NO

146 SEQ ID NO

152 SEQ ID NO

158 SEQ ID NO

164 SEQ ID NO

123 SEQ ID NO

129 SEQ ID NO

135 SEQ ID NO

141 SEQ ID NO

147 SEQ ID NO

153 SEQ ID NO

159 SEQ ID NO

165 SEQ ID NO

124 SEQ

130 SEQ

136 SEQ

142 SEQ

148 SEQ

154 SEQ

160 SEQ

166 SEQ

- 10 040502

ID NO: 167, SEQ ID NO: 168, SEQ

ID NO: 173, SEQ ID NO: 174, SEQ

ID NO: 179, SEQ ID NO: 180, SEQ

ID NO: 185, SEQ ID NO: 186, SEQ

ID NO: 191, SEQ ID NO: 192, SEQ

ID NO: 197, SEQ ID NO: 198, SEQ

ID NO: 274, SEQ ID NO: 275, SEQ

ID NO: 280, SEQ ID NO: 281, SEQ

ID NO: 286, SEQ ID NO: 287, SEQ

ID NO: 292, SEQ ID NO: 293, SEQ

ID NO: 298, SEQ ID NO: 299, SEQ

ID NO: 304, SEQ ID NO: 305, SEQ

ID NO: 310, SEQ ID NO: 311, SEQ

ID NO: 316, SEQ ID NO: 317, SEQ

ID NO: 322, SEQ ID NO: 323, SEQ

ID NO: 328, SEQ ID NO: 329, SEQ

ID NO: 334, SEQ ID NO: 335, SEQ

ID NO: 340, SEQ ID NO: 341, SEQ

ID NO: 379 or SEQ ID NO: 384.

In some embodiments, chimeric polypeptides are provided that contain portions of at least two different IPD090 polypeptides of the present invention.

In some embodiments, chimeric polypeptides are provided comprising portions of at least two different IPD090 polypeptides selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

117 SEQ ID NO

123 SEQ ID NO

129 SEQ ID NO

135 SEQ ID NO

141 SEQ ID NO

147 SEQ ID NO

153 SEQ ID NO

159 SEQ ID NO

165 SEQ ID NO

171 SEQ ID NO

177 SEQ ID NO

183 SEQ ID NO

189 SEQ ID NO

195 SEQ ID NO

201 SEQ ID NO

278 SEQ ID NO

284 SEQ ID NO

290 SEQ ID NO

296 SEQ ID NO

302 SEQ ID NO

308 SEQ ID NO

314 SEQ ID NO

320 SEQ ID NO

326 SEQ ID NO

332 SEQ ID NO

338 SEQ ID NO

118, SEQ ID NO 124, SEQ ID NO 130, SEQ ID NO 136, SEQ ID NO 142, SEQ ID NO 148, SEQ ID NO 154, SEQ ID NO 160, SEQ ID NO 166, SEQ ID NO 172, SEQ ID NO 178, SEQ ID NO 184, SEQ ID NO 190, SEQ ID NO 196, SEQ ID NO 202, SEQ ID NO 279, SEQ ID NO 285, SEQ ID NO 291, SEQ ID NO 297, SEQ ID NO 303, SEQ ID NO 309, SEQ ID NO 315, SEQ ID NO 321, SEQ ID NO 327, SEQ ID NO 333, SEQ ID NO 339, SEQ ID NO

344, SEQ ID NO: 377, SEQ ID NO: 379, and SEQ ID NO: 384.

In some embodiments, a chimeric IPD090 polypeptide is provided comprising

ID NO: 169, SEQ ID NO: 170, SEQ

ID NO: 175, SEQ ID NO: 176, SEQ

ID NO: 181, SEQ ID NO: 182, SEQ

ID NO: 187, SEQ ID NO: 188, SEQ

ID NO: 193, SEQ ID NO: 194, SEQ

ID NO: 199, SEQ ID NO: 200, SEQ

ID NO: 276, SEQ ID NO: 277, SEQ

ID NO: 282, SEQ ID NO: 283, SEQ

ID NO: 288, SEQ ID NO: 289, SEQ

ID NO: 294, SEQ ID NO: 295, SEQ

ID NO: 300, SEQ ID NO: 301, SEQ

ID NO: 306, SEQ ID NO: 307, SEQ

ID NO: 312, SEQ ID NO: 313, SEQ

ID NO: 318, SEQ ID NO: 319, SEQ

ID NO: 324, SEQ ID NO: 325, SEQ

ID NO: 330, SEQ ID NO: 331, SEQ

ID NO: 336, SEQ ID NO: 337, SEQ

ID NO: 342, SEQ ID NO: 343, SEQ

119, SEQ ID NO 125, SEQ ID NO 131, SEQ ID NO 137, SEQ ID NO 143, SEQ ID NO 149, SEQ ID NO 155, SEQ ID NO 161, SEQ ID NO 167, SEQ ID NO 173, SEQ ID NO 179, SEQ ID NO 185, SEQ ID NO 191, SEQ ID NO

197, SEQ ID NO 274, SEQ ID NO 280, SEQ ID NO 286, SEQ ID NO 292, SEQ ID NO 298, SEQ ID NO 304, SEQ ID NO 310, SEQ ID NO 316, SEQ ID NO 322, SEQ ID NO 328, SEQ ID NO 334, SEQ ID NO 340, SEQ ID NO

120 SEQ ID NO

126 SEQ ID NO

132 SEQ ID NO

138 SEQ ID NO

144 SEQ ID NO

150 SEQ ID NO

156 SEQ ID NO

162 SEQ ID NO

168 SEQ ID NO

174 SEQ ID NO

180 SEQ ID NO

186 SEQ ID NO

192 SEQ ID NO

198 SEQ ID NO

275 SEQ ID NO

281 SEQ ID NO

287 SEQ ID NO

293 SEQ ID NO

299 SEQ ID NO

305 SEQ ID NO

311 SEQ ID NO

317 SEQ ID NO

323 SEQ ID NO

329 SEQ ID NO

335 SEQ ID NO

341 SEQ ID NO

ID NO: 171, SEQ ID NO: 172, SEQ

ID NO: 177, SEQ ID NO: 178, SEQ

ID NO: 183, SEQ ID NO: 184, SEQ

ID NO: 189, SEQ ID NO: 190, SEQ

ID NO: 195, SEQ ID NO: 196, SEQ

ID NO: 201, SEQ ID NO: 202, SEQ

ID NO: 278, SEQ ID NO: 279, SEQ

ID NO: 284, SEQ ID NO: 285, SEQ

ID NO: 290, SEQ ID NO: 291, SEQ

ID NO: 296, SEQ ID NO: 297, SEQ

ID NO: 302, SEQ ID NO: 303, SEQ

ID NO: 308, SEQ ID NO: 309, SEQ

ID NO: 314, SEQ ID NO: 315, SEQ

ID NO: 320, SEQ ID NO: 321, SEQ

ID NO: 326, SEQ ID NO: 327, SEQ

ID NO: 332, SEQ ID NO: 333, SEQ

ID NO: 338, SEQ ID NO: 339, SEQ

ID NO: 344, SEQ ID NO: 377, SEQ

121, SEQ ID NO: 122, SEQ ID

127 SEQ ID NO: 128 SEQ ID

133, SEQ ID NO: 134, SEQ ID

139, SEQ ID NO: 140, SEQ ID

145 SEQ ID NO: 146 SEQ ID

151, SEQ ID NO: 152, SEQ ID

157 SEQ ID NO: 158 SEQ ID

163, SEQ ID NO: 164, SEQ ID

169, SEQ ID NO: 170, SEQ ID

175 SEQ ID NO: 176 SEQ ID

181, SEQ ID NO: 182, SEQ ID

187 SEQ ID NO: 188 SEQ ID

193 SEQ ID NO: 194 SEQ ID

199, SEQ ID NO: 200, SEQ ID

276 SEQ ID NO: 277 SEQ ID

282, SEQ ID NO: 283, SEQ ID

288 SEQ ID NO: 289 SEQ ID

294, SEQ ID NO: 295, SEQ ID

300, SEQ ID NO: 301, SEQ ID

306 SEQ ID NO: 307 SEQ ID

312, SEQ ID NO: 313, SEQ ID

318 SEQ ID NO: 319 SEQ ID

324, SEQ ID NO: 325, SEQ ID

330, SEQ ID NO: 331, SEQ ID

336 SEQ ID NO: 337 SEQ ID

342, SEQ ID NO: 343, SEQ ID

The N-terminal region of the first IPD090 polypeptide of the present invention, operably fused to

The C-terminal region of the second IPD090 polypeptide of the present invention.

In some embodiments, a chimeric IPD090 polypeptide is provided comprising

An N-terminal portion of a first IPD090 polypeptide operably fused to a C-terminal portion of a second IPD090 polypeptide, wherein the first and second IPD090 polypeptides are selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 10, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122,

SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128,

SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134,

SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140,

- 11 040502

SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO

SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO

SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO

SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO

SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO

SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO

SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO

SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO

SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO

SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO

SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO

SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO

SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID NO

SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO

SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO

SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO

SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID NO

SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID NO

SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID NO

SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID NO

SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO

SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO

143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146

149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152

155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158

161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164

167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170

173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176

179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182

185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188

191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194

197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200

274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277

280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283

286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289

292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295

298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301

304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307

310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313

316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319

322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325

328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331

334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337

340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343

SEQ ID NO: 344, SEQ ID NO: 377, SEQ ID NO: 379, and SEQ ID NO: 384.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids 1 to about 144, amino acids 1 to about 239, amino acids 1 to about 296, amino acids 1 to about 348, amino acids 1 to about 382, amino acids 1 to about 422, amino acids 1 to about 442 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids from about 146 to about 483, amino acids from about 241 to about 483, amino acids from about 297 to about 483, amino acids from about 349 to about 349 to about 483, about 383 to about 483 amino acids, about 423 to about 483 amino acids, or about 443 to about 483 amino acids of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids 1 to about 144 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids from about 146 to 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids 1 to about 239 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids from about 241 to 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids 1 to about 296 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids from about 297 to about 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids 1 to about 348 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids from about 349 to 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids 1 to about 382 of SEQ ID NO: 2 or SEQ

- 12 040502

ID NO: 4; and b) a C-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids from about 383 to 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids 1 to about 422 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids from about 423 to 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids 1 to about 442 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids from about 443 to 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region containing amino acids 1 to about 144, amino acids 1 to about 239, amino acids 1 to about 296, amino acids 1 to about 348, amino acids 1 to about 382 , amino acids 1 to about 422, amino acids 1 to about 442 of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; and b) a C-terminal region containing amino acids from about 146 to about 483, amino acids from about 241 to about 483, amino acids from about 297 to about 483, amino acids from about 349 to about 483, amino acids from about 383 to about 483, amino acids from about 423 to about 483 or amino acids from about 443 to about 483 of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

In some embodiments, the implementation of the chimeric IPD090 polypeptide contains a) an N-terminal region containing amino acids from 1 to about 144 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 146 to 483 of SEQ ID NO: 6.

In some embodiments, the implementation of the chimeric IPD090 polypeptide contains a) an N-terminal region containing amino acids from 1 to about 239 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 241 to 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region containing amino acids 1 to about 296 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 297 to about 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region containing amino acids 1 to about 348 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 349 to 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region containing amino acids 1 to about 382 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 383 to 483 of SEQ ID NO: 6.

In some embodiments, the implementation of the chimeric IPD090 polypeptide contains a) an N-terminal region containing amino acids from 1 to about 422 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 423 to 483 of SEQ ID NO: 6.

In some embodiments, the chimeric IPD090 polypeptide comprises a) an N-terminal region containing amino acids 1 to about 442 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 443 to 483 of SEQ ID NO: 6.

In other embodiments, the IPD090 polypeptide may be expressed as a precursor protein with an insert sequence that catalyzes multi-step post-translational splicing of the protein. Protein splicing involves excising insertion sequences from a polypeptide while adding flanking sequences to form a new polypeptide (Chong, et al., (1996) J. Biol. Chem., 271:22159-22168). This insertion sequence or protein splice element, called inteins, catalyzes its own excision through three concerted reactions at the N-terminal and C-terminal splicing boundaries: an acyl rearrangement of the N-terminal cysteine or serine; transesterification reactions between the two ends to form a branched ester or thioether intermediate and cleave the peptide bond conjugated to form a ring involving the C-terminal asparagine of the intein to release the intein (Evans, et al., (2000) J. Biol. Chem., 275 :9091-9094). The elucidation of the mechanism of protein splicing has led to a number of applications related to inteins (Comb et al., US patent No. 5496714; Comb et al., US patent No. 5834247; Camarero and Muir, (1999) J. Amer. Chem. Soc. 121 Chong, et al., (1997) Gene 192:271-281, Chong, et al., (1998) Nucleic Acids Res. 26:5109-5115 Chong, et al., (1998) J Biol Chem 273:10567-10577;

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Cotton, et al., (1999) J. Am. Chem. soc. 121:1100-1101; Evans, et al., (1999) J. Biol. Chem. 274:18359-18363; Evans, et al., (1999) J. Biol. Chem. 274:3923-3926; Evans, et al., (1998) Protein Sci. 7:2256-2264; Evans, et al., (2000) J. Biol. Chem. 275:9091-9094; Iwai and Pluckthun, (1999) FEBS Lett. 459:166-172; Mathys, et al., (1999) Gene 231:1-13; Mills, et al., (1998) Proc. Natl. Acad. sci. USA 95:3543-3548; Muir, et al., (1998) Proc. Natl. Acad. sci. USA 95:6705-6710; Otomo, et al., (1999) Biochemistry 38:16040-16044; Otomo, et al., (1999) J. Biolmol. NMR 14:105-114; Scott, et al., (1999) Proc. Natl. Acad. sci. USA 96:13638-13643; Severinov and Muir, (1998) J. Biol. Chem. 273:16205-16209; Shingledecker, et al., (1998) Gene 207:187-195; Southworth, et al., (1998) EMBO J. 17:918-926; Southworth, et al., (1999) Biotechniques 27:110-120; Wood, et al., (1999) Nat. Biotechnol. 17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. sci. USA 95:9226-9231; Wu, et al., (1998b) Biochim Biophys Acta 1387:422-432; Xu, et al., (1999) Proc. Natl. Acad. sci. USA 96:388-393; Yamazaki, et al., (1998) J. Am. Chem. Soc. 120:5591-5592). For the use of inteins in plant transgenes, see Yang, et al., (Transgene Res 15:583-593 (2006)) and Evans, et al., (Annu. Rev. Plant Biol. 56:375-392 (2005)) .

In another aspect, the IPD090 polypeptide can be encoded by two separate genes, whereby the intein of the precursor protein originates from two genes, it is called a split intein, and the two parts of the precursor are connected by forming a peptide bond. This peptide bond formation is mediated by intein-mediated transsplicing. For this purpose, the first and second expression cassettes containing two separate genes additionally encode inteins capable of mediating protein transsplicing. Using trans-splicing, proteins and polypeptides encoded by the first and second fragments can be connected by forming a peptide bond. The transsplicing inteins can be selected from the nuclear or organelle genome of various organisms, including eukaryotes, archaebacteria, and eubacteria. The inteins that can be used are listed on the website at neb.com/neb/inteins.html, which can be accessed via the World Wide Web using the prefix www). The nucleotide sequence encoding the intein can be divided into a 5'-part and a 3'-part, which encode respectively the 5'-part and the 3'-part of the intein. Portions of the sequence that are not necessary for intein splicing (eg, the homing endonuclease domain) may be deleted. The intein coding sequence is cleaved so that the 5' and 3' portions are capable of trans splicing. The considerations published in Southworth, et al., (1998) EMBO J. 17:918-926 can be followed to select an appropriate cleavage site for an intein coding sequence. When constructing the first and second expression cassettes, the 5'-sequence encoding intein is connected to the 3'-end of the first fragment encoding the N-terminal part of the IPD090 polypeptide, and the 3'-sequence encoding intein is connected to the 5'-end of the second fragment, encoding the C-terminal part of the IPD090 polypeptide.

In general, trans splicing partners can be engineered using any split intein, including any naturally occurring or artificially cleaved split intein. Several naturally occurring split inteins are known, for example, the split intein of the DnaE PCC6803 gene of Synechocystis sp. (See Wu, et al., (1998) Proc Natl Acad Sci USA. 95(16):9226-31 and Evans, et al., (2000) J Biol Chem. 275(13):9091-4) and gene DnaE from Nostoc punctiforme (see Iwai, et al., (2006) FEBS Lett. 580(7):1853-8) Non-split inteins have been artificially cleaved in the laboratory to create new split inteins, for example, the artificially cleaved Ssp DnaB intein (see Wu, et al., (1998) Biochim Biophys Acta. 1387:422-32), and the cleaved Sce VMA intein (see Brenzel, et al., (2006) Biochemistry. 45( 6):1571-8), and artificially digested fungal mini-intein (see Elleuche, et al., (2007) Biochem Biophys Res Commun. 355(3):830-4). Intein databases are also available, in known inteins (see, for example, the database available online at bioinformatics.weizmann.ac.il/~pietro/inteins/Inteinstable.html, which can be accessed via the World Wide Web using the prefix www).

Naturally occurring inteins that are not split inteins may have endonuclease or other enzymatic activities that can generally be eliminated by constructing an artificially cleaved split intein. Such mini inteins or minimized split inteins are well known in the art and are typically less than 200 amino acid residues in length (see Wu, et al., (1998) Biochim Biophys Acta. 1387:422-32 ). Suitable split inteins may have other scavenging polypeptide elements added to their structure, provided that such elements do not inhibit split intein splicing, or they are added in a manner that allows them to be removed prior to splicing. Protein splicing has been reported using proteins that contain bacterial intein-like (BIL) domains (see Amitai, et al., (2003) Mol Microbiol. 47:61-73) and hedgehog (Hog) self-processing domains (the latter when combined with inteins is referred to as the Hog/intein superfamily or the HINT family (see Dassa, et al., (2004) J Biol Chem. 279:32001-7), and domains such as these can also be used to prepare artificially cleaved inteins. in particular, non-spliced members of such families can be modified using molecular biology techniques to introduce or restore splicing activity in such related species.Recent studies show that splicing can be observed by providing a reaction of the N-terminal component

- 14 040502 split intein with the C-terminal component of split intein, while under natural conditions it is not its partner; for example, splicing has been observed using partners that are only 30-50% homologous to the natural splicing partner (see Dassa, et al., (2007) Biochemistry. 46(1):322-30). Other such mixtures of incompatible split intein partners have been shown not to react with each other (see Brenzel, et al., (2006) Biochemistry. 45(6):1571-8). However, it is within the skill of the art to determine whether a particular pair of polypeptides can bind to each other to provide a functional intein using standard techniques and without the use of inventive skills.

In some embodiments, the IPD090 polypeptide is a circular permutation variant. In certain embodiments, the IPD090 polypeptide is a circular permutation variant of the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384, or a variant thereof having an amino acid substitution, deletion, addition, or combinations thereof. The development of methods using recombinant DNA provided the opportunity to investigate the effects of sequence transposition on protein folding, structure and function. The approach taken to create new sequences is similar to that of naturally occurring pairs of proteins that bind by linear rearrangement of their amino acid sequences (Cunningham, et al., (1979) Proc. Natl. Acad. Sci. U.S.A. 76:3218 -3222; Teather and Erfle, (1990) J. Bacteriol 172:3837-3841; Schimming, et al., (1992) Eur. J. Biochem. 204:13-19; Yamiuchi and Minamikawa, (1991) FEBS Lett 260:127-130; MacGregor, et al., (1996) FEBS Lett. 378:263-266). The first in vitro application of this type of rearrangement in proteins was described by Goldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). When creating a variant with circular permutations, a new N-terminus is chosen at an internal site (dot break) of the original sequence, with the new sequence having the same amino acid order as the original, from the dot break until it reaches the amino acid that is at or near the original C-terminus. At this point, the new sequence is attached either directly or through an additional sequence (linker) to the amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is in an amino acid that was N-terminal to or near the point break site of the original sequence, this residue forming the new C-terminus of the chain. The amino acid sequence length of the linker can be chosen empirically, or based on structure information, or by using a combination of the two. If structure information is not available, small runs of linkers can be obtained for design testing, varying in length to cover the range from 0 to 50 A, and sequenced to match the availability of surface groups (Hydrophilicity, Hopp and Woods, ( 1983) Mol Immunol 20:483-489 Kyte and Doolittle (1982) J Mol Biol 157:105-132 Solvent accessible surface area Lee and Richards (1971) J Mol Biol 55:379-400) and the ability to assume the desired conformation without disrupting the configuration of the pesticidal polypeptide (conformationally flexible; Karplus and Schulz, (1985) Naturwissenschaften 72:212-213). Assuming a translation of an average residue length of 2.0-3.8 A, this would mean that the length to be tested would be from 0 to 30 residues, with 0-15 residues being the preferred range. An example of such an empirical series would be the design of linkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated n times, where n is 1, 2, 3, or 4. Those skilled in the art will appreciate that there are many such sequences, varying in length or composition, which can serve as linkers, primarily given that they are neither excessively long nor excessively short (see Sandhu, (1992) Critical Rev. Biotech. 12:437-462); moreover, if they are too long, then entropy effects are likely to destabilize the three-dimensional folding and can also make folding kinetically unfeasible, and if they are too short, they are likely to destabilize the molecule due to torsional or steric deformation. Those skilled in the analysis of protein structure information will appreciate that, when present, the distance between chain ends, defined as the distance between c-alpha carbons, can be used to determine the length of the sequence to be used, or at least to limit the number of possibilities that need to be tested. in the empirical selection of linkers. They will also understand that it is sometimes the case when the positions of the ends of a polypeptide chain are unclear in the structural models obtained from X-ray diffraction analysis or nuclear magnetic resonance spectroscopy, and in the case of such a situation, therefore, it must be taken into account for a correct estimation of the length required linker. Based on residues whose position is well defined, two residues are selected that are close in sequence to the ends of the chain, and the distance between their c-alpha carbon atoms is used to calculate the approximate length for the linker between them. Using the calculated length as preliminary data, linkers are then selected within the range of the number of residues (based on 2-3.8 A per residue). These linkers can be made up of the original sequence, truncated or extended as needed, and if extended, additional residues can be selected that are flexible and hydrophilic as described above; or optionally the original sequence can be replaced using a series of linkers, one example being the above mentioned Gly-Gly-Gly-Ser cassette approach; or optionally, a combination of the original sequence and a new sequence having a suitable total length may be used.

Sequences of pesticidal polypeptides capable of folding into biologically active states can be obtained by appropriate selection of initial (amino-terminus) and terminal (carboxyl-terminal) positions from the parent polypeptide chain, using the linker sequence as described above. The amino-terminus and carboxyl-terminus are selected from a common segment of the sequence, referred to as the puncture site, using the guidelines described below. Thus, a new amino acid sequence is obtained by selecting the amino terminus and carboxyl terminus from the same puncture site. In many cases, the choice of new ends will be such that the original position of the carboxyl terminus immediately precedes the position of the amino terminus. However, those skilled in the art will appreciate that the choice of ends anywhere within the region may have an effect, and that this will in fact result in either deletions or additions to the amino portion or carboxyl portion of the new sequence. The basic tenet of molecular biology is that the primary amino acid sequence of a protein determines the folding into the three-dimensional structure required for its biological function. Those skilled in the art are aware of methods for obtaining and interpreting three-dimensional structure information using X-ray diffraction analysis of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions. Examples of structural information that is suitable for identifying pinpoint break sites include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and antiparallel beta sheets, strand and loop reversals or turns; Kabsch and Sander, (1983) Biopolymers 22:2577-2637, the degree of solvent accessibility of amino acid residues, the extent and type of interactions of residues with each other (Chothia, (1984) Ann. Rev. Biochem. 53:537-572), and the static and dynamic distribution of conformations throughout polypeptide chain (Alber and Mathews, (1987) Methods Enzymol. 154:511-533. In some cases, additional information is known about the solvent accessibility of residues, one example being the post-translational carbohydrate attachment site, which is necessarily located on the surface of the protein. If experimental structure information is not available or cannot be obtained, methods are also available to analyze the primary amino acid research to make predictions about protein tertiary and secondary structure, solvent accessibility, and the presence of turns and loops. Biochemical methods are also sometimes applicable to empirically determine the availability of surface groups if direct methods of determining the structure are not possible; for example, the use of identification of depolymerization sites after limited proteolysis in order to infer the availability of surface groups (Gentile and Salvatore, (1993) Eur. J. Biochem. 218:603-621). Thus, by applying either experimental structure information or predictive methods (e.g., Srinivisan and Rose, (1995) Proteins: Struct., Funct. & Genetics 22:81-99), the original amino acid sequence is examined to classify regions. as to whether they are important in maintaining secondary and tertiary structures. The presence of sequences in regions known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) should be avoided. Similarly, regions of the amino acid sequence that are observed or predicted to have low solvent accessibility are most likely to be part of the so-called hydrophobic core of the protein and should also be avoided when choosing the amino or carboxyl terminus. In contrast, regions known or predicted to be in surface turns or loops, and in particular those regions known not to be required for biological activity, are preferred sites for the location of opposite ends of the polypeptide chain. . Preferred contiguous stretches of amino acid sequence based on the criteria above are referred to as a point break region. Polynucleotides encoding IPD090 circularly permuted polypeptides with a novel N-terminus/C-terminus, which contain a linker region separating the original C-terminus and N-terminus, can essentially be prepared according to the method described in Mullins, et al., ( 1994) J. Am. Chem. soc. 116:5529-5533. Several amplification steps by polymerase chain reaction (PCR) are used to rearrange the DNA sequence encoding the primary amino acid sequence of a protein. Polynucleotides encoding IPD090 circularly permuted polypeptides with a new N-terminus/C-terminus, which contain a linker region separating the original C-terminus and N-terminus, can be prepared based on the tandem repeat method described in Horlick, et al., (1992) ProteinEng. 5:427-431. Amplification by polymerase chain reaction (PCR) of genes with novel N-terminus/C-terminus is performed using a DNA template with tandem repeats.

In another embodiment, fusion proteins are provided, the amino acid sequence of which includes an amino acid sequence comprising an IPD090 polypeptide or a chimeric IPD090 polypeptide of the present invention. Methods for designing and constructing fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art. Polynucleotides encoding an IPD090 polypeptide can be fused to signal sequences that will direct the localization of the IPD090 polypeptide to specific compartments of a prokaryotic or eukaryotic cell and/or direct secretion of the IPD090 polypeptide, in embodiments, from a prokaryotic or eukaryotic cell. For example, in E. coli, it may be desirable to direct protein expression to the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) to which the IPD090 polypeptide can be fused to direct expression of the polypeptide to the bacterial periplasmic space include, but are not limited to, pelB signal sequence, maltose binding protein (MBP) signal sequence, MBP, signal sequence ompA, E. coli periplasmic heat-labile enterotoxin B subunit signal sequence, and alkaline phosphatase signal sequence. For the construction of fusion proteins, several vectors are commercially available that will direct protein localization, such as the pMAL vector series (particularly the pMAL-p series) available from New England Biolabs. In a specific embodiment, the IPD090 polypeptide can be fused to the pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such polypeptides in Gram-negative bacteria (see US Pat. Nos. 5,576,195 and 5,846,818). Plant plastid transit peptide/polypeptide fusions are well known in the art. Apoplastic transit peptides, such as the signal sequence for the secretion of rice or barley alpha-amylase, are also well known in the art. The plastid transit peptide is fused, typically at the N-terminus, to the polypeptide to be targeted (eg, a fusion partner). In one embodiment, the fusion protein consists essentially of a plastid transit peptide and an IPD090 polypeptide to be targeted. In another embodiment, the fusion protein consists of a plastid transit peptide and a polypeptide to be targeted. In such embodiments, the plastid transit peptide is preferably located at the N-terminus of the fusion protein. However, additional amino acid residues may be N-terminal relative to the plastid transit peptide, provided that the fusion protein is at least partially targeted to the plastid. In a particular embodiment, the plastid transit peptide is located at the N-terminal half, N-terminal third, or N-terminal quarter of the fusion protein. Typically, most or all of the plastid transit peptide is excised from the fusion protein after insertion into the plastid. The position of the cleavage may vary slightly between plant species, at different stages of plant development, as a result of specific intracellular conditions, or the particular combination of transit peptide/fusion partner used. In one embodiment, the cleavage site of the plastid transit peptide is homogeneous, whereby the cleavage site is identical within the fusion protein population. In another embodiment, the cleavage site of the plastid transit peptide is not homogeneous, whereby the cleavage site varies by 1-10 amino acids in the fusion protein population. The plastid transit peptide can be recombinantly fused to a second protein in one of several ways. For example, a restriction endonuclease recognition site can be introduced into the nucleotide sequence of the transit peptide at a position corresponding to its C-terminus, and the same or compatible site can be introduced by genetic engineering into the nucleotide sequence of the protein to be targeted at its N-terminus. When constructing these sites, care must be taken to ensure that the coding sequences for the transit peptide and the second protein are in-frame to ensure the synthesis of the desired fusion protein. In some cases, it may be preferable to remove the initiator methionine of the second protein when introducing a new restriction site. The introduction of restriction endonuclease recognition sites into both parent molecules and their subsequent connection by recombinant DNA techniques may result in the addition of one or more additional amino acids between the transit peptide and the second protein. Typically, this does not affect targeting activity because the cleavage site of the transit peptide remains accessible and the function of the second protein will not be affected by the addition of these additional amino acids to its N-terminus. Alternatively, a person skilled in the art can create a precise cleavage site between the transit peptide and the second peptide (with or without its initiator methionine) using gene synthesis (Stemmer, et al., (1995) Gene 164:49-53) or similar ways. In addition, the transition peptide fusion may intentionally include amino acids downstream of the cleavage site. Amino acids at the N-terminus of a mature protein can affect the ability of the transit peptide to target proteins to plastids and/or the efficiency of cleavage after protein import. This may depend on the protein to be targeted. See, for example, Comai, et al., (1988) J. Biol. Chem. 263(29):15104-9. In some embodiments, the IPD090 polypeptide is fused to a heterologous signal peptide or a heterologous transit peptide.

In some embodiments, fusion proteins are provided comprising an IPD090 polypeptide or a chimeric IPD090 polypeptide of the present invention, represented by the formula, you

- 17 040502 brane from the group consisting of R^L-Rf R2-LR ¹ , R ¹ -R ² or R2-R ¹ , where R ¹ is an IPD090 polypeptide or a chimeric IPD090 polypeptide of the present invention, and R ² is protein of interest. In some embodiments, R ¹ and R ² are an IPD090 polypeptide or a chimeric IPD090 polypeptide of the present invention. The R ¹ polypeptide is fused either directly or via a linker (L) segment to the R ² polypeptide. The term directly denotes fusions in which polypeptides are joined without a peptide linker. Thus, L is a chemical bond or polypeptide segment to which both R ¹ and R ² are fused in frame, most often L is a linear peptide to which R ¹ and R ² are linked via amide bonds linking the carboxy terminus R ¹ with the amino end of L and the carboxy end of L with the amino end of R ² . By in-frame fusion is meant that there is no translation termination site or gap between the R ¹ and R ² reading frames. The linker group (L) is usually a polypeptide ranging from 1 to 500 amino acids in length. Linkers connecting two molecules are preferably designed so that (1) they allow the two molecules to fold and act independently of each other, (2) they do not tend to develop an ordered secondary structure that may conflict with the functional domains of the two proteins, (3) that they have minimal hydrophobic or charging characteristics that may conflict with the functional domains of the protein, and (4) that they provide steric separation of R ¹ and R ² so that R ¹ and R ² can simultaneously interact with their respective receptors on the same cell. Generally, surface amino acids in the flexible regions of a protein include Gly, Asn, and Ser. As such, any permutation of amino acid sequences containing Gly, Asn and Ser is expected to satisfy the above criteria for a linker sequence. Other neutral amino acids such as Thr and Ala can also be used in the linker sequence. Additional amino acids can also be included in the linkers since the addition of unique restriction sites to the linker sequence facilitates the construction of fusions.

In some embodiments, the linkers contain sequences selected from the group of formulas: (Gly ₃ Ser) _n , (Gly ₄ Ser) _n , (Gly ₅ Ser) _n , (Gly _n Ser) _n or (AlaGlySer) n , where n is integer. One example of a very flexible linker is the spacer region rich (GlySer) present in the pIII protein of filamentous bacteriophages, such as M13 or fd bacteriophages (Schaller, et al., 1975). This region provides a long flexible spacer region between the two pIII surface protein domains. Also included are linkers that include an endopeptidase recognition sequence. Such a cleavage site can be very important in separating individual fusion components to determine if they are properly folded and active in vitro. Examples of various endopeptidases include, without limitation, plasmin, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostripain, chymosin, collagenase, Russell's viper venom protease, postproline cleavage enzyme, V8 protease, thrombin, and factor Xa. In some embodiments, the linker contains the amino acids EEKKN (SEQ ID NO: 376) from a multigene expression vehicle (MGEV) that is cleaved by vacuole proteases as disclosed in US Patent Application Publication No. 2007/0277263. In other embodiments, peptide linker segments from the IgG, IgA, IgM, IgD, or IgE immunoglobulin heavy chain hinge region provide for angular interaction between attached polypeptides. Particularly useful are those hinge regions in which cysteine residues are replaced by serine residues. The linkers of the present invention comprise sequences derived from a mouse IgG gamma 2b hinge region in which cysteine residues have been replaced with serine residues. Fusion proteins are not limited by the shape, size, or number of linker sequences used, and the only requirement for the linker is that it does not functionally adversely affect the folding and function of the individual fusion molecules.

Nucleic acid molecules, as well as their variants and fragments.

Isolated or recombinant nucleic acid molecules are provided containing nucleic acid sequences encoding IPD090 polypeptides or biologically active portions thereof, as well as nucleic acid molecules suitable for use as hybridization probes to identify nucleic acid molecules encoding proteins with sequence homology regions. As used herein, the term nucleic acid molecule refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA), as well as DNA analogs or RNA made using analogs. nucleotides. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

An isolated nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid (or DNA) sequence that is no longer in its natural environment, such as in vitro. A recombinant nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid (or DNA) sequence that is found in a recombinant bacterial or plant host cell. In some embodiments, the isolated or recombinant nucleic acid does not contain

- 18 040502 sequences (preferably protein-coding sequences) that naturally flank the nucleic acid (ie sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For the purposes of the present invention, the terms isolated or recombinant, when used to refer to nucleic acid molecules, exclude isolated chromosomes. For example, in various embodiments, a recombinant nucleic acid molecule encoding IPD090 polypeptides may comprise less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0 .5 t.o. or 0.1 t.o. of nucleic acid sequences that naturally flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.

In some embodiments, the isolated nucleic acid molecule encoding the IPD090 polypeptides has one or more nucleic acid sequence changes relative to the native or genomic nucleic acid sequence. In some embodiments, a change in the native or genomic nucleic acid sequence includes, without limitation, changes in the nucleic acid sequence due to degeneracy of the genetic code; changes in the nucleic acid sequence due to amino acid substitution, insertion, deletion and/or addition compared to the native or genomic sequence; removal of one or more introns; deletion of one or more regulatory regions located above or below; and deletion of the 5' and/or 3' untranslated region associated with the genomic nucleic acid sequence. In some embodiments, the nucleic acid molecule encoding the IPD090 polypeptide is a non-genomic sequence.

A number of polynucleotides are contemplated that encode for IPD090 polypeptides or related proteins. Such polynucleotides are useful for generating IPD090 polypeptides in host cells when they are operably linked to a suitable promoter, transcription terminator and/or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode IPD090 polypeptides or related proteins.

Polynucleotides encoding IPD090 polypeptides.

One source of polynucleotides that encode IPD090 polypeptides or related proteins is the bacterium of the genus Pseudomonas or Woodsholea, which contains the IPD090 polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 378, and SEQ ID NO: 380 encoding the IPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, and SEQ ID NO: 384, respectively. The polynucleotides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 378, or SEQ ID NO: 380 can be used to express IPD090 polypeptides in recombinant bacterial hosts, which include, but are not limited to, bacterial cells hosts of the genus Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas and Rhizobium. The polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode IPD090 polypeptides or related proteins. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from species of the genus Pseudomonas.

Polynucleotides that encode for IPD090 polypeptides can also be synthesized de novo from the sequence of an IPD090 polypeptide. The gene sequence of a polynucleotide can be deduced from the sequence of the IPD090 polypeptide using the genetic code. Computer programs such as BackTranslate (GCG™ Package, Acclerys, Inc., San Diego, CA) can be used to convert a peptide sequence into the corresponding nucleotide sequence encoding a peptide. Examples of IPD090 polypeptide sequences that can be used to obtain the corresponding nucleotide coding sequences include, but are not limited to, IPD090 polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, and SEQ ID NO: 384 In addition, synthetic IPD090 polynucleotide sequences of the present disclosure can be designed such that they are expressed in plants.

In some embodiments, the nucleic acid molecule encoding the IPD090 polypeptide is a polynucleotide with the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 378, or SEQ ID NO: 380, and its variants, fragments and sequences complementary to it. The term complementary sequence is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to that nucleic acid sequence such that a stable duplex is formed. The term polynucleotide sequence variants is used herein to refer to a nucleic acid sequence that, without considering the dissimilarity associated with the degeneracy of the genetic code, encodes the same polypeptide.

In some embodiments, the nucleic acid molecule encoding the IPD090 polypeptide is a non-genomic nucleic acid sequence. use

- 19 040502 As used herein, the terms non-genomic nucleic acid sequence or non-genomic nucleic acid molecule or non-genomic polynucleotide refers to a nucleic acid molecule that has one or more changes in the nucleic acid sequence compared to with native or genomic nucleic acid sequences. In some embodiments, a change with respect to a native or genomic nucleic acid molecule includes, without limitation, changes in the nucleic acid sequence due to the degeneracy of the genetic code; optimizing the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion and/or addition compared to the native or genomic sequence; removal of one or more introns associated with the genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions that are associated with the genomic nucleic acid sequence; insertion of one or more heterologous regulatory regions above or below; deletion of the 5' and/or 3' untranslated region associated with the genomic nucleic acid sequence; insertion of a heterologous 5' and/or 3' untranslated region; and modification of the polyadenylation site. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

In some embodiments, the nucleic acid molecule encoding the IPD090 polypeptide is a non-genomic polynucleotide with a nucleotide sequence that is at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO : 5, SEQ ID NO: 378 or SEQ ID NO: 380, where the IPD090 polypeptide is characterized by insecticidal activity.

In some embodiments, the nucleic acid molecule encodes an IPD090 polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384 with 1, 2, 3 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70.71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or more amino acid substitutions compared to the native amino acid at the corresponding position of SEQ ID NO: 2 , SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384.

In some embodiments, the nucleic acid molecule encodes an IPD090 polypeptide variant containing any one or more amino acid substitutions corresponding to positions 3, 4, 8, 12, 15, 16, 21, 23, 24, 26, 28, 30, 38, 46, 47, 50, 52, 55, 62, 63, 67, 68, 70, 73, 74, 75, 76, 80, 90, 91, 94, 99, 100, 108, 115, 127, 129, 161, 169, 175, 177, 178, 180, 185, 207, 213, 223, 240, 241, 247, 255, 266, 273, 275, 277, 278, 287, 288, 302, 306, 309, 310, 311, 312, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325,

326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,

348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,

370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 391, 392,

395, 397, 400, 401, 402, 405, 407, 410, 423, 425, 426, 431, 433, 434, 437, 438, 439, 440, 441, 442, 443, 444,

445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 457, 458, 459, 460, 468, and 471 SEQ ID NO: 2 in any combination.

In some embodiments, the nucleic acid molecule encodes an IPD090 polypeptide variant containing any one or more amino acid substitutions from Table 10 or 12.

Also contemplated are nucleic acid molecules that encode transcription and/or translation products that are further spliced to eventually form functional IPD090 polypeptides. Splicing may be carried out in vitro or in vivo and may include cis or trans splicing. The splicing substrate can be polynucleotides (eg, RNA transcripts) or polypeptides. An example of cis-splicing of a polynucleotide is when an intron inserted in a coding sequence is removed and two flanking exons are joined to form the sequence encoding the IPD090 polypeptide. An example of trans splicing would be where a polynucleotide is encoded to split the coding sequence into two or more fragments that can be transcribed separately and then joined to form a full length pesticidal coding sequence. The use of a splicing enhancer sequence that can be introduced into a construct can facilitate splicing, both cis and trans splicing of polypeptides (US Pat. Nos. 6,365,377 and 6,531,316). Thus, in some embodiments, the polynucleotides do not directly encode a full-length IPD090 polypeptide, but encode a fragment or fragments of an IPD090 polypeptide. These polynucleotides can be used to express a functional IPD090 polypeptide through a mechanism involving splicing, where splicing can occur on

- 20 040502 at the level of a polynucleotide (eg intron/exon) and/or polypeptide (eg intein/extein). This may be useful, for example, in controlling the expression of pesticidal activity, since a functional pesticidal polypeptide will only be expressed if all the required fragments are expressed in a medium that allows splicing processes to form a functional product. In another example, the introduction of one or more insertion sequences into a polynucleotide can facilitate recombination with a low homology polynucleotide; however, the use of an intron or intein in relation to the insertion sequence facilitates the removal of the insertion sequence, thereby restoring the function of the encoded variant.

Nucleic acid molecules that are fragments of these nucleic acid sequences encoding IPD090 polypeptides are also covered by the embodiments. As used herein, the term fragment refers to a portion of a nucleic acid sequence encoding an IPD090 polypeptide. A fragment of the nucleic acid sequence may encode the biologically active part of the IPD090 polypeptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods described below. Nucleic acid molecules that are fragments of a nucleic acid sequence encoding an IPD090 polypeptide contain at least about 150, 180, 210, 240, 270, 300, 330, or 360 contiguous nucleotides, or up to the number of nucleotides present in the full length nucleic acid sequence encoding the IPD090 polypeptide disclosed herein, depending on the intended use. The term adjacent nucleotides is used herein to refer to nucleotide residues that are directly adjacent to each other. Fragments of nucleic acid sequences according to embodiments will encode protein fragments that retain the biological activity of the IPD090 polypeptide and, accordingly, retain insecticidal activity. The term retains insecticidal activity is used herein to refer to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or more of the insecticidal activity of the full length polypeptide. IPD090Aa (SEQ ID NO: 2). In some embodiments, the implementation of the insecticidal activity is directed against species of Lepidoptera. In one embodiment, the insecticidal activity is activity against a beetle species. In some embodiments, the insecticidal activity is against one or more of the corn bug pests: western corn bug, Diabrotica virgifera; northern corn beetle, D. barberi: southern corn beetle or eleven-spotted flea beetle; Diabrotica undecimpunctata howardi, and the Mexican corn beetle, D. virgifera zeae. In one embodiment, the insecticidal activity is activity against a Diabrotica species.

In some embodiments, the IPD090 polypeptide is encoded by a nucleic acid sequence sufficiently homologous to the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 378, or SEQ ID NO: 380. The term is sufficiently homologous used herein to refer to an amino acid or nucleic acid sequence characterized by at least about 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or greater sequence homology when compared to a reference sequence using one of the alignment programs described herein using standard parameters. One of ordinary skill in the art will appreciate that these values can be adjusted accordingly to determine the respective homology of the proteins encoded by the two nucleic acid sequences, taking into account degeneracy, amino acid similarity, reading frame location, and the like. In some embodiments, sequence homology is relative to the full length sequence of the polynucleotide encoding the IPD090 polypeptide or to the full length sequence of the IPD090 polypeptide.

In some embodiments, the nucleic acid encodes an IPD090 polypeptide whose sequence is at least about 50, 55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99% or more sequence identical when compared with the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379 or SEQ ID NO: 384. In some embodiments, sequence identity is calculated using the ClustalW algorithm in the ALIGNX® module of the Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, CA) with all standard parameters. In some embodiments, sequence identity is calculated over the entire length of the polypeptide using the ClustalW algorithm in the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA) with all standard parameters.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for purposes of optimal comparison. The percent identity of two sequences is a function of the number of identical positions present in both sequences (ie, percent identity = number of identical positions/total number of positions (eg, overlapping positions) x 100). In one embodiment, the two sequences are the same length. In another embodiment, the comparison is made over the entire length of the reference sequence (eg, over the entire length of SEQ ID NO: 1). The percent identity of two sequences can be determined using techniques similar to those described below, which allow gaps or do not allow gaps. When calculating percentage identity, as a rule, exact matches are counted.

Another non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48(3):443-453 used in GAP version 10 software to determine sequence identity and similarity using the following standard parameters: percent identity and percent similarity for a nucleic acid sequence using a gap opening penalty of 50, and a gap extension penalty 3, and substitution matrices nwsgapdna.cmpii; percent identity or percent similarity for an amino acid sequence using gap opening penalty 8 and gap extension penalty 2, and the BLOSUM62 substitution program. You can also use equivalent programs. The term equivalent program is used herein to refer to any sequence comparison program that, for any two sequences under investigation, generates an alignment with identical nucleotide residue matches and identical percent sequence identity when compared to the corresponding alignment generated using GAP version 10.

In some embodiments, an IPD090 polynucleotide encodes an IPD090 polypeptide comprising an amino acid sequence that is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95%, 96%, 97%, 98%, 99% or more identical throughout the amino acid sequence to SEQ ID NO: 2.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides containing portions of at least two different IPD090 polypeptides of the present invention.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising portions of at least two different IPD0 90 polypeptides selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

115, SEQ ID NO 121, SEQ ID NO 127, SEQ ID NO 133, SEQ ID NO 139, SEQ ID NO 145, SEQ ID NO 151, SEQ ID NO 157, SEQ ID NO 163, SEQ ID NO 169, SEQ ID NO 175, SEQ ID NO 181, SEQ ID NO 187, SEQ ID NO 193, SEQ ID NO 199, SEQ ID NO 276, SEQ ID NO 282, SEQ ID NO 288, SEQ ID NO 294, SEQ ID NO 300, SEQ ID NO 306, SEQ ID NO 312, SEQ ID NO 318, SEQ ID NO 324, SEQ ID NO 330, SEQ ID NO 336, SEQ ID NO

116 SEQ ID NO

122 SEQ ID NO

128 SEQ ID NO

134 SEQ ID NO

140 SEQ ID NO

146 SEQ ID NO

152 SEQ ID NO

158 SEQ ID NO

164 SEQ ID NO

170 SEQ ID NO

176 SEQ ID NO

182 SEQ ID NO

188 SEQ ID NO

194 SEQ ID NO

200 SEQID NO

277 SEQ ID NO

283 SEQ ID NO

289 SEQ ID NO

295 SEQ ID NO

301 SEQ ID NO

307 SEQ ID NO

313 SEQ ID NO

319 SEQ ID NO

325 SEQ ID NO

331 SEQ ID NO

337 SEQ ID NO

117, SEQ ID NO 123, SEQ ID NO 129, SEQ ID NO 135, SEQ ID NO 141, SEQ ID NO 147, SEQ ID NO 153, SEQ ID NO 159, SEQ ID NO

165, SEQ ID NO 171, SEQ ID NO 177, SEQ ID NO 183, SEQ ID NO 189, SEQ ID NO 195, SEQ ID NO 201, SEQ ID NO 278, SEQ ID NO

284, SEQ ID NO 290, SEQ ID NO 296, SEQ ID NO 302, SEQ ID NO 308, SEQ ID NO 314, SEQ ID NO 320, SEQ ID NO 326, SEQ ID NO

332, SEQ ID NO 338, SEQ ID NO

118, SEQ ID NO 124, SEQ ID NO 130, SEQ ID NO 136, SEQ ID NO 142, SEQ ID NO 148, SEQ ID NO 154, SEQ ID NO 160, SEQ ID NO 166, SEQ ID NO 172, SEQ ID NO 178, SEQ ID NO 184, SEQ ID NO 190, SEQ ID NO 196, SEQ ID NO 202, SEQ ID NO 279, SEQ ID NO 285, SEQ ID NO 291, SEQ ID NO 297, SEQ ID NO 303, SEQ ID NO 309, SEQ ID NO 315, SEQ ID NO 321, SEQ ID NO 327, SEQ ID NO 333, SEQ ID NO 339, SEQ ID NO

119 SEQ ID NO

125 SEQ ID NO

131 SEQ ID NO

137 SEQ ID NO

143 SEQ ID NO

149 SEQ ID NO

155 SEQ ID NO

161 SEQ ID NO

167 SEQ ID NO

173 SEQ ID NO

179 SEQ ID NO

185 SEQ ID NO

191 SEQ ID NO

197 SEQ ID NO

274 SEQ ID NO

280 SEQ ID NO

286 SEQ ID NO

292 SEQ ID NO

298 SEQ ID NO

304 SEQID NO

310 SEQ ID NO

316 SEQ ID NO

322 SEQ ID NO

328 SEQ ID NO

334 SEQ ID NO

340, SEQ ID NO : 114, SEQ ID : 120, SEQ ID : 126, SEQ ID : 132, SEQ ID : 138, SEQ ID : 144, SEQ ID : 150, SEQ ID : 156, SEQ ID : 162, SEQ ID : 168, SEQ ID : 174, SEQ ID : 180, SEQ ID : 186, SEQ ID : 192, SEQ ID : 198, SEQ ID : 275, SEQ ID : 281, SEQ ID : 287, SEQ ID : 293, SEQ ID : 299, SEQ ID : 305, SEQ ID : 311, SEQ ID : 317, SEQ ID : 323, SEQ ID : 329, SEQ ID : 335, SEQ ID : 341, SEQ ID

342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 377, SEQ ID NO: 379, and SEQ ID NO: 384.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising the N-terminal portion of a first IPD090 polypeptide of the present invention operably fused to the C-terminal portion of a second IPD090 polypeptide of the present invention.

In some embodiments, polynucleotides encoding chimeric

- 22 040502 polypeptides containing the N-terminal region of the first polypeptide IPD090, functionally fused with

The C-terminal region of the second IPD090 polypeptide, where the IPD090 polypeptide is selected from SEQ ID NO: 2,

SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

NO

116 SEQ ID NO

122 SEQ ID NO

128 SEQ ID NO

134 SEQ ID NO

140 SEQ ID NO

146 SEQ ID NO

152 SEQ ID NO

158 SEQ ID NO

164 SEQ ID NO

170 SEQ ID NO

176 SEQ ID NO

182 SEQ ID NO

188 SEQ ID NO

194 SEQ ID NO

200 SEQID NO

277 SEQ ID NO

283 SEQ ID NO

289 SEQ ID NO

295 SEQ ID NO

301 SEQ ID NO

307 SEQ ID NO

313 SEQ ID NO

319 SEQ ID NO

325 SEQ ID NO

331 SEQ ID NO

337 SEQ ID NO

117 SEQ ID NO

123 SEQ ID NO

129 SEQ ID NO

135 SEQ ID NO

141 SEQ ID NO

147 SEQ ID NO

153 SEQ ID NO

159 SEQ ID NO

165 SEQ ID NO

171 SEQ ID NO

177 SEQ ID NO

183 SEQ ID NO

189 SEQ ID NO

195 SEQ ID NO

201 SEQ ID NO

278 SEQ ID NO

284 SEQ ID NO

290 SEQ ID NO

296 SEQ ID NO

302 SEQ ID NO

308 SEQ ID NO

314 SEQ ID NO

320 SEQ ID NO

326 SEQ ID NO

332 SEQ ID NO

338 SEQ ID NO

118 SEQ ID NO

124 SEQ ID NO

130 SEQ ID NO

136 SEQ ID NO

142 SEQ ID NO

148 SEQ ID NO

154 SEQ ID NO

160 SEQ ID NO

166 SEQ ID NO

172 SEQ ID NO

178 SEQ ID NO

184 SEQ ID NO

190, SEQ ID NO

196 SEQ ID NO

202 SEQ ID NO

279 SEQ ID NO

285 SEQ ID NO

291 SEQ ID NO

297 SEQ ID NO

303 SEQ ID NO

309 SEQ ID NO

315 SEQ ID NO

321 SEQ ID NO

327 SEQ ID NO

333 SEQ ID NO

339 SEQ ID NO

119 SEQ ID NO

125 SEQ ID NO

131 SEQ ID NO

137 SEQ ID NO

143 SEQ ID NO

149 SEQ ID NO

155 SEQ ID NO

161 SEQ ID NO

167 SEQ ID NO

173 SEQ ID NO

179 SEQ ID NO

185 SEQ ID NO

191 SEQ ID NO

197 SEQ ID NO

274 SEQ ID NO

280 SEQ ID NO

286 SEQ ID NO

292 SEQ ID NO

298 SEQ ID NO

304 SEQID NO

310 SEQ ID NO

316 SEQ ID NO

322 SEQ ID NO

328 SEQ ID NO

334 SEQ ID NO

340 SEQ ID NO

343, SEQ ID NO: 344, SEQ ID NO: 377, SEQ ID NO: 379, and SEQ ID NO

114, SEQ ID NO 120, SEQ ID NO 126, SEQ ID NO 132, SEQ ID NO 138, SEQ ID NO 144, SEQ ID NO 150, SEQ ID NO 156, SEQ ID NO 162, SEQ ID NO 168, SEQ ID NO 174, SEQ ID NO 180, SEQ ID NO 186, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 275, SEQ ID NO 281, SEQ ID NO 287, SEQ ID NO 293, SEQ ID NO 299, SEQ ID NO 305, SEQ ID NO 311, SEQ ID NO 317, SEQ ID NO 323, SEQ ID NO 329, SEQ ID NO 335, SEQ ID NO 341, SEQ ID NO 384.

115 SEQ ID

121 SEQ ID

127 SEQ ID

133 SEQ ID

139 SEQ ID

145 SEQ ID

151 SEQ ID

157 SEQ ID

163 SEQ ID

169 SEQ ID

175 SEQID

181, SEQ ID

187 SEQ ID

193 SEQID

199 SEQID

276 SEQ ID

282 SEQ ID

288 SEQ ID

294 SEQ ID

300 SEQ ID

306 SEQ ID

312 SEQ ID

318 SEQ ID

324 SEQ ID

330 SEQ ID

336 SEQ ID

342 SEQ ID

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region having at least 90% sequence identity with amino acid residues corresponding to amino acids 1 to about 144, amino acids 1 to about 239, amino acids 1 to about 296, amino acids 1 to about 348, amino acids 1 to about 382, amino acids 1 to about 422, amino acids 1 to about 442 SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids from about 146 to about 483, amino acids from about 241 to about 483, amino acids from about 297 to about 483, amino acids from about 349 to about 4 83, amino acids from about 383 to about 483, amino acids from about 423 to about 483, or amino acids from about 443 to about 483 of SEQ ID NO: 6.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids 1 to about 144 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids from about 146 to 483 of SEQ ID NO: 6.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids 1 to about 239 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids from about 241 to 483 of SEQ ID NO: 6.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids 1 to about 296 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids from about 297 to about 483 of SEQ ID NO: 6.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids 1 to about 348 of

SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids from about 349 to

483 of SEQ ID NO: 6.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids 1 to about 382 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids from about 383 to 483 of SEQ ID NO: 6.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids 1 to about 422 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids from about 423 to 483 of SEQ ID NO: 6.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids 1 to about 442 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region with at least 90% sequence identity to amino acid residues corresponding to amino acids from about 443 to 483 of SEQ ID NO: 6.

In some embodiments, provided are polynucleotides encoding chimeric polypeptides comprising a) an N-terminal region comprising amino acids 1 to about 144, amino acids 1 to about 239, amino acids 1 to about 296, amino acids 1 to about 348, amino acids from 1 to about 382, amino acids 1 to about 422, amino acids 1 to about 442 of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; and b) a C-terminal region containing amino acids from about 146 to about 483, amino acids from about 241 to about 483, amino acids from about 297 to about 483, amino acids from about 349 to about 483, amino acids from about 383 to about 483, amino acids from about 423 to about 483 or amino acids from about 443 to about 483 of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

In some embodiments, provided are polynucleotides encoding chimeric polypeptides comprising a) an N-terminal region containing amino acids 1 to about 144 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 146 to 483 of SEQ ID NO: 6.

In some embodiments, provided are polynucleotides encoding chimeric polypeptides comprising a) an N-terminal region containing amino acids 1 to about 239 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 241 to 483 of SEQ ID NO: 6.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region containing amino acids 1 to about 296 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 297 to about 483 of SEQ ID NO: 6.

In some embodiments, provided are polynucleotides encoding chimeric polypeptides comprising a) an N-terminal region containing amino acids 1 to about 348 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 349 to 483 of SEQ ID NO: 6.

In some embodiments, polynucleotides are provided encoding chimeric polypeptides comprising a) an N-terminal region containing amino acids 1 to about 382 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 383 to 483 of SEQ ID NO: 6.

In some embodiments, provided are polynucleotides encoding chimeric polypeptides comprising a) an N-terminal region containing amino acids 1 to about 422 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 423 to 483 of SEQ ID NO: 6.

In some embodiments, provided are polynucleotides encoding chimeric polypeptides comprising a) an N-terminal region containing amino acids 1 to about 442 of SEQ ID NO: 2 or SEQ ID NO: 4; and b) a C-terminal region containing amino acids from about 443 to 483 of SEQ ID NO: 6.

In some embodiments, the IPD090 polynucleotide encodes an IPD090 polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID

NO: 379 or SEQ ID NO: 384 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,

25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,

55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,

85, 85, 86, 87, 88, 89, 90 or more amino acid substitutions compared to the native amino acid at the corresponding position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384.

In some embodiments, the IPD090 polynucleotide encodes an IPD090 polypeptide comprising the amino acid sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49,

50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72 amino acid substitutions, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384.

In some embodiments, the IPD090 polynucleotide encodes an IPD090 polypeptide comprising the amino acid sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, or 48 amino acid substitutions, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO : 384.

In some embodiments, the IPD090 polynucleotide encodes an IPD090 polypeptide comprising the amino acid sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 20, 21, 22, 23, or 24 amino acid substitutions, in any combination, compared to the native amino acid at the corresponding position of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, or SEQ ID NO: 384.

In some embodiments, the IPD090 polynucleotide encodes an IPD090 polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID

NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID

NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID

NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID

NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID

NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID

NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID

NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID

NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID

NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID

NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID

NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID

NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID

NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID

NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID

NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, SEQ ID

NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID

NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID

NO: 298, SEQ ID NO: 299, SEQ ID NO: 300, SEQ ID NO: 301, SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID

NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, SEQ ID NO: 309, SEQ ID

NO: 310, SEQ ID NO: 311, SEQ ID NO: 312, SEQ ID NO: 313, SEQ ID NO: 314, SEQ ID NO: 315, SEQ ID

NO: 316, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 319, SEQ ID NO: 320, SEQ ID NO: 321, SEQ ID

NO: 322, SEQ ID NO: 323, SEQ ID NO: 324, SEQ ID NO: 325, SEQ ID NO: 326, SEQ ID NO: 327, SEQ ID

NO: 328, SEQ ID NO: 329, SEQ ID NO: 330, SEQ ID NO: 331, SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID

NO: 334, SEQ ID NO: 335, SEQ ID NO: 336, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID

NO: 340, SEQ ID NO: 341, SEQ ID NO: 342, SEQ ID NO: 343, SEQ ID NO: 344, SEQ ID NO: 377, SEQ ID

NO: 379, and SEQ ID NO: 384.

Embodiments also cover nucleic acid molecules encoding IPD090 polypeptide variants. The term variants of nucleic acid sequences encoding an IPD090 polypeptide includes sequences that encode for the IPD090 polypeptides disclosed herein, but which differ in conservative substitutions due to degeneracy of the genetic code, as well as sequences that are sufficiently identical, as discussed above. Naturally occurring allelic variants can be identified using well-known molecular biology techniques such as polymerase chain reaction (PCR) and hybridization techniques outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that

- 25 040502 were obtained, for example, using site-directed mutagenesis, but which still encode the disclosed IPD090 polypeptides discussed below.

The present invention provides isolated or recombinant polynucleotides that encode any of the IPD090 polypeptides disclosed herein. Those of ordinary skill in the art will readily appreciate that, due to the degeneracy of the genetic code, there are multiple nucleotide sequences encoding the IPD090 polypeptides of the present invention.

It will also be appreciated by one of ordinary skill in the art that changes can be introduced by mutating nucleic acid sequences that result in changes in the amino acid sequence of the encoded IPD090 polypeptides without altering the biological activity of the proteins. Thus, variant nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions and/or deletions to the corresponding nucleic acid sequence disclosed herein such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also covered by the present invention.

Alternatively, variant nucleic acid sequences can be generated by randomly introducing mutations in all or part of the coding sequence, such as by site-saturating mutagenesis, and the resulting mutants can be screened for their ability to confer pesticidal activity to identify mutants that retain activity. . After mutagenesis, the encoded protein can be recombinantly expressed and protein activity can be determined using standard assay techniques.

The polynucleotides of the present invention and fragments thereof are optionally used as substrates for a variety of recombination and recurrent recombination reactions, in addition to the standard cloning methods set forth in, for example, Ausubel, Berger and Sambrook, i. to obtain additional homologues of pesticidal polypeptides and their fragments with the desired properties. A number of such reactions are known, including those developed by the authors of the present invention and their collaborators. Methods for producing a variant of any nucleic acid provided herein involve recurrent recombination of such a polynucleotide with a second (or more) polynucleotide, thus obtaining a library of variant polynucleotides is also an embodiment of the present invention, as are the resulting libraries, cells containing libraries and any recombinant polynucleotide obtained by such methods. Additionally, such methods optionally provide for the selection of a variant polynucleotide from such libraries based on pesticidal activity, as is, where such recurrent recombination is carried out in vitro or in vivo.

A number of protocols for creating diversity, including protocols for recurrent recombination of nucleic acids, are available and are fully described in the prior art. To obtain one or more variants of a nucleic acid or set of nucleic acids, as well as variants of encoded proteins, the procedures can be used alone and/or in combination. Individually and collectively, these procedures provide reliable, widely used methods for generating diversified nucleic acids and sets of nucleic acids (including, for example, nucleic acid libraries) useful, for example, for the construction or rapid evolution of nucleic acids, proteins, metabolic pathways, cells. and/or organisms with new and/or improved characteristics.

Although the following discussion makes a distinction and classification for clarity, it will be appreciated that methodologies are often not mutually exclusive. Moreover, the various methods can be used singly or in combination, simultaneously or sequentially, to access different sequence variants.

Any of the diversification procedures described herein may result in the creation of one or more nucleic acids that can be selected or screened for nucleic acids that have or provide the desired properties, or nucleic acids that encode proteins that have the desired properties. or providing them. After diversification using one or more of the methods described herein or otherwise available to a person skilled in the art, any resulting nucleic acids can be selected for the desired activity or property, for example, pesticidal activity or such activity at the desired pH, etc. d. This may include identifying any activity that can be detected, eg in an automated or automated format, by any of the assays known in the art, see, for example, discussion of screening for insecticidal activity below. It is possible to evaluate a number of related (or even unrelated) properties sequentially or simultaneously, at the discretion of the practitioner.

Descriptions of a number of diversification procedures to generate modified nucleic acid sequences, such as sequences encoding polypeptides with pesticidal activity or fragments thereof, are found in the following publications and literature references,

- 26 040502 cited in them: Soong, et al., (2000) Nat Genet 25(4):436-439; Sterner, et al., (1999) Tumor Targeting 4:14; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang, et al., (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999) Curr Opin Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol 17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al., (1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS uSa 94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733; Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) Nat Biotechnol 14:315-319; Gates, et al., (1996) J Mol Biol 255:373386; Stemmer, (1996) Sexual PCR and Assembly PCR in The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457; Crameri and Stemmer, (1995) BioTechniques 18:194-195; Stemmer, et al. , (1995) Gene, 164:49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995) Bio/Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 and Stemmer, (1994) PNAS USA 91:10747-10751.

Mutational methods for creating diversity include, for example, site-directed mutagenesis (Ling, et al., (1997) Anal Biochem 254 (2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374 ; Smith, (1985) All Rev Genet 19:423-462; Botstein and Shortle, (1985) Science 229:1193-1201; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) The efficiency of oligonucleotide directed mutagenesis in Nucleic Acids & Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin)); mutagenesis using uracil-containing matrices (Kunkel, (1985) PNAS USA 82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass, et al., (1988) Science 242:240- 245); Oligonucleotide directed mutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500; Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller and Smith, (1982) Nucleic Acids Res 10: 6487-6500), phosphorothioate-modified DNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764; Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988) Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res 16:803-814) ; mutagenesis using gapped duplex DNA (Kramer, et al., (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol 154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, et al. (1988) Nucl Acids Res 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, et al., (1984) Cell 38:879-887), mutagenesis using repair deficient host strains (Carter, et al., (1985) Nucl Acids Res 13 :4431-4443 and Carter, (1987) Methods in Enzymol 154:382-403), deletion mutagenesis (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115), restriction-selection and restriction-purification (Wells, et al ., (1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total gene synthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar and Khorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene 34:315-323 and Grundstrom, et al., (1985) Nucl Acids Res 13:3305-3316), double strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech 4:450-455). Additional information on many of the above methods can be found in Methods Enzymol, Volume 154, which also provides helpful troubleshooting guides for various mutagenesis methods.

Additional details regarding the various ways to create diversity can be found in the following US Patents, PCT Publications, and EPO Publications: US Patent No. 5,723,323; US Patent No. 5,763,192; US Patent No. 5,814,476; US Patent No. 5,817,483; , US Pat. No. 5,976,862, US Pat. No. 5,605,793, US Pat. No. 5,811,238, US Pat. No. 5,830,721, US Patent No. 5,834,252, US Pat. WO 1999/41402 WO 1999/41383 WO 1999/41369 WO 1999/41368 EP 752008 EP 0932670 WO 1999/23107 WO 1999/21979 27230, WO 2000/00632, WO 2000/09679, WO 1998/42832, WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO 2000/18906/0WO 2000 42561, WO 2000/42559, WO 2000/42560, WO 2001/23401 and PCT/US01/06775.

Nucleotide sequences according to embodiments can also be used to isolate corresponding sequences from a bacterial source, including but not limited to species of the genus Pseudomonas. Thus, methods such as PCR, hybridization, etc. can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Embodiments cover sequences selected based on their sequence identity to the complete sequences set forth herein, or fragments thereof. Such sequences include sequences that are orthologues of the disclosed sequences. The term orthologues refers to genes derived from a common ancestral gene and found in different species due to speciation. Genes found in different species are considered orthologues if their nucleotide sequences and/or the protein sequences they encode have a significant degree of identity, as defined elsewhere in this document. Ortholog functions are often highly conserved across species.

In the case of a PCR based approach, oligonucleotide primers can be designed for use in PCR reactions to amplify appropriate DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Ways

- 27 040502 PCR primer design and PCR cloning are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York, hereinafter Sambrook. See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene specific primers, vector specific primers, partially mismatched primers, and the like.

To identify potential IPD090 polypeptides from bacterial collections, cell lysates can be screened with antibodies raised against IPD090 polypeptides and/or IPD090 polypeptides using Western blot and/or ELISA methods. This type of analysis can be performed in a high throughput manner. Positive samples can be further analyzed using various techniques such as purification and identification of proteins using antibodies. Methods for producing antibodies are well known in the art, as discussed below.

Alternatively, a mass spectrometry-based protein identification method using protocols from the literature can be used to identify IPD090 polypeptide homologues (Scott Patterson, (1998), 10.22, 1-24, Current Protocol in Molecular Biology, published by John Wiley & Son Inc. ). In particular, the LC-MS/MS-based protein identification method is used to link MS data of specified cell lysates or samples enriched in molecules of the desired molecular weight (cut from an SDS-PAGE gel with bands of molecular weight corresponding to IPD090 polypeptides), with sequence information for IPD090 polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 and their homologues. Any match in peptide sequences indicates the possibility of homologous proteins in the samples. Additional techniques (protein purification and molecular biology techniques) can be used to isolate the protein and identify homologue sequences.

Hybridization methods for screening cDNA or genomic libraries may use all or part of the pesticidal nucleic acid sequence. Methods for constructing such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra. The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides and may be labeled with a detectable group such as 32P or any other detectable marker such as other radioactive isotopes, a fluorescent compound, an enzyme or a cofactor. enzyme. Hybridization probes can be generated by labeling synthetic oligonucleotides based on the known nucleic acid sequence encoding the IPD090 polypeptide disclosed herein. Additionally, degenerate primers designed based on conservative nucleotide or amino acid residues in the nucleic acid sequence or encoded amino acid sequence can be used. Typically, a probe contains a region of a nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of the sequence. a nucleic acid encoding an IPD090 polypeptide of the present invention, or a fragment or variant thereof. Methods for making probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra, incorporated herein by reference.

For example, the entire nucleic acid sequence encoding an IPD090 polypeptide disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing to corresponding nucleic acid sequences encoding sequences similar to an IPD090 polypeptide and messenger RNAs. To achieve specific hybridization under various conditions, such probes include sequences that are unique and preferably are at least about 10 nucleotides in length, or at least about 20 nucleotides in length. Such probes can be used to amplify appropriate pesticide sequences from a selected organism by PCR. This technique can be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of seeded DNA libraries (either plaques or colonies; see e.g. Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N.Y.).

Hybridization of such sequences can be carried out under stringent conditions. Gesture terms

- 28 040502 Strict or stringent hybridization conditions are used herein to refer to conditions under which a probe will hybridize to its target sequence to a clearly greater extent than to other sequences (e.g., at least 2 times more than the background sequence). ). Stringent conditions are sequence dependent and will differ under different circumstances. By controlling the stringency of the hybridization and/or wash conditions, target sequences that are 100% complementary to the probe can be identified (homologous probe hybridization). Alternatively, the stringency conditions can be adjusted to provide some mismatch in the sequences in order to detect lower degrees of similarity (hybridization with a heterologous probe). In general, the probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

Compositions.

Also included are compositions containing at least one IPD090 polypeptide or chimeric IPD090 polypeptide of the present invention. In one embodiment, the composition comprises an IPD090 polypeptide of the present invention and an agriculturally acceptable carrier.

One embodiment of the present invention relates to a composition comprising an IPD090 polypeptide of the present invention and a strain of an entomopathogenic fungus selected from Metarhizium robertsii and Metarhizium anisopliae. In certain embodiments, the fungal entomopathogen is a spore, microsclerotium, or conidium. In some embodiments, the implementation of the fungal entomopathogen is characterized by insecticidal activity.

In one embodiment, the present invention relates to a composition for increasing resistance to a pest, pathogen or insect pest of a plant, or for improving the health and/or yield of a plant, comprising an IPD090 polypeptide of the present invention and one or more strains of entomopathogenic fungi selected from the group, consisting of Metarhizium anisopliae 15013-1 (NRRL 67073), Metarhizium robertsii 23013-3 (NRRL 67075), Metarhizium anisopliae 3213-1 (NRRL 67074) or any combination thereof. In another embodiment, the present invention provides a composition comprising an IPD090 polypeptide of the present invention, an agriculturally acceptable carrier, and a fungal entomopathogen selected from the group consisting of Metarhizium anisopliae 15013-1, Metarhizium robertsii 23013-3, Metarhizium anisopliae 3213- 1 or any combination thereof. In a further embodiment, the fungal entomopathogen is a spore, conidium, or microsclerotium. In another embodiment, the present invention relates to a composition comprising an IPD090 polypeptide of the present invention and one or more strains of entomopathogenic fungi selected from the group consisting of Metarhizium anisopliae 15013-1 (NRRL 67073), Metarhizium robertsii 23013-3 (NRRL 67075), Metarhizium anisopliae 3213-1 (NRRL 67074), mutants of these strains, a metabolite or combination of metabolites produced by a strain disclosed herein that has insecticidal activity against a plant pest, pathogen or insect, or any combination thereof.

Antibodies.

Also covered are antibodies to an IPD090 polypeptide according to embodiments, or variants or fragments thereof. The antibodies of the present invention include polyclonal and monoclonal antibodies, as well as fragments thereof, which retain their ability to bind to the IPD090 polypeptide found in the insect gut. An antibody, monoclonal antibody, or fragment thereof is said to be capable of binding a molecule if it is capable of specifically reacting with the molecule, thereby binding the molecule to the antibody, monoclonal antibody, or fragment thereof. The term antibody (Ab) or monoclonal antibody (Mab) is intended to include intact molecules as well as fragments or binding sites or domains (such as, for example, Fab and F(ab)2 fragments) that are capable of binding a hapten. Typically, such fragments are obtained by proteolytic cleavage, for example with papain or pepsin. Alternatively, hapten-binding fragments can be generated by the use of recombinant DNA technology or by synthetic chemistry. As a rule, methods for obtaining antibodies according to the present disclosure of the invention from the prior art. For example, see Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory, N.Y. (1988), as well as the references cited therein. Standard reference works outlining the general principles of immunology include Klein, J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Sons, N.Y. (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyzes, Plenum Press, N.Y. (1980) and Campbell, Monoclonal Antibody Technology, in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon, et al., (eds.), Elsevier, Amsterdam (1984). See also US Pat. Nos. 4,196,265; 4609893; 4713325; 4714681; 4716111; 4,716,117 and 4,720,459. Antibodies to IPD090 polypeptides, or antigen-binding portions thereof, can be generated by a number of techniques, including conventional monoclonal antibody techniques, such as the standard somatic cell hybridization technique of Kohler and Milstein, (1975) Nature 256:495. You can also use other

- 29 040502 techniques for obtaining a monoclonal antibody, such as viral or oncogenic transformation of B-lymphocytes. The animal-based system for producing hybridomas is a mouse-based system. Immunization protocols and procedures for isolating splenocytes from immunized animals for fusion are known in the art. Fusion partners (eg, mouse myeloma cells) and fusion procedures are also known. The antibody and monoclonal antibodies of the present invention can be obtained by using the IPD090 polypeptide as antigens.

A kit is provided to detect the presence of an IPD090 polypeptide or to detect the presence of a nucleotide sequence encoding an IPD090 polypeptide in a sample. In one embodiment, the kit contains antibody-based reagents to detect the presence of an IPD090 polypeptide in a tissue sample. In another embodiment, the kit contains labeled nucleic acid probes used to detect the presence of one or more polynucleotides encoding an IPD090 polypeptide. The kit also contains the appropriate reagents and controls for performing the detection method, as well as instructions for using the kit.

Identification and isolation of the receptor.

Also covered are receptors for an IPD090 polypeptide according to embodiments, or variants or fragments thereof. Methods for identifying receptors are well known in the art (see Hofmann, et. al., (1988) Eur. J. Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. 27277 -27282) can be used to identify and isolate the receptor that recognizes the IPD090 polypeptide using brush-border membrane vesicles from susceptible insects. In addition to the radioactive labeling method given in the references mentioned, the IPD090 polypeptide can be labeled with a fluorescent dye and other standard labels such as streptavidin. Brush border membrane vesicles (BBMVs) of susceptible insects such as soybean armyworm and stink bugs can be prepared according to the protocols given in the references and separated on SDS-PAGE gel and transferred to a suitable membrane. A labeled IPD090 polypeptide can be incubated with a membrane containing blotted BBMVs, and the labeled IPD090 polypeptide can be identified using labeled reporters. Identification of the band(s) of the protein(s) that interact with the IPD090 polypeptide can be performed by gas phase sequencing of the N-terminal amino acid residues or by a mass spectrometry-based protein identification method (Patterson, (1998) 10.22, 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc). Once the protein has been identified, the corresponding gene can be cloned from a genomic DNA or cDNA library of susceptible insects and the binding affinity can be assessed directly with the IPD090 polypeptide. The receptor function in conferring the insecticidal activity of the IPD090 polypeptide can be confirmed by performing the RNAi gene knockout method (Rajagopal, et al., (2002) J. Biol. Chem. 277:4684946851).

Nucleotide constructs, cassettes and expression vectors.

The use of the term nucleotide constructs herein is not intended to limit the embodiments to nucleotide constructs containing DNA. Those skilled in the art will appreciate that nucleotide constructs, in particular polynucleotides and oligonucleotides consisting of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be used in the methods disclosed herein. Nucleotide constructs, nucleic acids, and nucleotide sequences according to embodiments further encompass all complementary forms of such constructs, molecules, and sequences. In addition, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences that can be used in the methods of the embodiments to transform plants, including, but not limited to, those consisting of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides have both naturally occurring molecules and synthetic counterparts. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

An additional embodiment relates to a transformed organism, such as an organism selected from plant or insect cells, bacteria, yeast, baculovirus, protozoa, nematodes, and algae.

The transformed organism contains a DNA molecule according to embodiments, an expression cassette containing a DNA molecule, or a vector containing an expression cassette, which can be stably integrated into the genome of the transformed organism.

The sequences of the embodiments are provided as part of DNA constructs for expression in the organism of interest. The constructs will include 5' and 3' regulatory sequences operably linked to the sequence of the embodiments. The term operably linked as used in this document refers to

- 30 040502 to a functional link between the promoter and the second sequence, where the promoter sequence initiates and mediates the transcription of the DNA sequence corresponding to the second sequence. In general, operably linked means that the linked nucleic acid sequences are contiguous and optionally join two protein-coding regions in the same reading frame. The construct may further comprise at least one additional gene to be introduced into the organism by co-transformation. Alternatively, additional gene(s) may be provided in multiple DNA constructs.

Such a DNA construct is provided with multiple restriction sites to insert the sequence of the gene encoding the IPD090 polypeptide of the present invention, the transcription of which will be regulated by regulatory regions. The DNA construct may further comprise selectable marker genes.

Typically, in the direction of transcription from the 5' to the 3' end, the DNA construct will include: a transcription and translation initiation site (i.e., a promoter), a DNA sequence according to embodiments, and a transcription and translation termination site (i.e., a termination site). ) that function in the host organism. The transcription initiation site (ie, promoter) may be native, analogous, foreign, or heterologous with respect to the host organism and/or sequence according to the embodiments. In addition, the promoter may be a natural sequence or, alternatively, a synthetic sequence. The term foreign, as used herein, indicates that the promoter is not found in the native organism into which the promoter is introduced. If the promoter is foreign or heterologous to the sequence according to the embodiments, then it is assumed that the promoter is not a native or naturally occurring promoter for the operably linked sequence according to the embodiments. A chimeric gene, as used herein, contains a coding sequence operably linked to a transcription initiation site that is heterologous to the coding sequence. If the promoter is a native or naturally occurring sequence, then the expression of the operably linked sequence is altered from that of the wild type, resulting in a change in phenotype.

In some embodiments, the DNA construct contains a polynucleotide encoding an IPD090 polypeptide according to the embodiments.

In some embodiments, the DNA construct contains a polynucleotide encoding a chimeric IPD090 polypeptide according to the embodiments.

In some embodiments, the DNA construct comprises a polynucleotide encoding a fusion protein comprising an IPD090 polypeptide according to the embodiments.

In some embodiments, the DNA construct comprises a polynucleotide comprising a first coding sequence encoding the N-terminal region of a first IPD090 polypeptide of the present invention and a second coding sequence encoding the C-terminal region of a second IPD090 polypeptide of the present invention.

In some embodiments, the DNA construct comprises a polynucleotide encoding the polypeptide of SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, or SEQ ID NO: 388. In some embodiments, the DNA construct comprises a polynucleotide of SEQ ID NO: : 381, SEQ ID NO: 382 or SEQ ID NO: 383.

In some embodiments, the DNA construct may also include a transcriptional enhancer sequence. As used herein, the term enhancer refers to a DNA sequence that can stimulate the activity of a promoter, which can be a characteristic element of the promoter or a heterologous element inserted to increase the level of activity or tissue specificity of the promoter. Various enhancers are known in the art, for example, introns with properties to improve gene expression in plants can also be used (U.S. Patent Application Publication No. 2009/0144863, ubiquitin intron (i.e., maize ubiquitin intron 1 (see, for example, the NCBI sequence S94464)), omega enhancer or omega bar enhancer (Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9:1685-96), and the enhancers of US Pat. No. 7,803,992, each of which is incorporated by reference. U.S. Patent No. US8785612 discloses a sugar cane badnavirus (SCBV) transcriptional enhancer.

The above list of transcriptional enhancers is not intended to be limiting. In embodiments, any suitable transcriptional enhancer can be used.

The termination site may be native to the transcription initiation site, may be native to the operably linked DNA sequence of interest, may be native to the host plant, or may be derived from another source (i.e. foreign or heterologous to the promoter, sequence of interest). interest, host plant, or any combination thereof).

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Suitable termination sites are available from the A. tumefaciens Ti plasmid, such as the termination sites for the octopine synthase and nopaline synthase genes. See also Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) Nucleic Acid Res. 15:9627-9639. Other suitable transcription terminators for transgene expression in plants include the MYB2, KTI1, PIP1, EF1A2 and MTH1 transcription terminators from US8741634.

If necessary, the nucleic acid can be optimized to enhance expression in the host organism. Thus, if the host organism is a plant, synthetic nucleic acids can be synthesized using plant-preferred codons to enhance expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-11, which discusses the use of the preferred host. For example, while the nucleic acid sequences of the embodiments can be expressed in both monocot and dicot plant species, the sequences can be modified to accommodate monocot or dicot specific preferences and GC content preferences if preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the maize preference for a particular amino acid can be derived from the known maize gene sequences. 4 from Murray, et al., cited above. Methods for synthesizing plant-preferred genes are available in the art. See, for example, Murray, et al., (1989) Nucleic Acids Res. 17:477-498 and Liu H et al. Mol Bio Rep 37:677-684, 2010, incorporated herein by reference. A table of maize uses can also be found on the website at kazusa.or.jp//cgi-bin/show.cgi?species=4577, which can be accessed using the prefix www.

A Glycine max application chart can be found on the website at kazusa.or.jp//cgibin/show.cgi?species=3847&aa=l&style=N, which can be accessed using the prefix www.

In some embodiments, the recombinant nucleic acid molecule encoding the IPD090 polypeptide has codons optimized for maize.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include the elimination of sequences encoding false polyadenylation signals, exon and intron splicing site signals, transposon-like repeats, and other well-characterized sequences that can be detrimental to gene expression. The content of GC in the sequence can be adjusted to levels that are average for a given cellular host, calculated taking into account known genes expressed in the host cell. The term host cell as used herein refers to a cell that contains a vector and that supports replication and/or expression of putative expression vectors. The host cells can be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells, or monocot or dicot plant cells. An example of a monocot plant host cell is a maize host cell. If possible, the sequence is modified to avoid the formation of predicted mRNA hairpin secondary structures.

Expression cassettes may additionally contain 5' leader sequences. Such leader sequences can enhance translation. Translation leader sequences are known in the art and include picornavirus leader sequences, for example the EMCV (5' non-coding region of encephalomyocarditis virus) leader sequence (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126 -6130); potyvirus leader sequences, e.g. TEV (Tobacco Etching Virus) leader (Gallie, et al., (1995) Gene 165(2):233-238), MDMV (maize dwarf mosaic virus) leader, heavy chain binding protein sequence human immunoglobulin (BiP) (Macejak, et al., (1991) Nature 353:90-94); an untranslated leader sequence from alfalfa mosaic virus envelope protein mRNA (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus (TMV) leader (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and maize chlorosis mosaic virus (MCMV) leader (Lommel, et al., (1991) Virology 81:382-385). See also Delia Cioppa et al. (1987) Plant Physiology 84:965968. Such constructs may also contain a signal sequence or leader sequence to facilitate translationally coupled or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus.

The term signal sequence as used herein refers to a sequence known or expected to result in translational-coupled or post-translational transport of a peptide across a cell membrane. In eukaryotes, this usually involves secretion in the Golgi vesicles, with glycosylation occurring to some extent.

- 32 040502 degrees. Bacterial insecticidal toxins are often synthesized as protoxins that are activated by proteolysis in the gut of the target pest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments, the implementation of the signal sequence is located in the native sequence or can be obtained from the sequence according to the variants of implementation. The term leader sequence as used herein refers to any sequence that, when translated, results in an amino acid sequence capable of driving translation-coupled transport of a peptide chain into an intracellular organelle. Thus, this provides for leader sequences that target transport and/or glycosylation through passage to the endoplasmic reticulum, passage to vacuoles, plastids, including chloroplasts, mitochondria, and the like. Nuclear-encoded proteins targeted to the chloroplast thylakoid cavity compartment have a characteristic double transit peptide consisting of a stroma-targeting signal peptide and a cavity-targeting signal peptide. Information for targeting stroma is located in the part of the transit peptide closest to the amino-terminus. The signal peptide targeting the cavity is located in the part of the transit peptide closest to the carboxy-terminus and contains all the information for targeting the cavity. Recent studies in higher plant chloroplast proteomics have been successful in identifying numerous nuclear-encoded cavity proteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al. Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta 1503:350-356, 2001), while their signal peptide targeting the cavity can potentially be used in accordance with the present invention. Approximately 80 proteins from Arabidopsis, as well as homologous proteins from spinach and garden peas, are reported in Kieselbach et al., Photosynthesis Research, 78:249-264, 2003. In particular, in Table. 2 of this publication, which is incorporated herein by reference, discloses 85 chloroplast cavity proteins identified by their accession number (see also US Patent Application Publication No. 2009/09044298). In addition, a recently published preliminary version of the rice genome (Goff et al, Science 296:92-100, 2002) is a suitable source for information on a cavity-targeting signal peptide that can be used in accordance with the present invention.

Suitable chloroplast transit peptides (CTPs), well known to those skilled in the art, also include chimeric CTs containing, but not limited to, the N-terminal domain, central domain, or C-terminal domain of a CTP from Oryza 1-deoxy-D-xylose-5-phosphate synthase. sativa, superoxide dismutase Oryza sativa, soluble starch synthase Oryza sativa, NADP-dependent enzyme acting on malic acid, Oryza sativa, phospho-2-dehydro-3-deoxyheptonaldolase 2 Oryza sativa, L-ascorbate peroxidase 5 Oryza sativa, phosphoglucan-water-dikinase Oryza sativa, ssRUBISCO Zea Mays, Zea Mays beta-glucosidase, Zea Mays malate dehydrogenase, Zea Mays M-type thioredoxin (US Patent 9150625); chloroplast transit peptide from US Patent Application Publication No. US20130210114.

The gene for the IPD090 polypeptide to be targeted to the chloroplast can be optimized for expression in the chloroplast, taking into account differences in utilization between the plant nucleus and this organelle. Thus, nucleic acids of interest can be synthesized using chloroplast-preferred sequences.

Upon receipt of the expression cassette with different DNA fragments, manipulations can be carried out so as to obtain DNA sequences in the proper orientation and, if necessary, in the proper reading frame. To this end, adapters or linkers may be used to join DNA fragments, or other manipulations may be employed to provide suitable restriction sites, remove excess DNA, remove restriction sites, and the like. To this end, in vitro mutagenesis, repair with primers, restriction, hybridization, repeated substitutions, such as transitions and transversions, can be used.

In the practice of the embodiments, a number of promoters can be used. Promoters can be selected based on the desired result. Nucleic acids can be combined with constitutive, tissue-preferred, inducible, or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant cell host include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999/43838 and US Pat. No. 6,072,050; 35S CaMV basic promoter (Odell, et al., (1985) Nature 313:810-812); rice actin promoter (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin promoter (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730), US8168859, US8420797; a group of ubiquitin transcriptional regulatory elements and transcriptional expression regulatory elements disclosed in US9062316; an ALS promoter (US Pat. No. 5,659,026); and the like. The constitutive ADF1 soybean promoter is disclosed in US Patent Application Publication US20150184174. The constitutive soybean CCP1 promoter is disclosed in US Patent Application Publication US20150167011. Other constitutive promoters include, for example, those disclosed in US Pat. Nos. 5,608,149; 5608144; 5604121; 5569597; 5466785; 5399680;

- 33 040502

5268463; 5,608,142 and 6,177,611. Transcription initiation sites isolated from blueberry red ring spot virus (BRRV) are disclosed in US Patent US8895716. Transcription initiation sites isolated from cocoa shoot strain virus (CSSV) are disclosed in US Pat. No. US8962916.

Depending on the desired result, it may be useful to express the gene with an inducible promoter. Of particular interest in regulating the expression of nucleotide sequences according to embodiments in plants are wound-inducible promoters. Such wound-inducible promoters can respond to insect feeding damage and include the potato proteinase inhibitor (pin II) gene promoter (Ryan, (1990) All. Rev. Phytopath. 28:425-449; Duan, et al., ( 1996) Nature Biotechnology 14:494-498); wun1 and wun2, US Pat. No. 5,428,148; win1 and win2 (Stanford, et al., (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992) Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol. 22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); the MPI gene (Corderok, et al., (1994) Plant J. 6(2):141-150) and the like, incorporated herein by reference.

In addition, pathogen-inducible promoters can be used in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include promoters from pathogenesis-associated proteins (PR proteins) that are induced upon infection by a pathogen; e.g. PR proteins, SAR proteins, beta-1,3-glucanase, chitinases, etc. See, for example, Redolfi, et al., (1983) Neth. J. Plant Pathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and Van Loon, (1985) Plant Mol. Virol. 4:111-116. See also WO 1999/43819, incorporated herein by reference.

Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. sci. USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 and Yang, (1996) Proc. Natl. Acad. sci. USA 93:14972-14977. See also Chen, et al., (1996) Plant J. 10:955-966; Zhang, et al. , (1994) Proc. Natl. Acad. sci. USA 91:25072511; Warner, et al., (1993) Plant J. 3:191-201; Siebertz, et al., (1989) Plant Cell 1:961-968; US patent No. 5750386 (induced by nematodes); as well as the sources cited in these documents. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemically regulated promoters can be used to modulate gene expression in a plant through the use of an exogenous chemical regulator. Depending on the purpose, the promoter can be a chemically inducible promoter, where the application of the chemical induces gene expression, or a chemically repressed promoter, where the application of the chemical represses gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophiles that are used as preemergence herbicides, and the tobacco PR-1a promoter, which activated by salicylic acid. Other chemically regulated promoters of interest include steroid sensitive promoters (see, e.g., glucocorticoid inducible promoter in Schena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz, et al., (1991) Mol. Gen. Genet. 227:229 -237 and US Pat. Nos. 5,814,618 and 5,789,156), incorporated herein by reference.

Tissue-preferred promoters can be used to target increased expression of the IPD090 polypeptide in a particular plant tissue. Tissue-preferred promoters include those discussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gene Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ. 20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. sci. USA 90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Additional tissue-specific promoters are known in the art, including those from US Pat. Nos. US8816152 and US9150624. Such promoters can be modified, if necessary, to obtain weak expression.

Leaf-preferred promoters are known in the art. See, for example, Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994) Plant Physiol. 105:357-67;

Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993) Proc. Natl. Acad. sci. USA 90(20):9586-9590.

Preferred promoters or root-specific promoters for the US patent application are known and may be selected from many available in the literature or de novo isolated from

- 34 040502 different compatible types. See, for example, Hire, et al., (1992) Plant Mol. Biol. 20(2):207-218 (soy root-specific glutamine synthetase gene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (root-specific regulatory element in bean GRP 1.8 gene); Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene from Agrobacterium tumefaciens) and Miao, et al., (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in soybean roots and root nodules). See also Bogusz, et al., (1990) Plant Cell 2(7): 633-641, which describes two root-specific promoters isolated from hemoglobin genes from the non-legume nitrogen-fixing plant Parasponia andersonii and related Trema tomentosa, a non-legume and non-nitrogen fixing plant. The promoters of these genes were linked to the β-glucuronidase reporter gene and introduced into both the non-legume Nicotiana tabacum and the legume Lotus corniculatus, and in both cases the root-specific promoter activity was maintained. Leach and Aoyagi (1991) describe their analysis of promoters that provide high expression of the roIC and roID genes of Agrobacterium rhizogenes that induce root outgrowth (see Plant Science (Limerick) 79(1):69-76). They concluded that in these promoters, the enhancer and tissue-preferred DNA determinants are separated. Teeri et al. (1989) used a gene fusion with lacZ to show that the gene from Agrobacterium T-DNA encoding octopine synthase is particularly active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and is upregulated upon injury. in leaf tissue, which is a particularly desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8 (2): 343-350). The TR1' gene fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772) and the rolB promoter (Capana, et al., (1994) Plant Mol Biol 25(4):681-691 See also U.S. Patent Nos. 5837876, 5750386, 5633363, 5459252, 5401836, 5110732 and 5023179. US Patent No. US20160097054 discloses a sorghum root-preferred PLTP promoter US Patent Application Publication No. US20160145634 discloses a sorghum root-preferred TIP2-3 promoter US8916377 discloses a copro root-preferred RCc3 promoter.

Seed-preferred promoters include both seed-specific promoters (promoters that are active during seed development, such as seed storage protein promoters) and seed germination promoters (promoters that are active during seed germination). See Thompson, et al., (1989) BioEssays 10:108, incorporated herein by reference. Such seed-preferred promoters include, without limitation, Cim1 (cytokinin inducible transcript); CZ19B1 (19 kDa maize zein) and milps (myo-inositol-1 phosphate synthase); (See US patent No. 6225529, incorporated in this document by reference). Gammazein and Glb-1 are endosperm-specific promoters. In the case of dicotyledonous plants, seed-specific promoters include, but are not limited to, the Kunitz-type trypsin-3 inhibitor (KTi3) gene promoter (Jofuku and Goldberg, (1989) Plant Cell 1:1079-1093), bean β-phaseolin, napin, β-conglycinin , glycinin 1, soy lectin, cruciferin, etc. In the case of monocot plants, seed-specific promoters include, but are not limited to, promoters from the genes for zein 15 kDa, zein 22 kDa, zein 27 kDa, g-zein, waxy, shrunken 1, shrunken 2, maize globulin 1, etc. d. See also WO 2000/12733 which discloses seed-preferred promoters from the end1 and end2 genes, incorporated herein by reference. In dicotyledonous plants, seed-specific promoters include, but are not limited to, the seed coat protein gene promoter from Arabidopsis, pBAN; as well as promoters of early seed development genes from Arabidopsis, p26, p63 and p63tr (US Pat. Nos. 7,294,760 and 7,847,153). A promoter that is preferentially expressed in a particular tissue is expressed in that tissue to a greater extent than in at least one other plant tissue. Some tissue-preferred promoters have been shown to be expressed almost exclusively in a particular tissue.

If a low level of expression is desired, weak promoters will be used. In general, as used herein, the term weak promoter refers to a promoter that drives the expression of a coding sequence at a low level. By low expression is meant levels from about 1/1000 transcripts to about 1/100000 transcripts to about 1/500000 transcripts. Alternatively, it is understood that the term weak promoters also encompasses promoters that drive expression in only a small number of cells and do not drive others, while providing an overall low level of expression. If a promoter drives expression at unacceptably high levels, then portions of the promoter sequence can be removed or modified to reduce expression levels.

Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 1999/43838 and US Pat. No. 6,072,050), the core 35S promoter of CaMV, and the like. Other constitutive promoters include, for example, those disclosed in US Pat. Nos. 5,608,149; 5608144; 5604121;

5569597; 5466785; 5399680; 5268463; 5608142, 6177611 and 8697857, incorporated herein by reference.

Chimeric or hybrid promoters are also known in the art, including those disclosed in US Pat. Nos. US8846892, US8822666 and US9181560.

The above list of promoters is not intended to be limiting. In embodiments, any suitable promoter may be used.

Typically, the expression cassette will contain a selectable marker gene for selecting transformed cells. Selectable marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as ammonium glufosinate, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate. (2,4-D). Additional examples of suitable selectable marker genes include, without limitation, genes encoding chloramphenicol resistance (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820); streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5:131137); bleomycin (Hille, et al., (1990) Plant Mol. Biol. 1:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol. 15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423); glyphosate (Shaw, et al., (1986) Science 233:478-481 and US Patent Applications Serial Nos. 10/004357 and 10/427692); phosphinothricin (DeBlock, et al., (1987) EMBO J. 6:2513-2518). See, in general, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. microbiol. 6:2419-2422; Barkley, et al., (1980) in The Operon, pp. 177220; Hu, et al., (1987) Cell 48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713722; Deuschle, et al., (1989) Proc. Natl. Acad. sci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. sci. USA 86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen, (1993) PhD thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. sci. USA 90:1917-1921; Labow, et al., (1990) Mol. cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. sci. USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. struc. Biol. 10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993) PhD thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill, et al., (1988) Nature 334:721-724. Such disclosures are incorporated herein by reference.

The above list of selectable marker genes is not intended to be limiting. In embodiments, any selectable marker gene can be used.

Plant transformation.

Methods according to embodiments include introducing the polypeptide or polynucleotide into a plant. Introduction, as used herein, means incorporating a polynucleotide or polypeptide into a plant such that the sequence enters the interior of the plant cell. The methods of the embodiments do not depend on the specific method of introducing the polynucleotide or polypeptide into the plant, it is only necessary that the polynucleotide or polypeptides penetrate into at least one cell of the plant. Methods for introducing a polynucleotide and/or polypeptides into plants are known in the art, including, without limitation, stable transformation methods, transient transformation methods, and virus-mediated transformation methods.

Stable transformation, as used herein, means that a nucleotide construct introduced into a plant is integrated into the plant's genome and can be inherited by its offspring. Transient transformation, as used herein, means that a polynucleotide is introduced into a plant and it does not integrate into the plant's genome, or a polypeptide is introduced into the plant. The term plant as used herein refers to whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds, plant cells, plant parts for vegetative propagation, embryos, and their progeny. Plant cells may be differentiated or undifferentiated (eg, callus cells, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

Transformation protocols, as well as protocols for introducing nucleotide sequences into plants, may vary depending on the type of plant or plant cell, ie the class of monocots or dicots targeted for transformation. Suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium mediated transformation (US Pat.

- 36 040502 No. 5563055 and 5981840), direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for example, US patents No. 4945050; 5879918; 5886244 and 5932782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al., (1988) Biotechnology 6: 923-926) and transformation of the Lecl gene (WO 00/28058). For potato transformation, see Tu, et al., (1998) Plant Molecular Biology 37:829838 and Chong, et al., (2000) Transgenic Research 9:71-78.

Additional transformation procedures can be found in Weissinger, et al., (1988) All. Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-674 (soy); McCabe, et al., (1988) Bio/Technology 6:923-926 (soy).

Finer and McMullen, (1991) TL Vitro Cell Dev. Biol. 27P:175-182 (soy); Singh, et al., (1998) Theor. Appl. Genet. 96:319-324 (soy); Datta, et al., (1990) Biotechnology 8:736-740 (Figure).

Klein, et al., (1988) Proc. Natl. Acad. sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize).

US Patents No. 5240855; 5322783 and 5324646; Klein, et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al. , (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 311:763-764; US patent No. 5736369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al. , (1992) Theor. Appl. Genet. 84:560-566 (transformation mediated by piercing cells by shaking them in a microneedle suspension); D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li, et al. , (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (fig); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize with Agrobacterium tumefaciens); all of which are incorporated herein by reference.

In specific embodiments, the sequences of the embodiments can be introduced into a plant using a variety of transient transformation techniques. Such transient transformation methods include, without limitation, introducing an IPD090 polynucleotide or variants and fragments thereof directly into a plant, or introducing an IPD090 polypeptide transcript into a plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science 107:775-784, all of which are incorporated herein by reference. Alternatively, the plant can be transiently transformed with the IPD090 polynucleotide using techniques known in the art. Such techniques include the use of a viral vector system and precipitation of the polynucleotide in a manner that avoids subsequent release of the DNA. Thus, particle-bound DNA can be transcribed, but the frequency with which it is released for integration into the genome is greatly reduced. Such methods include the use of polyethyleneimine (PEI; Cat # Sigma P3143) coated particles.

Methods for the targeted insertion of a polynucleotide at a specific location in the plant genome are known in the art. In one embodiment, insertion of a polynucleotide at a desired location in the genome is performed using a site-specific recombination system. See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855 and WO 1999/25853, all of which are incorporated herein by reference. Briefly, a polynucleotide according to embodiments may be contained in a transfer cassette flanked by two non-identical recombination sites. The transfer cassette is introduced into a plant having a stably inserted target site in its genome that is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated into the target site. The polynucleotide of interest is thus integrated into a particular chromosomal position in the plant's genome.

Plant transformation vectors may consist of one or more DNA vectors necessary to achieve plant transformation. For example, it is common practice in the art to use plant transformation vectors that consist of more than one contiguous DNA segment. In the prior art, these vectors are often referred to as binary vectors. Binary vectors, as well as vectors with helper plasmids, are most commonly used in Agrobacterium-mediated transformation, where the size and complexity of the DNA segments required to achieve efficient transformation are very large, and separation of functions on single DNA molecules is preferred. Binary vectors typically contain a plasmid vector that contains cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is designed to be expressed in a plant cell, and a gene of interest (a gene designed to be expressed in a plant cell from which transgenic plants are to be obtained). Also in this plasmid vector are the sequences necessary for replication in bacteria. Operating in cis

- 37 040502 position sequences are arranged so as to enable efficient transfer to and expression in plant cells. For example, a selectable marker gene and a pesticide gene are located between the left and right borders. Often the second plasmid vector contains trans-acting factors that mediate the transfer of T-DNA from Agrobacterium into plant cells. This plasmid often contains virulence functions (Vir genes) that allow infection of Agrobacterium plant cells and DNA transfer by border cleavage and vir-mediated DNA transfer as understood in the art (Hellens and Mullineaux, (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains can be used for plant transformation (eg LBA4404, GV3101, EHA101, EHA105, etc.). The second plasmid vector is not necessary for plant transformation by other means such as microparticle bombardment, microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve the transfer of heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by the application of appropriate selection with a maximum threshold (depending on the selectable marker gene) to isolate transformed plant cells from the group of ^-transformed cell mass. Once the heterologous foreign DNA has been integrated into the plant cells, appropriate high threshold media selection is then applied to kill non-transformed cells and separate and propagate putatively transformed cells that survive this selection treatment by regularly transferring to fresh media. Through continued passaging and validation by appropriate selection, cells that have been transformed with the plasmid vector are identified and expanded. Molecular and biochemical methods can then be used to confirm the presence of an integrated heterologous gene of interest in the genome of the transgenic plant.

As a rule, the explants are transferred to a fresh portion of the same medium and cultured in the usual way. Subsequently, the transformed cells differentiate into shoots after being placed in a regeneration medium supplemented with a selection agent with a maximum threshold. The shoots are then transferred to a selective plant growth medium to produce rooted shoots or seedlings. The transgenic seedling is then grown to a mature plant and fertile seeds are obtained (eg, Hiei, et al., (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology 14:745-750). As a rule, the explants are transferred to a fresh portion of the same medium and cultured in the usual way. For a general description of techniques and methods for producing transgenic plants, see Ayres and Park, (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformed material contains a plurality of cells, both transformed and non-transformed cells are present in any part of the exposed target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limiting factor for the rapid production of transformed plant cells and the successful cultivation of transgenic plants.

Plants can be grown from cells that have been transformed according to conventional methods. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated, either using the same transformed line or other lines, and produce a hybrid with constitutive or inducible expression of the desired identified phenotypic characteristic. Two or more generations can be grown to ensure that the expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds are harvested to ensure that the expression of the desired phenotypic characteristic has been achieved.

Nucleotide sequences according to embodiments can be provided in a plant by bringing the plant into contact with a virus or viral nucleic acids. Typically, such methods involve inserting the nucleotide construct of interest into a viral DNA or RNA molecule. It is understood that the recombinant proteins of the embodiments may be initially synthesized as part of a viral polyprotein, which may later be processed by in vivo or in vitro proteolysis to produce the desired IPD090 polypeptide. It is also understood that such a viral polyprotein containing at least a portion of the IPD090 amino acid sequence according to the embodiments may have the desired pesticidal activity. Such viral polyproteins and the nucleotide sequences that encode them are encompassed by the embodiments. Methods for obtaining plants with nucleotide constructs and producing encoded proteins in plants that include viral DNA or RNA molecules are known in the art. See, for example, US Pat. Nos. 5,889,191; 5889190; 5866785; 5589367 and 5316931; incorporated into this document by reference.

- 38 040502

Methods for transforming chloroplasts are known in the art. See, for example, Svab, et al., (1990) Proc. Natl. Acad. sci. USA 87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. sci. USA 90:913917; Svab and Maliga, (1993) EMBO J. 12:601-606. The method is based on gene gun delivery of DNA containing a selectable marker and targeting the DNA into the plastid genome using homologous recombination. In addition, transformation of plastids can be accomplished by transactivating a silent transgene found in plastids by expressing, in a particular tissue, an RNA polymerase encoded by the nuclear genome and functioning in the plastid. Such a system has been reported in McBride, et al., (1994) Proc. Natl. Acad. sci. USA 91:7301-7305.

The embodiments further refer to the propagating material of the transformed plant of the embodiments, including but not limited to seeds, tubers, corms, bulbs, leaves, and root and shoot cuttings.

Embodiments can be used to transform any plant species, including but not limited to monocots and dicots. Examples of plants of interest include, without limitation, corn (Zea mays), Brassica sp. (e.g. B. napus, B. rapa, B. juncea), in particular Brassica species suitable as sources of plant seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum ( Sorghum bicolor, Sorghum vulgare), millet (e.g., pennisetum glaucum, millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius) ), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut species (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee tree (Coffea spp.), coconut tree (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), chocolate tree (Theobroma cacao), tea bush (Camellia sinensis), banana ( Musa spp.), avocado (Persea americana), fig (F icus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet varieties ( Beta vulgaris), sugarcane (Saccharum spp.), oat varieties, barley, vegetables, ornamentals and conifers.

Vegetables include tomato varieties (Lycopersicon esculentum), lettuce (e.g. Lactuca sativa), green bean varieties (Phaseolus vulgaris), lima bean varieties (Phaseolus limensis), pea varieties (Lathyrus spp.) and members of the Cucumis genus such as cucumber (C . sativus), cantaloupe (C. cantalupensis) and musk melon (C. melo). Ornamental plants include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum. Conifers that may be used in the practice of the embodiments include, for example, pine species such as fox pine (Pinus taeda), Elliot pine (Pinus elliotii), yellow pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and radiant (Pinus radiata); Pseudotsuga Menzies (Pseudotsuga menziesii); Canadian hemlock (Tsuga canadensis); blue spruce (Picea glauca); sequoia (Sequoia sempervirens); fir varieties such as sweet fir (Abies amabilis) and balsam fir (Abies balsamea), and thuja varieties such as giant thuja (Thuja plicata) and nootkatensis callitropsis (Chamaecyparis nootkatensis). Plants according to embodiments include crop plants (eg, corn, alfalfa, sunflower, brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.) such as corn and soybean plants.

Lawn grass varieties include, but are not limited to: annual bluegrass (Roa appia); annual ryegrass (Lolium multiflorum); oblate bluegrass (Poa compressa); red fescue Chuinga (Festuca rubra); bent grass (Agrostis tenuis); marsh bent grass (Agrostis palustris); desert wheatgrass (Agropyron desertorum); comb wheatgrass (Agropyron cristatum); long-leaved fescue (Festuca longifolia); meadow bluegrass (Poa pratensis); hedgehog team (Dactylis glomerata); perennial chaff (Lolium perenne); red fescue (Festuca rubra); white bent grass (Agrostis alba); common bluegrass (Poa trivialis); sheep fescue (Festuca ovina); awnless bonfire (Bromus inermis); cane fescue (Festuca arundinacea); meadow timothy (Phleum pratense); bent grass (Agrostis canina); spaced skewers (Puccinellia distans); Smith's wheatgrass (Agropyron smithii); pig fingered (Cynodon spp.); one-sided narrow furrower (Stenotaphrum secundatum); Zoysia (Zoysia spp.); noticeable buckwheat (Paspalum notatum); Athenian axonopus (Axonopus affinis); serpentine eremochloa (Eremochloa ophiuroides); Kikuyu (Pennisetum clandesinum); paspalum vaginal (Paspalum vaginatum); mosquito grass (Bouteloua gracilis); bison grass (Buchloe dactyloids); side oats (Bouteloua curtipendula).

Plants of interest include cereal plants which produce seeds of interest, oil plants and leguminous plants. Seeds of interest include seeds of cereals such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor bean, olive, etc. Leguminous plants include varieties of beans and varieties of peas. Beans include guar, carob, fenugreek, soybeans, common beans, cowpea, mung beans, lima beans, green beans, lentils, chickpeas, etc.

Assessment of plant transformation.

After introducing the heterologous foreign DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method for screening transformed cells, tissue, or shoots for the presence of an inserted gene at an early stage before planting in soil (Sambrook and Russell, (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY). PCR is carried out using oligonucleotide primers specific for the gene of interest or the original Agrobacterium-based vector sequences, etc.

Plant transformation can be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, (2001) supra). In general, total DNA is extracted from the transformant, cut with the appropriate restriction enzymes, fractionated on an agarose gel, and transferred to a nitrocellulose or nylon membrane. The membrane or blot is then analyzed with a probe, such as a 32P radioactively labeled target DNA fragment, to confirm integration of the introduced gene into the plant genome according to standard techniques (Sambrook and Russell, (2001) supra).

In Northern blot analysis, RNA is isolated from specific transformant tissues, fractionated on an agarose gel containing formaldehyde, and transferred to a nylon filter according to standard procedures commonly used in the art (Sambrook and Russell, (2001) supra). Then, the expression of the RNA encoded by the pesticide gene was tested by hybridization of the filter with a radioactive probe derived from the pesticide gene by methods known in the art (Sambrook and Russell, (2001) supra).

Western blots, biochemical assays, and the like can be performed on transgenic plants to confirm the presence of the protein encoded by the pesticide gene using standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more of the epitopes present. on the IPD090 polypeptide.

Ways to implement genome editing technologies in relation to plants.

In some embodiments, compositions based on the disclosed IPD090 polynucleotide can be introduced into the plant genome using genome editing technologies, or previously introduced IPD090 polynucleotides in the plant genome can be edited using genome editing technologies. For example, the disclosed polynucleotides can be introduced at a desired location in the plant genome by double strand break techniques such as TALEN, the use of meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. For example, the disclosed polynucleotides can be introduced at a desired location in the genome using the CRISPR-Cas system to create a site-specific insert. The desired region in the plant genome can be any desired target site for insertion, such as a region of the genome suitable for crossing; or it may be a target site located in the genomic window with a pre-existing feature of interest. An existing trait of interest can be either an endogenous trait or a previously introduced trait.

In some embodiments, where the disclosed IPD090 polynucleotide has been previously introduced into the genome, genome editing techniques can be used to alter or modify the introduced polynucleotide sequence. Site-specific modifications that can be introduced into compositions based on the disclosed IPD090 polynucleotide include those modifications made by any method of introducing a site-specific modification, including, without limitation, the use of oligonucleotides for gene repair (e.g., US Patent Publication 2013/0019349) , or the use of double strand break introducing technologies such as TALEN, the use of meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Such techniques can be used to modify a previously introduced polynucleotide by inserting, deleting, or substituting nucleotides within the introduced polynucleotide. Alternatively, double strand break techniques can be used to add additional nucleotide sequences to the injected polynucleotide. Additional sequences that may be added include additional expression elements such as enhancer and promoter sequences. In another embodiment, genome editing techniques can be used to place additional proteins with insecticidal activity in close proximity to compositions based on the disclosed IPD090 polynucleotide disclosed herein within the plant genome to create molecular packages of proteins with insecticidal activity.

The term altered target site, altered target sequence, modified target site, and modified target sequence, used interchangeably herein, means a target sequence disclosed herein that contains at least one change from an unchanged target sequence. Such changes include, for example, (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

Trait Bundling in a Transgenic Plant Transgenic plants may comprise a bundle of one or more of the insecticidal polynucleotides disclosed herein, with one or more additional polynucleotides resulting in the production or suppression of multiple polypeptide sequences.

Transgenic plants containing packets of polynucleotide sequences can be obtained either by traditional breeding methods, or by genetic engineering methods, or both. These methods include, without limitation, propagating individual lines each containing a polynucleotide of interest, transforming a transgenic plant containing the gene disclosed herein with an additional gene, and co-transforming genes from a single plant cell. As used herein, the term bundled refers to a condition in which multiple traits are present in a single plant (i.e. both traits are included in the nuclear genome, one trait is included in the nuclear genome and one trait is included in the plastid genome, or both traits are included in the plastid genome ). In one non-limiting example, packaged features provide a molecular package in which the sequences are physically adjacent to each other. The term trait as used herein refers to the phenotype associated with a particular sequence or groups of sequences.

Co-transformation of genes can be carried out using single transformation vectors containing multiple genes, or multiple vectors that carry single genes. If the sequences are packaged by plant genetic transformation, then the polynucleotide sequences of interest can be combined at any time and in any order. Features can be administered simultaneously in a co-transformation protocol with polynucleotides of interest represented by any combination of transformation cassettes. For example, if two sequences are to be administered, the two sequences may be contained in separate transformation cassettes (trans) or contained in the same transformation cassette (cis). Expression of these sequences may be driven by the same promoter or by different promoters. In certain cases, it may be necessary to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. It can be combined with any combination of other suppression or overexpression cassettes to produce the desired combination of traits in the plant. In addition, it is believed that polynucleotide sequences can be packaged at the desired location in the genome using a system for site-specific recombination. See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855 and WO 1999/25853, all of which are incorporated herein by reference.

In some embodiments, polynucleotides encoding an IPD090 polypeptide disclosed herein, alone or packaged with one or more additional insect resistance features, can be packaged with one or more additional input features (e.g., herbicide resistance, fungal resistance, resistance to viruses, stress tolerance, disease resistance, male sterility, stem vigor, etc.), or traits affecting production (e.g. increased yield, modified starch varieties, improved oil profile, balanced amino acids, high lysine content, or methionine, increased digestibility, improved fiber quality, drought tolerance, etc.). Thus, embodiments of a polynucleotide can be used to provide a complete set of agronomic traits that provide improved crop quality, with the ability to control any number of pests in a flexible and cost effective manner.

Suitable transgenes for bundling include, without limitation, those listed below.

1) Transgenes which confer resistance to insects or diseases and which encode the following:

(A) Plant disease resistance genes. Plant defense mechanisms are often activated through a specific interaction between the product of a disease resistance (R) gene in the plant and the product of the corresponding avirulence gene (Avr) in the pathogen. A plant variety can be transformed with a cloned resistance gene to develop plants that are resistant to specific pathogen strains. See, for example, Jones, et al., (1994) Science 266:789 (cloning of tomato Cf-9 gene conferring resistance to Cladosporium fulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene conferring resistance to tomato Pseudomonas syringae pv. encodes a protein kinase); Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene conferring resistance to Pseudomonas syringae), McDowell and Woffenden, (2003) Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) Transgenic Res. 11(6):567-82. plant resistant to disease

- 41 040502 nii, is a plant that is more resistant to a pathogen compared to a wild-type plant;

(B) genes encoding a Bacillus thuringiensis protein, a derivative thereof, or a synthetic polypeptide modeled on its basis. See, for example, Geiser, et al., (1986) Gene 48:109 which discloses the cloning and nucleotide sequence of the Bt delta-endotoxin gene. In addition, DNA molecules encoding delta-endotoxin genes are available from the American Type Culture Collection (Rockville, Maryland), for example, under ATCC® accession numbers 40098, 67136, 31995, and 31998. Other non-limiting examples of Bacillus thuringiensis transgenes designed with genetic engineering techniques are cited in the following patents and patent applications and are hereby incorporated by reference for this purpose: US Pat. Nos. 5,188,960; 5689052; 5880275; 5986177; 6023013, 6060594, 6063597, 6077824, 6620988, 6642030, 6713259, 6893826, 7105332; 7179965, 7208474; 7227056, 7288643, 7323556, 7329736, 7449552, 7468278, 7510878, 7521235, 7544862, 7605304, 7696412, 7629504, 7705216, 7772465, 7790846, 7858849, 9546378; publication of US patent application US20160376607 and WO 1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581 and WO 1997/40162.

Genes encoding pesticidal proteins can also be packaged, including but not limited to insecticidal proteins from Pseudomonas sp. protegens strain (formerly fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386: GenBank accession no. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric. Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals of Microbiology 59:45-50 and Li , et al., (2007) Plant Cell Tiss Organ Cult 89:159-168); insecticidal proteins from Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxinology Journal, 3:101118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069), U.S. Patent No. 6,048,838 and U.S. Patent No. 6,379,946 ; PIP-1 polypeptide from US Patent Application Publication US20140007292; AfIP-1A and/or AfIP-1B polypeptides from US Patent Application Publication US20140033361; PHI-4 polypeptide from US Patent Application Publication US20140274885 and US20160040184; PIP-47 polypeptide from PCT Publication No. WO2015/023846, PIP-72 polypeptide from US Patent Publication No. US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide from PCT Publication No. WO2015/120270; the PtIP-83 polypeptide from PCT Publication No. WO2015/120276; the PtIP-96 polypeptide from PCT Publication Serial No. PCT/US15/55502; polypeptide IPD079 from PCT Publication No. WO2017/023486; polypeptide IPD082 from patent document serial no. PCT/US16/65531; the IPD093 polypeptide from US Patent Document Serial No. 62/434020; the IPD080 polypeptide from US Patent Document Serial No. US62/411318; and δ-endotoxins, including but not limited to the δ-endotoxin genes of the Cry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18 classes , Cry19, Cry20, Cry21, Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry 54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, and Cry72 and B. thuringiensis cytolytic toxin genes Cyt1 and Cyt2. Representatives of these classes of insecticidal proteins from B. thuringiensis are well known to those skilled in the art (see Crickmore, et al., Bacillus thuringiensis toxin nomenclature (2011), available at lifesci.sussex.ac.uk/home/Neil Crickmore/Bt/, which can be accessed on the World Wide Web using the prefix www).

Examples of δ-endotoxins also include, without limitation, the Cry1A proteins of US Pat. mutant Cry1Ac from US9512187; DIG-3 or DIG-11 toxin (Cry protein variants with N-terminal deletion of α-helix 1 and/or α-helix 2, such as Cry1A) from US Pat. Cry1B from US Patent Application Publication Serial No. 10/525318, US Patent Application Publication No. US20160194364 and US Patent Nos. 9404121 and 8772577; Cry1B variants from PCT Publication No. WO2016/61197 and Serial No. PCT/US17/27160; Cry1C from US Pat. No. 6,033,874; Cry1F from US patents No. 518 8 960, 6218188; Cry1AF chimeras from US Pat. No. 7,070,982; 6962705 and 6713063); a Cry2 protein such as the Cry2Ab protein of US Pat. No. 7,064,249); a Cry3a protein, including but not limited to a genetically engineered hybrid insecticidal protein (eHIP) obtained by fusing unique combinations of variable regions and conserved units of at least two different Cry proteins, such as Cry3A with Cry1Aa or Cry1Ab (U.S. Patent No. US8309516 and US9522937); Cry4 protein; protein Cry5; Cry6 protein; Cry8 proteins from US Pat. Nos. 7,329,736; 7,449,552; 7,803,943; Cry9 protein, such as members of the Oy9A, Oy9B, Cry9C, Cry9D, Cry9E, and Cry9F families, including the Cry9 proteins of US Pat. Nos. 9,000,261 and 8,802,933 and US Pat. Cry15 protein from Naimov, et al., (2008) Applied and Environmental Microbiology 74:7145-7151; Cry22, Cry34Ab1 protein from US Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; truncated Cry34 protein from US Pat. No. US8816157; CryET33 and CryET34 protein from US Pat. homologues of CryET33 and CryET34 from the publication of patent applications

- 42 040502

US No. 2006/0191034, 2012/0278954 and PCT Publication No. WO 2012/139004; protein Cry35Ab1 from US patent No. 6083499, 6548291 and 6340593; Cry46 protein from US Pat. No. 9,403,881, Cry51 protein, Cry binary toxin; TIC901 or related toxin; TIC807 from US 2008/0295207; TIC853 from US Patent US8513493; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 from PCT US 2006/033867; engineered hemipteran toxic proteins from US Patent Application Publication No. US20160150795; TIC1498, TIC1415, TIC1497, TIC1886, TIC1925, TIC1414, TIC1885, TIC1922, TIC1422, TIC 1974, TIC2032, TIC2120, TIC1362 from US Patent US9238678; TIC2463 type protein from US Patent Application Publication No. US20150274786; TIC3668 type protein from US Patent Application Publication No. US20160319302; AXMI-027, AXMI-036 and AXMI-038 from US Patent No. 8236757; AXMI-031, AXMI039, AXMI-040, AXMI-049 from US7923602; AXMI-018, AXMI-020 and AXMI-021 from WO 2006/083891; AXMI-010 from WO 2005/038032; AXMI-003 from WO 2005/021585; AXMI-008 from US 2004/0250311; AXMI-006 from US 2004/0216186; AXMI-007 from US 2004/0210965; AXMI-009 from US 2004/0210964; AXMI-014 from US 2004/0197917; AXMI-004 from US 2004/0197916; AXMI-028 and AXMI-029 from WO 2006/119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 from WO 2004/074462; AXMI-150 from US Pat. No. 8,084,416; AXMI-205 from US20110023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI063 and AXMI-064 from US 2011/02 63488; AXMI-R1 and related proteins from US 2010/0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z from WO 2011/103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 from US Patent No. US9156895; AXMI-115, AXMI-113, AXMI-005, AXMI-163, and AXMI-184 from US Pat. No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 from US 2010/0298211; AXMI-066 and AXMI-076 from US2009/0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170 AXMI171 AXMI172 AXMI173 AXMI174 AXMI175 AXMI176 AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098,

AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109,

AXMI110, AXMI111, AXMI112, AXMI114, AXM116, AXMI117, AXMI118, AXMI119, AXMI120,

AXMI121, AXMI122, AXMI123, AXMI124, AXMI125, AXMI126, AXMI127, AXMI129, AXMI164,

AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 from US8461421; AXMI192 from US Patent US8461415; AXMI234 and AXMI235 from US Patent Application Publication No. US20150218583; AXMI281 from US Patent Application Publication No. US20160177332; AXMI422 from US Patent No. US8252872; and Cry proteins such as Cry1A and Oy3A with modified proteolytic cleavage sites from US Pat. No. 8,319,019; modified Cry3 from US Pat. No. US9109231; and Cry1Ac, Cry2Aa and Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication No. 2011/0064710. Cry proteins MP032, MP049, MP051, MP066, MP068, MP070, MP091S, MP109S, MP114, MP121, MP134S, MP183S, MP185S, MP186S, MP195S, MP197S, MP208S, MP209S, MP212S, MP234S,2 MP25 MP237S, MP242S, МР243, МР248, MP249S, МР251М, MP252S, МР253, MP259S, MP287S, MP288S, MP295S, MP296S, MP297S, MP300S, MP304S, MP306S, MP310S, MP312S, MP314S, MP319S, MP325S, MP326S, MP327S, MP328S,

MP334S, MP337S, MP342S, MP349S, MP356S, MP359S, MP360S, MP437S, MP451S, MP452S, MP466S,

MP468S, MP476S, MP482S, MP522S, MP529S, MP548S, MP552S, MP562S, MP564S, MP566S, MP567S,

MP569S, MP573S, MP574S, MP575S, MP581S, MP590, MP594S, MP596S, MP597, MP599S, MP600S, MP601S, MP602S, MP604S, MP626S, MP629S, MP630S, MP631S, MP632S, MP633S, MP6354S,

MP639S, MP640S, MP644S, MP649S, MP651S, MP652S, MP653S, MP661S, MP666S, MP672S, MP696S,

MP704S, MP724S, MP729S, MP739S, MP755S, MP773S, MP799S, MP800S, MP801S, MP802S, MP803S,

MP805S, MP809S, MP815S, MP828S, MP831S, MP844S, MP852, MP865S, MP879S, MP887S, MP891S,

MP896S, MP898S, MP935S, MP968, MP989, MP993, MP997, MP1049, MP1066, MP1067, MP1080, MP1081, MP1200, MP1206, MP1233, and MP1311 from US Patent Document Serial No. 62/429426. Other Cry proteins are well known to those skilled in the art (see Crickmore, et al., Bacillus thuringiensis toxin nomenclature (2011), web site at lifesci.sussex.ac.uk/home/Neil Crickmore/Bt/, accessed which can be accessed on the World Wide Web using the prefix www). The insecticidal activity of Cry proteins is well known to those skilled in the art (for a review, see van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cry proteins as traits of a transgenic plant is well known to those skilled in the art, and transgenic plants with Cry, including but not limited to Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A (US Patent US7276583), Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A (US Patent US7276583), Cry9c and CBI-Bt have been approved by regulatory authorities (see Sanahuja, (2011) Plant Biotech Journal 9:283-300 and CERA (2010) and the Center for Environmental Risk Assessment (CERA) GM Plant Database, ILSI Research Foundation, Washington, DC at cera-gmc.org/index.php?action=gm_crop_database, which can be accessed get in

- 43 040502 of the World Wide Web using the prefix www). Plants may also express two or more pesticidal proteins well known to the person skilled in the art, such as Vip3Ab and Cry1Fa (US2012/0317682), Cry1BE and Cry1F (US2012/0311746), Cry1CA and Cry1AB (US2012/0311745), Cry1F and CryCa (US2012/0317681), Cry1DA and Cry1BE (US2012/0331590), Cry1DA and Cry1Fa (US2012/0331589), Cry1AB and Cry1BE (US2012/0324606), and Cry1Fa and Cry2Aa, Cry1I or Cry1E (US2012/0324605). Pesticide proteins also include insecticidal lipases, including lipid acyl hydrolases from US Pat. Pesticide proteins also include VIP toxins (Vegetative Insecticide Proteins) of US Pat. .sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html, which can be accessed via the World Wide Web using the prefix www). Pesticide proteins also include toxin complex (TC) proteins, which can be obtained from organisms such as Xenorhabdus, Photorhabdus, and Paenibacillus (see US Pat. Nos. 7,491,698 and 8,084,418). Some TS proteins have independent insecticidal activity, while other TS proteins increase the activity of independent toxins produced by the same specified organism. The toxicity of a standalone TC protein (eg, from Photorhabdus, Xenorhabdus, or Paenibacillus) can be increased by one or more TC proteins, enhancers derived from a source organism from a different genus. There are three main types of TC proteins. As set forth herein, Class A proteins (Protein A) are toxins in their own right. Class B proteins (protein B) and class C proteins (protein C) increase the toxicity of class A proteins. Examples of class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi. Examples of class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticide proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include, but are not limited to, lycotoxin 1 peptides and mutant forms thereof (US Pat. No. 8,334,366).

(C) A polynucleotide encoding an insect-specific hormone or pheromone such as ecdysteroid and juvenile hormone, a variant thereof, a mimetic thereof, or an antagonist or agonist thereof. See, for example, the disclosure of Hammock, et al., (1990) Nature 344:458 baculovirus expression of cloned juvenile hormone esterase, juvenile hormone inactivator.

(D) A polynucleotide encoding an insect-specific peptide that, when expressed, disrupts the physiology of the affected insect. For example, see the disclosures of Regan, (1994) J. Biol. Chem. 269:9 (expression cloning results in DNA encoding the insect diuretic hormone receptor); Pratt, et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (allostatin found in Diploptera puntata);

Chattopadhyay, et al., (2004) Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J Nat Prod 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon 40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 and Vasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also US Pat. No. 5,266,317 to Tomalski, et al., which discloses genes encoding insect-specific toxins.

(E) A polynucleotide encoding an enzyme responsible for the overaccumulation of a monoterpene, sesquiterpene, steroid, hydroxamic acid, phenylpropanoid derivative, or other non-protein molecule with insecticidal activity.

(F) A polynucleotide encoding an enzyme involved in modification, including post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase, and a glucanase, either natural or synthetic. See PCT application WO 1993/02197 to Scott, et al., which discloses the nucleotide sequence of the callase gene. DNA molecules that contain sequences encoding chitinase can be obtained, for example, from ATCC® accession numbers 39637 and 67152. See also Kramer, et al., (1993) Insect Biochem. Molec. Biol. 23:691, which contains information about the nucleotide sequence of the cDNA encoding the chitinase of tobacco hawthorn, and Kawalleck, et al., (1993) Plant Molec. Biol. 21:673, which proposes the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and US Pat. Nos. 6,563,020; 7145060 and 7087810.

(G) A polynucleotide encoding a molecule that stimulates signal transduction. For example, see the disclosure in Botella, et al., (1994) Plant Molec. Biol. 24:757 regarding the nucleotide sequences of mung bean calmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol. 104:1467, which presents the nucleotide sequence of the maize calmodulin cDNA clone.

(H) Polynucleotide encoding a peptide with a hydrophobic moment. See PCT application WO 1995/16776 and U.S. Patent No. 5,580,852 disclosing peptide derivatives of tachyplesin that inhibit fungal plant pathogens and PCT application WO 1995/18855 and U.S. Patent No. 5,607,914 (which discloses synthetic antimicrobial peptides that confer disease resistance).

(I) A polynucleotide encoding a membrane permease, channel former or channel blocker.

- 44 040502

For example, see the disclosure in Jaynes, et al., (1993) Plant Sci. 89:43 heterologous expression of a cecropin-beta lytic peptide analogue to confer resistance to Pseudomonas solanacearum in transgenic tobacco plants.

(J) A gene encoding a viral invasive protein or a complex toxin derived from it. For example, accumulation of viral envelope proteins in transformed plant cells confers resistance to viral infection and/or disease development caused by the virus from which the envelope protein gene is derived, as well as related viruses. See Beachy, et al., (1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistance was conferred on transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus, and tobacco mosaic virus. There.

(K) A gene encoding an insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody that targets a critical metabolic function in the gut of an insect will inactivate the targeted enzyme, killing the insect. See also Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymatic inactivation in transgenic tobacco through the production of single chain antibody fragments).

(L) Gene encoding an antibody specific for a virus. See, for example, Tavladoraki, et al., (1993) Nature 366:469 which shows that transgenic plants expressing recombinant antibody genes are protected from virus attack.

(M) A polynucleotide encoding a developmental arrest protein naturally produced by a pathogen or parasite. Thus, fungal endo-alpha-1,4O-polygalacturonases facilitate fungal colonization and release of plant nutrients by solubilizing plant cell wall homoalpha-1,4O-galacturonase. See Lamb, et al., (1992) Bio/Technology 10:1436. Cloning and characterization of a gene that encodes a bean endopolygalacturonase inhibitory protein is described in Toubart, et al., (1992) Plant J. 2:367.

(N) A polynucleotide encoding a developmental arrest protein naturally produced by a plant. For example, Logemann, et al., (1992) Bio/Technology 10:305 showed that transgenic plants expressing a barley ribosome inactivating gene had increased resistance to fungal diseases.

(O) Genes involved in systemic acquired resistance response (SAR) and/or genes associated with pathogenesis. Briggs, (1995) Current Biology 5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64, and Somssich, (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol. 101:709-712, and Parijs, et al., (1991) Planta 183:258-264, and Bushnell, et al., (1998) Can. J. of Plant Path 20(2):137-149 Also see U.S. Patent Application Publications Serial Nos. 09/950933; 11/619645; 11/657710; 11/748994; 11/774121 Nos. 6891085 and 7306946. LysM receptor-like kinases for recognition of chitin fragments as a first step in plant defense against fungal pathogens (US 2012/0110696).

(Q) Detoxification system genes such as those encoding fumonisin, beauvericin, moniliformin, and zearalenone and their structurally related derivatives. For example, see US Pat. Nos. 5,716,820; 5792931; 5798255; 5846812; 6083736; 6538177; 6388171 and 6812380.

(R) Polynucleotide encoding cystatin and cysteine proteinase inhibitors. See US Pat. No. 7,205,453.

(S) Defensin genes. See WO 2003/000863 and US Pat. Nos. 6,911,577; 6855865; 6777592 and 7238781.

(T) Genes conferring nematode resistance. See, for example, PCT application WO 1996/30517; PCT application WO 1993/19181, WO 2003/033651 and Urwin, et al., (1998) Planta 204:472479, Williamson, (1999) Curr Opin Plant Bio. 2(4):327-31; US Pat. Nos. 6,284,948 and 7,301,069; and miR164 genes (WO 2012/058266).

(U) Genes that confer resistance to Phytophthora root rot such as Rps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes. See, for example, Shoemaker, et al., Phytophthora Root Rot Resistance Gene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif. (1995).

(V) Genes that confer resistance to brown stem rot, such as those described in US Pat. No. 5,689,035, incorporated by reference for this purpose.

(W) Genes that confer resistance to Colletotrichum, such as those described in US Patent Application Publication US 2009/0035765, incorporated by reference for this purpose. They include the Rcg locus, which can be used as a single locus conversion.

2) Transgenes that confer herbicide resistance, such as those listed below.

(A) A polynucleotide encoding resistance to a herbicide that inhibits growth cone or meristem formation, such as imidazolinone or a sulfonylurea. Illustrative genes

- 45 040502 in this category encode the mutant enzyme ALS and AHAS, as described, for example, respectively in

Lee, et al., (1988) EMBO J. 7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449. See also US Pat. Nos. 5,605,011; 5013659; 5141870; 5767361; 5731180; 5304732; 4761373; 5331107; 5928937 and 5378824; US Patent Application Serial No. 11/683737; and International Publication WO 1996/33270.

(B) A polynucleotide encoding a protein for resistance to glyphosate (resistance is conferred by mutant genes for 5-enolpriruvil-3-phosphoshikimate synthase (EPSP) and aroA, respectively) and other phosphonic compounds such as glufosinate (genes for phosphinothricin acetyltransferase (PAT) and phosphinothricin acetyltransferase (bar) Streptomyces hygroscopicus), and pyridineoxy or phenoxypropionic acids and cyclohexones (genes encoding an ACCase inhibitor). See, for example, US Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSPS that can confer resistance to glyphosate. US Pat. No. 5,627,061 to Barry, et al. also describes genes encoding EPSPS enzymes. See also US Pat. Nos. 6,566,587; 6338961; 6248876; 6040497; 5804425; 5633435; 5145783; 4971908; 5312910; 5188642; 5094945, 4940835; 5866775; 6225114; 6130366; 5310667; 4535060; 4769061; 5633448; 5510471; Re. 36449; RE 37287 E and 5491288 and publications of international applications EP 1173580; WO2001/66704; EP 1173581 and EP 1173582, which are incorporated herein by reference for this purpose. Glyphosate resistance is also conferred on plants that express the gene encoding the enzyme glyphosate oxidoreductase, as described in more detail in US Pat. In addition, glyphosate resistance can be conferred on plants through overexpression of genes encoding glyphosate K-acetyltransferase. See, for example, US Pat. Nos. 7,462,481; 7,405,074 and US Patent Application Publication No. US 2008/0234130. A DNA molecule encoding a mutant aroA gene is available under Accession No. ATCC® 39256, and the nucleotide sequence of the mutant gene is disclosed in US Pat. No. 4,769,061 to Comai. In the application EP No. 0333033 issued by Kumada, et al. and US Pat. No. 4,975,374 to Goodman, et al. disclose glutamine synthetase gene nucleotide sequences conferring resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of the phosphinothricin acetyltransferase gene is presented in EP applications No. 0242246 and 0242236, Leemans, et al., De Greef, et al., (1989) Bio/Technology 7:61, which describes the production of transgenic plants that express chimeric bar genes encoding activity. See also US Pat. Nos. 5,969,213; 5489520; 5550318; 5874265; 5919675; 5561236; 5648477; 5646024; 6177616 and 5879903, which are incorporated herein for this purpose by reference. Exemplary genes conferring resistance to phenoxypropionic acids and cyclohexones such as sethoxydim and haloxyfop are the Acc1-S1, Accl-S2 and Acc1-S3 genes described in Marshall, et al., (1992) Theor. Appl. Genet. 83:435.

(C) A polynucleotide encoding a protein conferring resistance to a herbicide that inhibits photosynthesis, such as triazine (psbA and gs+ genes) and benzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell 3:169 describe transformation of Chlamydomonas with plasmids encoding mutant psbA genes. The nucleotide sequences of nitrilase genes are disclosed in US Pat. et al., (1992) Biochem. J. 285:173.

(D) A polynucleotide encoding a protein conferring resistance to acetohydroxy acid synthase has been introduced into a number of plants, which has been found to render plants that express this enzyme resistant to several types of herbicides (see, e.g., Hattori, et al., (1995) Mol Gen Genet 246:419). Other genes that confer herbicide resistance include the gene encoding the rat cytochrome P4507A1 chimeric protein and yeast NADPH cytochrome P450 oxidoreductase (Shiota, et al., (1994) Plant Physiol 106:17), genes encoding glutathione reductase and superoxide dismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687), and genes encoding various phosphotransferases (Datta, et al., (1992) Plant Mol Biol 20:619).

(E) A polynucleotide encoding resistance to a herbicide that targets protoporphyrinogen oxidase (protox), which is required for chlorophyll production. The protox enzyme serves as a target for a number of herbicidal compounds. These herbicides also inhibit the growth of all the various plant species present, causing their complete destruction. The development of plants with altered protox activity that are resistant to these herbicides is described in US Pat. Nos. 6,288,306; 628283 and 5767373 and international publication WO2001/12825.

(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodes the aryloxyalkanoate dioxygenase (AAD-1) protein. The feature provides tolerance to herbicides based on 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate (commonly referred to as fop herbicides such as quizalofop). The aad-1 gene as such conferring herbicide tolerance in plants was first disclosed in WO 2005/107437 (see also US 2009/0093366). The aad-12 gene, derived from Delftia acidovorans, which encodes the aryloxyalkanoate dioxygenase (AAD-12) protein resulting in herbicide tolerance based on 2,4-

- 46 040502 dichlorophenoxyacetic acid and pyridyloxyacetate by inactivating some herbicides with an aryloxyalkanoate moiety, including phenoxy auxin (eg 2,4-D, MCPA) as well as pyridyloxy auxin species (eg fluroxypyr, triclopyr).

(G) A polynucleotide encoding herbicide-resistant dicamba monooxygenase disclosed in US Patent Application Publication 2003/0135879 to confer tolerance on dicamba.

(H) A polynucleotide molecule encoding bromoxynyl nitrilase (Bxn) disclosed in US Pat. No. 4,810,648 to confer bromoxynil tolerability.

(I) Polynucleotide molecule encoding phytoene (crtl) described in Misawa, et al., (1993) Plant J. 4:833-840 and Misawa, et al., (1994) Plant J. 6:481-489 , for tolerability of norflurazone.

3) Transgenes that provide or contribute to altered grain characteristics. For example.

(A) Modified fatty acid content, eg using the below.

(1) Suppression of stearoyl-ACP to increase the content of stearic acid in the plant. See Knultzon, et al., (1992) Proc. Natl. Acad. sci. USA 89:2624 and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn).

(2) Increasing oleic acid by modifying the FAD-2 gene and/or reducing linolenic acid by modifying the FAD-3 gene (See US Pat.

(3) Changes in the content of conjugated linolenic or linoleic acid, such as in WO 2001/12800.

(4) Changes in LEC1, AGP, Dek1, Superall, mil ps and various Ipa genes such as Ipa1, Ipa3, hpt or hggt. For example, see WO 2002/42424, WO 1998/22604, WO 2003/011015, WO 2002/057439, WO 2003/011015, US Patent Nos. 6423886, 6197561, 6825397 and US Patent Application Publication Nos. 2003/0204870 and Rivera-Madrid, et al., (1995) Proc. Natl. Acad. sci. 92:5620-5624.

(5) Genes coding for delta-8 desaturase to produce long-chain polyunsaturated fatty acids (US Pat. Nos. 8,058,571 and 8,338,152), delta-9 desaturase to reduce saturated fat (US Pat. -3 fatty acids.

(6) Isolated nucleic acids and proteins associated with the regulation of lipid and sugar metabolism, in particular the lipid metabolism protein (LMP), used in methods for producing transgenic plants and modulating the levels of seed storage substances, including lipids, fatty acids, starch species or seed storage proteins, and their use in methods for modulating seed size, seed number, seed weight, root length and leaf size of plants (EP 2404499).

(7) Changes in the expression of a highly expressed sugar inducible 2 (HSI2) protein in a plant to increase or decrease the expression of HSI2 in the plant. Increasing HSI2 expression increases oil content, while decreasing HSI2 expression reduces abscisic acid susceptibility and/or increases drought tolerance (US Patent Application Publication No. 2012/0066794).

(8) Expression of cytochrome b5 (Cb5) alone or together with FAD2 to modulate plant seed oil content, in particular to increase omega-3 fatty acid levels and improve the ratio of omega-6 to omega-3 fatty acids (U.S. Patent Application Publication No. 2011/0191904).

(9) Nucleic acid molecules encoding wrinkledl-like polypeptides for modulating sugar metabolism (US Pat. No. 8,217,223).

(B) Modified phosphorus content, for example, using the following.

(1) The introduction of a gene encoding a phytase will improve the breakdown of phytate, resulting in more free phosphate in the transformed plant. For example, see Van Hartingsveldt, et al., (1993) Gene 127:87 for disclosure of the nucleotide sequence of the Aspergillus niger phytase gene.

(2) Modulation of a gene that reduces phytate content. In maize, this can be done, for example, by cloning and then reintroducing DNA associated with one or more alleles, such as the LPA alleles found in maize mutants characterized by low levels of phytic acid, such as in WO 2005/113778 , and/or by changing the activity of inositolkinase, as in WO 2002/059324, US Patent Application Publication No. 2003/0009011, WO 2003/027243, US Patent Application Publication No. 2003/0079247, WO 1999/05298, US Patent No. 6197561 , US Pat. No. 6,291,224;

(C) Altered carbohydrates that have been affected, for example, by altering a gene encoding an enzyme that affects the branching pattern of starch, or a gene that alters thioredoxin, such as NTR and/or TRX (see US patent No. 653164 8, which is included for this purpose by reference), and/or knocking out gamma-zein or using a mutant such as cs27 or TUSC27 or en27 (see U.S. Patent No. 6,858,778 and U.S. Patent Application Publication No. 2005/0160488, U.S. Patent Application Publication 2005/0204418, which are incorporated for this purpose by reference).

- 47 040502

See Shiroza, et al., (1988) J. Bacteriol. 170:810 (nucleotide sequence of the mutant Streptococcus fructosyltransferase gene), Steinmetz, et al., (1985) Mol. Gen. Genet. 200:220 (Nucleotide sequence of Bacillus subtilis levansucrase gene), Pen, et al., (1992) Bio/Technology 10:292 (Production of transgenic plants that express Bacillus licheniformis alpha-amylase), Elliot, et al., (1993) Plant Molec. Biol. 21:515 (nucleotide sequences of tomato invertase genes), S0gaard, et al., (1993) J. Biol. Chem. 268:22480 (site-directed mutagenesis of the barley alpha-amylase gene) and Fisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starch branch enzyme II), WO 1999/10498 (Improved digestibility and/or starch recovery by modifying UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1, HCHL, C4H), US Pat. No. 6,232,529 (method for producing high oil seeds by modifying starch levels (AGP)). The fatty acid modification genes mentioned herein can also be used to influence starch content and/or composition through the interplay of starch and oil metabolic pathways.

(D) Altered content or composition of antioxidants, such as altered tocopherol or tocotrienols. For example, see US Pat. No. 6,787,683, US Patent Application Publication No. 2004/0034886 and WO 2000/68393 for manipulation of antioxidant levels, and WO 2003/082899 by altering the homogentisategeranylgeranyltransferase (hggt).

(E) Altered content of essential amino acids in the seed. For example, see U.S. Patent No. 6,127,600 (method for increasing the accumulation of essential amino acids in seeds), U.S. Patent No. 6,080,913 (binary methods for increasing the accumulation of essential amino acids in seeds), U.S. Patent No. 5,990,389 (high lysine content), WO 1999/40209 seed composition), WO 1999/29882 (Methods for altering the amino acid composition of proteins), US Pat. U.S. Patent No. 5,885,801 (High Threonine), U.S. Patent No. 6,664,445 (Plant Enzymes for Amino Acid Biosynthesis), U.S. Patent No. 6,459,019 (Increased Lysine and Threonine), U.S. Patent No. 6,441,274 (Beta Subunit of Plant Tryptophan Synthase), U.S. Patent No. methionine metabolism enzymes), US Pat. No. 5,939,599 (high sulfur), US Pat. amino acid biosynthetic enzymes), WO 1998/45458 (genetically engineered seed protein having a higher percentage of essential amino acids), WO 1998/42831 (increased lysine content), US Pat. No. 5559223 (synthetic storage proteins with a specific structure containing programmed levels of essential amino acids to improve the nutritional value of plants), WO 1996/01905 (increased threonine content), WO 1995/15392 (increased lysine content), US Patent Application Publication No. 2003/ 0163838, US Patent Application Publication No. 2003/0150014, US Patent Application Publication No. 2004/0068767, US Patent No. 6803498, WO 2001/79516.

4) Genes that control male sterility.

Several methods of providing genetic male sterility are available, such as multiple mutant genes at specific positions within the genome that confer male sterility, as disclosed in US Pat. Nos. 4,654,465 and 4,727,219 to Brar, et al. in US Pat. Nos. 3,861,709 and 3,710,511. In addition to these methods, Albertsen, et al. US Pat. No. 5,432,068 describes a nuclear male sterility system that includes: identifying a gene that is critical to male fertility; silencing of this native gene, which is extremely important for male fertility; removing a native promoter from a gene important for male fertility and replacing it with an inducible promoter; inserting this genetically engineered gene back into the plant, and thereby creating a plant that is male sterile because the inducible promoter is not included, whereby the male fertility gene is not transcribed. Fertility is restored by inducing or turning on a promoter, which in turn transcribes the male fertility gene.

(A) Introduction of the deacetylase gene under the control of a tapetum-specific promoter and using the chemical N-Ac-PPT (WO 2001/29237).

(B) Introduction of various stamen-specific promoters (WO 1992/13956, WO 1992/13957).

(C) Introduction of barnase and barstar gene (Paul, et al., (1992) Plant Mol. Biol. 19:611-622).

For additional examples of nuclear male and female sterility systems and genes, see also US Pat. Nos. 5,859,341; 6297426; 5478369; 5824524; 5850014 and 6265640, all of which are hereby incorporated by reference.

5) Genes that create a site for site-specific DNA integration.

This implies the introduction of FRT sites that can be used in the FLP/FRT system and/or Lox sites that can be used in the Cre/Loxp system. For example, see Lyznik, et al., (2003) Plant Cell Rep 21:925-932 and WO 1999/25821, which are hereby incorporated by reference. Other systems that can be used include the Gin recombinase from the Mu phage (Maeser, et al., (1991) Vicki Chandler, The

Maize Handbook ch. 118 (Springer-Verlag 1994), the Pin recombinase from E. coli (Enomoto, et al., 1983), and the R/RS system from the pSRi plasmid (Araki, et al., 1992).

6) Genes that affect resistance to abiotic stress.

Including, but not limited to, flowering, ear and seed development, improved nitrogen use efficiency, altered nitrogen reactivity, drought tolerance or tolerance, cold tolerance or tolerance, and salinity tolerance or tolerance, and increased yields when stress.

(A) For example, see WO 2000/73475 where the water use efficiency is changed by changing the malate content; US patents No. 5892009, 5965705, 5929305, 5891859, 6417428, 6664446, 6706866, 6717034, 6801104, Wo 2000/060089, WO 2001/026459, Wo 2001/035725, Wo 2001/034726, Wo 2001/0326, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, Wo5726, WO , Wo 2001/036597, Wo 2001/036598, Wo 2002/015675, Wo 2002/017430, Wo 2002/077185, Wo 2002/079403, Wo 2003/013227, Wo 2003/013228, Wo 2003/014327, Wo 2004/031349 WO 2004/076638, WO 199809521.

(B) WO 199938977, which describes genes, including CBF genes, and transcription factors effective in reducing the negative effects of freezing, high salinity and drought on plants, as well as providing other positive effects on plant phenotype.

(C) US Patent Application Publication No. 2004/0148654 and WO 2001/36596, where the abscisic acid content of plants is altered, resulting in an improved plant phenotype, such as increased yield and/or increased abiotic stress tolerance.

(D) WO2000/006341, WO2004/090143, US Pat. See also WO 2002/02776, WO 2003/052063, JP 2002/281975, US Pat. No. 6,084,153, WO 2001/64898, US Pat. No. 6,177,275 and US Pat. No. 6,107,547 (improved nitrogen utilization and altered nitrogen reactivity).

(E) With respect to the change in ethylene content, see US Patent Application Publication No. 2004/0128719, US Patent Application Publication No. 2003/0166197 and WO 2000/32761.

(F) For plant transcription factors or transcription regulators associated with abiotic stress response, see, for example, US Patent Application Publication No. 2004/0098764 or US Patent Application Publication No. 2004/0078852.

(G) Genes that increase the expression of vacuolar pyrophosphatase, such as AVP1 (US patent No. 8058515), for increased yield; a nucleic acid encoding HSFA4 or HSFA5 polypeptides (heat shock factor class A4 or A5), an oligopeptide transporter protein-like polypeptide (OPT4-like); Plastochron-2-like (PLA2-like) polypeptide or Wuschel-related homeobox 1-like (WOX1-like) polypeptide (U.S. Patent Application Publication No. US 2011/0283420).

(H) Suppression of polynucleotides encoding poly-(ADP-ribose) polymerase (PARP) proteins for modulating programmed cell death (US Pat. No. 8,058,510) to increase power.

(I) A polynucleotide encoding DTP21 polypeptides for drought tolerance (US Patent Application Publication No. US 2011/02 77181).

(J) Nucleotide sequences encoding ACC synthase 3 (ACS3) proteins for modulating development, modulating stress response, and modulating stress tolerance (U.S. Patent Application No. US 2010/0287669).

(K) Polynucleotides encoding proteins that result in a drought tolerant (DTP) phenotype for drought tolerance (WO 2012/058528).

(L) Tocopherol cyclase (TC) genes for drought and salinity tolerance (US Patent Application Publication No. 2012/0272352).

(M) Proteins of the protease family targeting CAAX-terminal amino acids to confer stress tolerance (US Pat. No. 8,338,661).

(N) Mutations in the gene encoding SAL1 were characterized by increased stress tolerance, including increased drought tolerance (US Patent Application Publication No. 2010/0257633).

(O) Expression of a nucleic acid sequence that encodes a polypeptide selected from the group consisting of: GRF polypeptide, RAA1-like polypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptide enhancing yield-related traits (U.S. Patent Application Publication No. 2011 /0061133).

(P) Modulation of expression in a plant of a nucleic acid encoding a class III trehalose phosphate phosphatase (TPP) polypeptide to improve yield-related traits in plants, in particular increased seed yield (U.S. Patent Application Publication No. 2010/0024067).

Other genes and transcription factors that affect plant growth and agronomic traits such as yield, flowering, plant growth and/or plant structure can be introduced or

- 49 040502 introgress into plants, see for example WO 1997/49811 (LHY), WO 1998/56918 (ESD4), WO

1997/10339 and US Patent No. 6573430 (TFL), US Patent No. 6713663 (FT), WO 1996/14414 (CON), WO

1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO 1999/49064 (GI), WO 2000/46358 (FR1), WO 1997/29123, US Pat. No. 6,794,560, US Pat. No. 6,307,126 (GAI), WO 1999/09174 (D8 and

Rht) and WO 2004/076638 as well as WO 2004/031349 (transcription factors).

7) Genes that provide increased yield.

(A) A transgenic crop plant transformed with a nucleic acid encoding a 1-aminocyclopropane-1-carboxylate deaminase-like polypeptide (ACCDP), wherein expression of the nucleic acid sequence in the crop plant results in increased root growth and/or increased yield, and/ or increased stress tolerance under the influence of environmental factors in the plant, compared with a variety of wild-type plants (US patent No. 8097769).

(B) Overexpression of the maize zinc finger protein (Zm-ZFP1) gene using a promoter active predominantly in seeds has been shown to enhance plant growth, increase the number of grains, and increase the total grain weight per plant (U.S. Patent Application Publication No. 2012/0079623 ).

(C) Constitutive overexpression of the lateral organ boundary (LOB) domain protein (Zm-LOBDP1) of maize has been shown to increase the number of grains and the total grain weight per plant (U.S. Patent Application Publication No. 2012/0079622).

(D) Improving yield-related traits in plants by modulating plant expression of a nucleic acid encoding a VIM1 (methylation variant 1)-like polypeptide or VTC2-like (GDP-L-galactose phosphorylase) polypeptide or polypeptide DUF1685, or ARF6-like (auxin receptive factor) polypeptide (WO 2012/038893).

(E) Modulation of the expression in a plant of a nucleic acid encoding a Ste20-like polypeptide or homologue thereof allows plants to provide increased yield relative to control plants (EP 2431472).

(F) Genes encoding nucleoside diphosphate kinase (NDK) polypeptides and their homologues for plant root modification (US Patent Application Publication No. 2009/0064373).

8) Genes that provide plant digestibility.

(A) Changing the level of xylan present in the plant cell wall by modulating xylan synthase expression (US Pat. No. 8,173,866).

In some embodiment, a packaged trait may be a trait or transgenic entity that has received regulatory approval, including, but not limited to, regulatory approved transgenic entities that are well known to one of skill in the art and can be found on the Center's website. environmental risk assessment (cera-gmc.org/?action=gm_crop_database, which can be accessed using the prefix www) and the website of the International Service for the Acquisition of Agrobiotechnology Applications (isa.org/gmapprovaldatabase/default.asp, which can be accessed can be obtained using the prefix www).

Gene silencing.

In some embodiments, the packaged trait may be in a form useful for silencing one or more polynucleotides of interest, resulting in the suppression of one or more target pest polypeptides. In some embodiments, silencing is achieved through the use of a suppression DNA construct.

In some embodiments, one or more polynucleotides encoding polypeptides of an IPD090 polypeptide, or fragments or variants thereof, may be packaged with one or more polynucleotides encoding one or more polypeptides having the insecticidal activity or agronomic traits set forth above, and may optionally further provide one or multiple polynucleotides provided for gene silencing of one or more target polynucleotides, as discussed below.

A suppression DNA construct is a recombinant DNA construct that, when transformed or stably integrated into the genome of a plant, results in silencing of the target gene in the plant. The target gene may be endogenous or transgenic to the plant. The term silencing, as used herein in relation to a target gene, generally refers to providing suppression of the levels of mRNA or protein/enzyme expressed by the target gene and/or the level of enzyme activity or protein function. The term suppression includes reduction, reduction, aggravation, reduction, suppression, elimination, and prevention. Silencing or gene silencing does not specify a mechanism and includes, without limitation, antisense oligonucleotide-mediated suppression, co-suppression, virus-mediated suppression, hairpin-mediated suppression, stem-loop mediated suppression, RNAi-based approaches, and small molecular weight RNA-based approaches.

The suppression DNA construct may contain a region derived from the target gene of interest and may contain all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest. Depending on the approach to be used, the region may be 100% identical or less than 100% identical (e.g., at least 50% or any integer from 51 to 100% identical) of the entire semantic strand (or antisense strand) of the gene of interest, or a portion thereof.

Suppression DNA constructs are well known in the art, are readily constructed once a target gene of interest has been selected, and include, but are not limited to, co-suppression constructs, antisense constructs, viral suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, constructs producing double-stranded RNA, and more generally RNAi (RNA interference) constructs and small RNA constructs such as siRNA (short interfering RNA) and miRNA (microRNA) constructs.

The term antisense suppression refers to the production of antisense RNA transcripts capable of providing suppression of the expression of a target protein.

The term antisense RNA refers to an RNA transcript that is complementary to all or part of the target primary transcript or mRNA and that blocks the expression of a fragment of the target isolated nucleic acid. The complementarity of the antisense RNA can be with any part of a particular gene transcript, i.e. with a 5' non-coding sequence, a 3' non-coding sequence, introns, or a coding sequence.

The term cosuppression refers to the production of sense RNA transcripts capable of providing suppression of the expression of a target protein. The term sense RNA refers to an RNA transcript that includes mRNA and can be translated into a protein in a cell or in vitro. Plant cosuppression constructs have previously been developed with a focus on overexpressing nucleic acid sequences with native mRNA homology in sense orientation, resulting in reduced levels of all RNAs with homology to the overexpressed sequence (see Vaucheret, et al., (1998) Plant J. 16:651-659 and Gura, (2000) Nature 404:804-808).

In another embodiment, the use of plant virus sequences to control the suppression of proximal mRNA-coding sequences is described (PCT Publication WO 1998/36083).

Recent work has described the use of hairpin structures that include all or part of an mRNA coding sequence in complementary orientation, resulting in a potential stem-loop structure in the expressed RNA (PCT publication WO 1999/53050). In this case, the stem is formed by polynucleotides corresponding to the gene of interest in sense or antisense orientation with respect to the promoter, and the loop is formed by several polynucleotides of the gene of interest that do not have a complementary sequence in the construct. This increases the frequency of co-suppression or silencing in regenerated transgenic plants. For a review of hairpin suppression, see Wesley, et al., (2003) Methods in Molecular Biology, Plant Functional Genomics: Methods and Protocols 236:273-286.

A construct in which the stem is formed by at least 30 nucleotides from the gene to be suppressed and the loop is formed by a random nucleotide sequence has also been effectively used for suppression (PCT publication WO 1999/61632).

The use of poly-T and poly-A sequences to produce a stem in a stem-loop structure has also been described (PCT Publication WO 2002/00894).

Another option involves the use of synthetic repeats to promote stem formation in a stem-loop structure. Transgenic organisms produced with such recombinant DNA fragments have been shown to have reduced levels of the protein encoded by the nucleotide fragment that forms the loop, as described in PCT publication WO 2002/00904.

RNA interference refers to a sequence-specific process of post-transcriptional gene silencing in animals that is mediated by short interfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806).

The corresponding process in plants is commonly referred to as post-transcriptional gene silencing (PTGS) or RNA-mediated silencing, and is also referred to as downregulation in fungi. The process of post-transcriptional gene silencing is considered to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes, and is widespread in various plant and animal phyla (Fire, et al., (1999) Trends Genet. 15:358). This defense against foreign gene expression may have evolved in response to the production of double-stranded RNA (dsRNA), derived from viral infection or from the random integration of transposon elements into the host genome, by a cellular response that specifically degrades the homologous single-stranded RNA of the viral genomic RNA. The presence of dsRNA in cells induces an RNAi response through a mechanism that has not yet been fully characterized.

The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme called dicer. Dicer is involved in dsRNA processing to form short dsRNA fragments called short interfering RNAs (siRNAs) (Berstein et al., Nature 409:363, 2001). Typically, short interfering RNAs resulting from Dicer activity are about 21 to about 23 nucleotides in length, and include duplexes of about 19 base pairs (Elbashir, et al., (2001) Genes Dev. 15:188). In addition, Dicer has been implicated in the excision of 21- and 22-nucleotide small transient RNAs (stRNA) from conserved precursor RNA, which are involved in translational control (Hutvagner, et al., (2001) Science 293:834). The RNAi response is also characterized by an endonuclease complex, commonly referred to as the RNA-induced silencing complex (RISC), which mediates the cleavage of single-stranded RNA whose sequence is complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir, et al., (2001) Genes Dev. 15:188). In addition, RNA interference may also imply gene silencing mediated by small RNAs (e.g., miRNAs), presumably through cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see, e.g., Allshire, (2002) Science 297:1818-1819; Volpe, et al., (2002) Science 297:1833-1837; Jenuwein, (2002) Science 297:2215-2218 and Hall, et al., (2002) Science 297:2232-2237 ). Thus, the miRNA molecules of the present invention can be used to mediate gene silencing by interacting with RNA transcripts, or alternatively by interacting with specific gene sequences, where such interaction results in gene silencing either at the transcriptional or post-transcriptional level.

Additionally, methods and compositions are provided that enable the level of RNAi produced by the silencing element to be increased. In such embodiments, the methods and compositions use a first polynucleotide comprising a silencing element for a target pest sequence operably linked to a promoter active in a plant cell; and a second polynucleotide comprising a suppression-enhancing element comprising the target pest sequence or an active variant or fragment thereof operably linked to a promoter active in the plant cell. Combined expression of a silencing element with a suppression-enhancing element leads to an increase in the amplification of inhibitory RNAs generated from the silencing element beyond that achieved by expression of the silencing element alone. In addition to increased amplification of specific RNAi molecular species per se, the methods and compositions further allow the generation of a distinct group of RNAi molecular species that can improve the efficiency of disrupting target gene expression. Thus, when a suppression-enhancing element is expressed in a plant cell in combination with a silencing element, the methods and compositions can allow the systemic production of RNAi molecules throughout the plant; the production of larger numbers of molecules for RNAi than would be observed in the case of only designing the silencing element separately; and improved loading of RNAi molecules into the plant phloem, thus providing better control of phloem-feeding insects with the RNAi approach. Thus, various methods and compositions provide improved methods for delivering inhibitory RNAs to a target organism. See, for example, US Patent Application Publication 2009/0188008.

As used herein, the term suppression-enhancing element refers to a polynucleotide comprising a target sequence to be suppressed, or an active fragment or variant thereof. It is understood that the suppression-enhancing element need not be identical to the target sequence, but rather, the suppression-enhancing element may contain a variant of the target sequence, provided that the sequence of the suppression-enhancing element is sufficiently identical to the target sequence to allow an increased level of RNAi produced by the element silencing, beyond the level that is achieved when only the silencing element is expressed. Similarly, the suppression-enhancing element may comprise a fragment of the target sequence, where the fragment is of sufficient length to allow an increased level of RNAi produced by the silencing element beyond that achieved by expression of the silencing element alone.

It is understood that multiple suppression-enhancing elements from the same target sequence, or from different target sequences, or from different regions of the same target sequence, may be used. For example, the suppression-enhancing elements used may contain fragments of the target sequence derived from another portion of the target sequence (ie, from the 3'UTR, coding sequence, intron, and/or 5'UTR). In addition, the suppression-enhancing element may be contained in an expression cassette as described elsewhere herein, and in certain embodiments, the suppression-enhancing element is on one or the other DNA vector or construct relative to the silencing element. The suppression-enhancing element may be operably linked to the promoter disclosed herein. It is recognized that the suppressor-enhancing element may be expressed constitutively, or alternatively it may be produced in a stage-dependent manner using various inducible, or tissue-preferred, or developmentally regulated promoters, which are discussed elsewhere. place of this document.

In certain embodiments, when using both a silencing element and a suppression-enhancing element, systemic production of molecules for RNAi occurs throughout the plant. In additional embodiments, the plant or plant parts of the present invention have an increased load of molecules for RNAi in the plant phloem, which could be observed when expressing the silencing element construct alone, and thus better control of phloem-feeding insects with the RNAi approach. In certain embodiments, the plants, plant parts, and plant cells of the present invention can be further characterized as being capable of producing a variety of RNAi molecule species that can improve the efficiency of disrupting target gene expression.

In certain embodiments, the combined expression of the silencing element and the suppression-enhancing element increases the concentration of inhibitory RNAs in the plant cell, plant, plant part, plant tissue, or phloem beyond that achieved by expressing the silencing element alone.

As used herein, an increased level of inhibitory RNAs refers to any statistically significant increase in the level of molecules for RNAi produced in a plant with combined expression compared to a corresponding control plant. For example, an increase in the level of RNAi in a plant, plant part, or plant cell may include at least about 1%, about 1-5%, about 510%, about 10-20%, about 20-30%, about 30-40%, approximately 4050%, approximately 50-60%, approximately 60-70%, approximately 70-80%, approximately 8090%, approximately 90-100% or greater increase in RNAi levels in a plant, plant part, plant cell, or phloem compared to the corresponding control. In other embodiments, the increase in RNAi levels in a plant, plant part, plant cell, or phloem may be at least about 1-fold, about 1-5-fold, about 5-10-fold, about 10-20-fold, about 20 -30 times, about 30-40 times, about 40-50 times, about 50-60 times, about 60-70 times, about 70-80 times, about 80-90 times, about 90-100 times a fold or greater increase in the level of RNAi in a plant, plant part, plant cell, or phloem compared to an appropriate control. Examples of combined expression of a silencing element with a suppression-enhancing element for control of stink bugs and Lygus can be found in US Patent Application Publication 2011/0301223 and US Patent Application Publication 2009/0192117.

Some embodiments relate to the downregulation of target gene expression in insect pest species by interfering ribonucleic acid (RNA) molecules. PCT publication WO 2007/074405 describes methods for suppressing the expression of target genes in invertebrate pests, including the Colorado potato beetle. PCT publication WO 2005/110068 describes methods for suppressing the expression of target genes in invertebrate pests, including in particular the western corn bug, as a means of controlling insect infestation. In addition, PCT publication WO 2009/091864 describes compositions and methods for suppressing target genes from insect pest species, including pests from the genus Lygus. Nucleic acid molecules, including molecules for RNAi to target the H subunit of vacuolar ATPase, are useful for population control and infestation by beetles, as described in US Patent Application Publication 2012/0198586. PCT publication WO 2012/055982 describes a ribonucleic acid (RNA or double-stranded RNA) that represses or downregulates a target gene that encodes: an insect ribosomal protein such as L19 ribosomal protein, L40 ribosomal protein, or S27A ribosomal protein; an insect proteasome subunit such as Rpn6 protein, Pros 25, Rpn2 protein, proteasome subunit beta 1 protein, or Pros beta 2 protein; β-coatomer of an insect COPI vesicle, γ-coatomer of a COPI vesicle, β'-coatomer protein, or ζ-coatomer of a COPI vesicle; the insect tetraspanin 2A protein, which is a putative transmembrane domain protein; an insect protein belonging to the actin family, such as actin 5C; insect ubiquitin-5E protein; insect Sec23 protein, which is an activator of GTPases involved in intracellular protein transport; the crinkled insect protein, which is a non-standard myosin that is involved in locomotor activity; the insect crooked neck protein, which is involved in the regulation of nuclear alternative mRNA splicing; insect vacuolar H+-ATPase G subunit protein; and insect Tbp-1, such as Tat-binding protein. PCT publication WO 2007/035650 describes a ribonucleic acid (RNA or double-stranded RNA) that represses or down-regulates the expression of a target gene encoding Snf7. US Patent Application Publication 20150176009 describes polynucleotide silencing elements that target Rnapii-140, which confer resistance to beetle pests. US Patent Application Publication 2011/0054007 describes

- 53 040502 polynucleotide silencing elements targeting RPS10. US Patent Application Publications 2014/0275208 and US2015/0257389 describe polynucleotide silencing elements that target RyanR and PAT3. U.S. Patent Application Publications 2012/029750, US 20120297501 and 2012/0322660 describe interfering ribonucleic acids (RNA or double-stranded RNA) that function when ingested by insect pest species to down-regulate expression of a target gene in said pest, wherein the RNA contains at least one silencing element, where the silencing element is a section of double-stranded RNA containing hybridized complementary strands, of which one strand contains or consists of a nucleotide sequence that is at least partially complementary to the target nucleotide sequence within the target gene. U.S. Patent Application Publication 2012/0164205 describes potential targets for interfering double-stranded ribonucleic acids for invertebrate pest control, including: Chd3 homologous sequence, beta-tubulin homologous sequence, V-ATPase 40 kDa homologous sequence, EF1a homologous sequence, EF1a homologous sequence, 26S proteasome p28 subunit sequence, juvenile hormone epoxide hydrolase homologous sequence, swelling-dependent chloride channel protein homologous sequence, glucose-6-phosphate-1-dehydrogenase protein homologous sequence, Act42A protein homologous sequence, ADP-ribosylation factor 1 homologous sequence, homologous sequence transcription factor IIB protein, chitinase homologous sequences, ubiquitin conjugating enzyme homologous sequence, glyceraldehyde-3-phosphate dehydrogen homologous sequence zy, homologous sequence of ubiquitin B, homolog of juvenile hormone esterase and homologous sequence of alpha-tubulin.

Application in pesticide control.

General methods are known in the art for using strains containing the nucleic acid sequence of the embodiments, or a variant thereof, in pesticide control or in the construction of other organisms as pesticidal agents.

Host microorganisms can be selected that are known to colonize the phytosphere (phylloplane, phyllosphere, rhizosphere and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so that they can successfully compete with wild-type microorganisms in a particular environment, provide stable maintenance and expression of the gene expressing the IPD090 polypeptide, and desirably provide improved protection of the pesticide from degradation and inactivation in the environment.

Alternatively, the IPD090 polypeptide was generated by introducing a heterologous gene into the host cell. Expression of a heterologous gene results, directly or indirectly, in the production and maintenance of the pesticide within the cell. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of the target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated IPD090 polypeptides can then be formulated according to conventional techniques for application to the environment of the target pest, such as soil, water, and plant foliage. See, for example, EPA 0192319 and the references cited therein.

pesticide compositions.

In some embodiments, the active ingredients may be applied in the form of a composition, and may be applied to the cultivated area or plant to be treated, simultaneously or sequentially with other compounds. These compounds can be fertilizers, weed control agents, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and/or formulations with a sustained release or biodegradable carrier that provides a long lasting dosage. application in the target area after a single application of the composition. They may also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bactericides, nematocides, molluscicides, or mixtures of several of these preparations, if necessary together with additional agriculturally acceptable carriers, surfactants. or application aids conventionally used in the formulation art. Suitable carriers and adjuvants may be solid or liquid and correspond to those commonly used in formulation technology, such as natural or reclaimed minerals, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Similarly, formulations can be prepared as edible baits or molded into pest traps that cause the pesticide formulation to be consumed or ingested by the target pest.

Methods for applying an active ingredient or agrochemical composition that contains at least one of the IPD090 polypeptides produced by bacterial strains include applying

- 54 040502 for leaves, seed coating and soil application. The number of applications and the application rate depend on the intensity of the damage by the corresponding pest.

The composition can be formulated as a powder, a dust, a pellet, a granule, a spray solution, an emulsion, a colloid, a solution, and the like, and can be obtained by such conventional methods as drying, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation or concentration of cell culture containing the polypeptide. In all such compositions that contain at least one such pesticidal polypeptide, the polypeptide may be present at a concentration of from about 1 to about 99% by weight.

Pests of the Lepidoptera, Diptera, Bedbugs, Nematodes, Hemiptera, or Coleoptera groups can be killed or reduced in a specified area using the methods of this disclosure, or the agents can be applied prophylactically to an area in the environment to prevent infestation by a susceptible pest.

Preferably, the pest ingests or comes into contact with a pesticidally effective amount of the polypeptide. The term pesticidally effective amount, as used herein, refers to an amount of a pesticide that can result in the death of at least one pest or markedly reduced growth, nutrition, or normal physiological development of the pest. This amount will vary depending on factors such as, for example, the specific target pest to be controlled, the specific environment, location, plant, crop or agricultural area to be treated, environmental conditions and method, rate, concentration, stability and number of applications. pesticidally effective polypeptide composition. The formulations may also vary depending on climatic conditions, environmental considerations, and/or frequency of application and/or severity of pest infestation.

The described pesticidal compositions can be obtained by formulating either a suspension of bacterial cells, crystals and/or spores, or an isolated protein component with the desired agriculturally acceptable carrier. Compositions may be formulated prior to administration by appropriate means such as lyophilization, freeze drying, drying, or in an aqueous vehicle, medium, or suitable solvent such as saline or other buffer. The formulated compositions may be in the form of a dust, or granular material, or suspension in oil (vegetable or mineral), or aqueous or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural use. . Suitable agriculturally acceptable carriers may be solid or liquid and are well known in the art. The term agriculturally acceptable carrier encompasses all adjuvants, inerts, dispersants, surfactants, tackifiers, binders, etc. that are commonly used in pesticide formulation technology; which are well known to those skilled in the art of formulating pesticides. Formulations can be mixed with one or more solid or liquid adjuvants and obtained using various methods, for example, by uniform mixing, mixing and/or milling the pesticide composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described in US Pat. No. 6,468,523, incorporated herein by reference. Plants may also be treated with one or more chemical compositions, including one or more herbicides, insecticides, or fungicides. Illustrative chemical compositions include the following. Fruit/Vegetable Herbicides: Atrazine, Bromacyl, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Gowan's Halosulfuron, Paraquat, Propizamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam; fruit/vegetable insecticides: aldicarb, Bacillus thuriengiensis, carbaryl, carbofuran, chlorpyrifos, cypermethrin, deltamethrin, diazinon, malathion, abamectin, cyfluthrin/betacyfluthrin, esfenvalerate, lambda-cyhalothrin, acequinocil, biphenazate, methoxylopacloside, novalotenuron, crotimafenuron, crotimafenuron , fluacripirim, tolfenpyrad, clothianidin, spirodiclofen, gamma-cyhalothrin, spiromesifen, spinosad, rinakipyr, cyazipyr, spinoteram, triflumuron, spirotetramat, imidacloprid, flubendiamide, thiodicarb, metaflumizone, sulfoxaflor, cyflumethofen, cyanopirafene, imidacloprid, , flonicamid, methiocarb, emamectin benzoate, indoxacarb, fortiazate, fenamiphos, cadusafos, pyriproxyfen, fenbutatin oxide, hexthiazox, methomyl, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan -2(5H)-one; fruit/vegetable fungicides: carbendazim, chlorthalonil, EBDC, sulfur, thiophanate methyl, azoxystrobin, cymoxanil, fluazinam, fosetil, iprodione, kresoxim-methyl, metalaxyl/mefenoxam, trifloxystrobin, ethaboxam, iprovalicarb, trifloxystrobin, fenhexamide, oxpoconazole fumarate, cyazofamide, phenhexamide , zoxamide, Zorvec™, picoxystrobin, pyraclostrobin, cyflufenamid, boscalid; herbicides for cereals: isoproturon, bromoxynil, ioxynil, phenoxy compounds, chlorsulfuron, clodinafop, diclofop, diflufenican, fenoxaprop, florasulam, fluroxypyr, metsulfuron, triasulfuron, flucarbazone, iodosulfuron, propoxycarbazone, picolinafen, mesosulfuron, beflusulfuron, fenoxabutamide, pinoxa-dosulfuron methyl, tribenuron, flupyrsulfuron, sulfosulfuron, pyrasul- 55 040502 photol, pyroxsulam, flufenacet, tralkoxydim, pyroxasulfone; fungicides for cereals: carbendazim, chlorothalonil, azoxystrobin, cyproconazole, cyprodinil, fenpropimorph, epoxiconazole, kresoximmethyl, quinoxyfen, tebuconazole, trifloxystrobin, simeconazole, picoxystrobin, pyraclostrobin, dimoxystrobin, prothioconazole, fluoxastrobin; cereal insecticides: dimethoate, lambdacyhalothrin, deltamethrin, alpha-cypermethrin, β-cyfluthrin, bifenthrin, imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinetofuran, chlorpyrifos, methamidophos, oxydemeton-methyl, pyrimicarb, methiocarb; maize herbicides: atrazine, alachlor, bromoxynil, acetochlor, dicamba, clopyralide, (S-)dimethenamid, glufosinate, glyphosate, isoxaflutol, (S-)metolachlor, mesotrione, nicosulfuron, primisulfuron, Revulin Q®; rimsulfuron, sulcotrione, foramsulfuron, topramesone, tembotrione, saflufenacil, thiencarbazone, flufenacet, pyroxasulfone; insecticides for maize: carbofuran, chlorpyrifos, bifenthrin, fipronil, imidacloprid, lambda-cyhalothrin, tefluthrin, terbufos, thiamethoxam, clothianidin, spiromesifen, flubendiamide, triflumuron, rinaxipir, deltamethrin, thiodicarb, β-cyfluthrin, cyperomethrin, triflufenutrin, biflufemoron tefluthrin, tebupyrimphos, ethiprole, cyazipyr, thiacloprid, acetamiprid, dinetofuran, avermectin, methiocarb, spirodiclofen, spirotetramat; fungicides for maize: fenitropan, thiram, prothioconazole, tebuconazole, trifloxystrobin; herbicides for rice: butachlor, propanil, azimsulfuron, bensulfuron, cyhalofop, daimuron, fentrazamide, imazosulfuron, mefenacet, oxaziclomefon, pyrazosulfuron, pyributicarb, quinclorac, thiobencarb, indanophane, fluphenacet, fentrazamide, halosulfuron, oxazicycloxuloximide, benzobicyclomide oxadiargyl, ethoxysulfuron, pretilachlor, mesotrione, tefuryltrione, oxadiazon, fenoxaprop, pyrimisulfan; rice insecticides: diazinon, fenitrothion, fenobucarb, monocrotophos, benfuracarb, buprofezin, dinotefuran, fipronil, imidacloprid, isoprocarb, thiacloprid, cromafenoside, thiacloprid, dinotefuran, clothianidin, ethiprol, flubendiamide, rinaxipyr, deltamethrin, acetamiprid, sadocytamiprid, sadocytamiprid, spinotoram, emamectin benzoate, cypermethrin, chlorpyrifos, cartap, methamidophos, etofenprox, triazophos, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan2(5H)-one, carbofuran , benfuracarb; fungicides for rice: thiophanate-methyl, azoxystrobin, carpropamide, edifenphos, ferimzone, iprobenfos, isoprothiolan, pencicuron, probenazole, pyroquilon, tricyclazole, trifloxystrobin, diclocimet, fenoxanil, simeconazole, thiadinil; herbicides for cotton: diuron, fluometuron, MSMA, oxyfluorfen, promethrin, trifluralin, carfentrazone, cletodym, fluazifopbutyl, glyphosate, norflurazon, pendimethalin, pyrithiobac-sodium, trifloxysulfuron, tepraloxydim, glufosinate, flumioxazine, thidiazuron; insecticides for cotton: acephate, aldicarb, chlorpyrifos, cypermethrin, deltamethrin, malathion, monocrotophos, abamectin, acetamiprid, emamectin benzoate, imidacloprid, indoxacarb, lambda-cyhalothrin, spinosad, thiodicarb, gamma-cyhalothrin, spiromesifen, triflubennicamid, pyridalil , rinakipir, beta-cyfluthrin, spirotetramat, clothianidin, thiamethoxam, thiacloprid, dinetofuran, flubendiamide, cyazipyr, spinosad, spinotoram, gammacyhalothrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino ]furan-2(5H)-one, thiodicarb, avermectin, flonicamid, pyridalil, spiromesifen, sulfoxaflor, profenofos, triazophos, endosulfan; fungicides for cotton: etridiazol, metalaxyl, quintozene; soybean herbicides: alachlor, bentazone, trifluralin, chlorimuron-ethyl, chloransulam-methyl, fenoxaprop, fomesafen, fluazifop, glyphosate, imazamox, imazakvin, imazethapyr, (S-)metolachlor, metribuzin, pendimethalin, tepraloxydim, glufosinate; soy insecticides: lambda-cyhalothrin, methomyl, parathion, thiocarb, imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinetofuran, flubendiamide, rinaxipyr, cyazipyr, spinosad, spinotoram, emamectin benzoate, fipronil, ethiprol, deltamethrin, β-cyfluthrin, gamma- and lambda-cyhalothrin, 4-[[(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one, spirotetramat, spinodiclofen, triflumuron, flonicamid, thiodicarb, beta-cyfluthrin; soybean fungicides: azoxystrobin, cyproconazole, epoxiconazole, flutriafol, pyraclostrobin, tebuconazole, trifloxystrobin, prothioconazole, tetraconazole; sugar beet herbicides: chloridazone, desmedifam, etofumezat, phenmedifam, triallat, clopyralid, fluazifop, lenacil, metamitron, quinmerac, cycloxidim, triflusulfuron, tepraloxydim, quizalofop; sugar beet insecticides: imidacloprid, clothianidin, thiamethoxam, thiacloprid, acetamiprid, dinetofuran, deltamethrin, β-cyfluthrin, gamma/lambda-cyhalothrin, 4-[[(6-chloropyridin-3yl)methyl](2,2-difluoroethyl)amino ]furan-2(5H)-one, tefluthrin, rinaxipyr, cyaxipyr, fipronil, carbofuran; canola herbicides: clopyralid, diclofop, fluazifop, glufosinate, glyphosate, metazachlor, trifluralin, etametsulfuron, quinmerac, quizalofop, clethodim, tepraloxydim; canola fungicides: azoxystrobin, carbendazim, fludioxonil, iprodione, prochloraz, vinclozolin; canola insecticides: carbofuran organophosphorus compounds, pyrethroids, thiacloprid, deltamethrin, imidacloprid, clothianidin, thiamethoxam, acetamiprid, dinetofuran, β-cyfluthrin, gamma and lambdacyhalothrin, tau-fluvaleriate, etiprole, spinosad, spinotoram, flubendiamide, cyrinapyzipyr, 4[ [(6-chloropyridin-3-yl)methyl](2,2-difluoroethyl)amino]furan-2(5H)-one.

In some embodiments, the herbicide is atrazine, bromacyl, diuron, chlorsulfuron, metsulfuron, thifensulfuron-methyl, tribenuron, acetochlor, dicamba, isoxaflutol, nicosulfuron, rimsulfuron, pyrithiobac-sodium, flumioxazine, chlorimuron-ethyl, metribuzin, quizalofop, S-metolachlor , hexazinon, or a combination thereof.

- 56 040502

In some embodiments, the insecticide is esfenvalerate, chlorantraniliprole, methomyl, indoxacarb, oxamyl, or a combination thereof.

pesticidal and insecticidal activity.

The pest includes, without limitation, insects, fungi, bacteria, nematodes, mites, ixodid ticks, and the like. Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., in particular Lepidoptera and Coleoptera.

Those skilled in the art will appreciate that not all compounds are equally effective against all pests. The compounds of the embodiments exhibit activity against insect pests, which may include economically important pests of agronomic, forestry, greenhouse products, ornamental nursery products, food and fiber products, human and animal health products, household products. and commercial structure, household goods and storage products.

Larvae of the order Lepidoptera include, but are not limited to, cutworms, cutworms, moths, and heliotines of the family Noctuidae Spodoptera frugiperda JE Smith (grass cutworm); S. exigua Hubner (small cutworm); S. litura Fabricius (tobacco cutworm, inflorescence-eating caterpillar); Mamestra configurata Walker (Bert's cutworm); M. brassicae Linnaeus (cabbage scoop); Agrotis ipsilon Hufnagel (upsilon cutworm); A. orthogonia Morrison (rectangular cutworm); A. subterranea Fabricius (grain cutworm); Alabama argillacea Hubner (American cotton bollworm); Trichoplusia ni Hubner (Ni scoop); Pseudoplusia includens Walker (soy worm); Anticarsia gemmatalis Hubner (velvet bean scoop); Hypena scabra Fabricius (clover cutworm); Heliothis virescens Fabricius (tobacco leaflet); Pseudaletia unipuncta Haworth (meadow cutworm); Athetis mindara Barnes and Mcdunnough (rough cutworm); Eihoa messoria Harris (black-sided cutworm); Earias insulana Boisduval (Egyptian cotton bollworm); E. vittella Fabricius (spotted cutworm); Helicoverpa armigera Hubner (Bristle-legged mop cutworm); N. zea Boddie (corn bollworm or cotton bollworm); Melanchra picta Harris (scoop caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); moths, sheath-bearers, spider nest-building butterflies, cone butterflies and leaf-skeletal pests of the family Pyralidae Ostrinia nubilalis Hubner (corn stem moth); Amyelois transitella Walker (caterpillars that damage citrus hilums); Anagasta kuehniella Zeller (mill moth); Cadra cautella Walker (dried fruit moth); Chilo suppressalis Walker (yellow rice moth); C. partellus, (sorghum moth); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn moth); C. teterrellus Zincken (bluegrass moth); Cnaphalocrocis medinalis Guenee (rice leaflet); Desmia funeralis Hubner (grape leaflet); Diaphania hyalinata Linnaeus (melon moth); D. nitidalis Stoll (cucumber pickle moth); Diatraea grandiosella Dyar (southwestern corn moth), D. saccharalis Fabricius (sugarcane moth); Eoreuma loftini Dyar (Mexican rice moth); Ephestia elutella Hubner (grain moth (cocoa)); Galleria mellonella Linnaeus (large wax moth); Herpetogramma licarsisalis Walker (grass moth); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (small corn moth); Achroia grisella Fabricius (small wax moth); Loxostege sticticalis Linnaeus (meadow moth); Orthaga thyrisalis Walker (tea moth); Maruca testulalis Geyer (Acacia moth); Plodia interpunctella Hubner (Indian flour moth); Scirpophaga incertulas Walker (stem rice moth); Udea rubigalis Guenee (rusty brown moth); as well as leafworms, budworms, codling moths and caterpillar pests of fruits from the family Tortricidae Acleris gloverana Walsingham (western black-headed leafworm); A. variana Fernald (eastern black-headed leafworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller) and other species of Archips, Adoxophyes orana Fischer von Rosslerstamm (mesh leaf roller); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (codling moth); C. pomonella Linnaeus (codling moth); Platynota flavedana Clemens (variable leaf roller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermuller (European grape leaflet); Spilonota ocellana Denis & Schiffermuller (bud leaf roller); Endopiza viteana Clemens (vine leaflet); Eupoecilia ambiguella Hubner (bunch leaf roller); Bonagota salubricola Meyrick (Brazilian apple leaflet); Grapholita molesta Busck (eastern peach codling moth); Suleima helianthana Riley (sunflower leaf roller); Argyrotaenia spp.; Choristoneura spp.

Other selected pests from the order Lepidoptera include, without limitation, Alsophila pometaria Harris (autumn fruitworm); Anarsia lineatella Zeller (fruit stripe moth); Anisota senatoria J.E. Smith (saturnia orange oak); Antheraea pernyi Guerin-Meneville (Chinese oak silkworm); Bombyx mori Linnaeus (silkworm); Bucculatrix thurberiella Busck (crooked cotton moth); Colias eurytheme Boisduval (alfalfa jaundice); Datana integerrima Grote & Robinson (nut corydalis); Dendrolimus sibiricus Tschetwerikov (Siberian silkworm), Ennomos subsignaria Hubner (elm moth); Erannis tiliaria Harris (linden moth); Euproctis chrysorrhoea Linnaeus (golden silkworm); Harrisina americana Guerin-Meneville (American pyromorph); Hemileuca oliviae Cockrell (Saturnia butterfly caterpillar); Hyphantria cunea Drury (American white butterfly); Keiferia lycopersicella Walsingham (tomato moth); Lambdina fiscellaria fiscellaria Hulst (moth moth

- 57 040502 hemlock oriental); L. fiscellaria lugubrosa Hulst (Western Hemlock Moth); Leucoma salicis Linnaeus (willow waveweed); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five-spot hawk, tomato hawk hawk); M. sexta Haworth (tomato hawk hawk, tobacco hawk hawk); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring moth); Papilio cresphontes Cramer (cresfontes sailfish, orange dog); Phryganidia californica Packard (California ringed cocoon moth); Phyllocnistis citrella Stainton (citrus miner); Phyllonorycter blancardella Fabricius (bottom moth); Pieris brassicae Linnaeus (large cabbage white); P. rapae Linnaeus (small cabbage white); R. napi Linnaeus (rutabaga whitefish); Platyptilia carduidactyla Riley (Artichoke Fingerwing); Plutella xylostella Linnaeus (cabbage moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval and Leconte (checkered whitefish); Sabulodes aegrotata Guenee (omnivorous moth); Schizura concinna J.E. Smith (corydalis); Sitotroga cerealella Olivier (Angumois barley moth); Thaumetopoea pityocampa Schiffermuller (Pine walking silkworm); Tineola bisselliella Hummel (room moth); Tuta absoluta Meyrick (tomato moth); Yponomeuta padella Linnaeus (ermine fruit moth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults from the order Coleoptera, including weevils from the families Anthribidae, Bruchidae and Curculionidae (including but not limited to Anthonomus grandis Boheman (cotton weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus (granary weevil) ; S. oryzae Linnaeus (rice weevil); Hypera punctata Fabricius (point weevil); Cylindrocopturus adspersus LeConte (stem sunflower weevil); Smicronyx fulvus LeConte (red sunflower weevil); S. sordidus LeConte (gray sunflower weevil); Sphenophorus maidis Chittenden ( maize weevil)); ground beetles, cucumber leaf beetles, root worms, leaf beetles, potato beetles and leaf miners of the family Chrysomelidae (including but not limited to Leptinotarsa decemlineata Say (Colorado potato beetle); Diabrotica virgifera virgifera LeConte (western corn beetle); D. barberi Smith and Lawrence (northern corn beetle); D. undecimpunctata howardi Barber (southern corn beetle); Chaetocnema pulicaria Melsheimer (ground corn beetle); Phyllotreta crueferae Goeze (cruciferous beetle); Phyllotreta strlolata (striped beetle); Colaspis brunnea Fabricius (vine leaf beetle); Oulema melanopus Linnaeus (red-breasted beetle); Zygogramma exclamatlonls Fabricius (sunflower leaf beetle)); beetles from the family Coccinellidae (including but not limited to Epilachna varivestis Mulsant (Mexican bean ladybug)); beetles and other beetles from the family Scarabaeidae (including, without limitation: Popillia japonica Newman (Japanese beetle); Cyclocephala borealis Arrow (Northern double-beam, common beetle); C. immaculata Olivier (Southern double-beetle, common beetle); Rhlzotrogus majalls Razoumowsky (European beetle) ; Phyllophaga crinita Burmeister (beetle larva); Ligyrus gibbosus De Geer (carrot beetle)); skin beetles of the family Dermestidae; wireworms from the family Elateridae, Eleodes spp., Melanotus spp.; Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae; and beetles from the family Tenebrionidae.

Of interest are adults and immature specimens of the order Diptera, including leafminers Agromyza parvicornis Loew (corn leaf miner); gall midges (including but not limited to Contarinia sorghicola Coquillett (varietal gall midge); Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Gehin (grass orange midge); Neolasioptera murtfeldtiana Felt, (sunflower midge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (fruit flies); flower flies (including but not limited to: Delia platura Meigen (sprout fly); D. coarctata Fallen (winter fly) and other Delia spp., Meromyza americana Fitch (American meromys); Musca domestica Linnaeus (houseflies); Fannia canicularis Linnaeus , F. femoralis Stein (small house flies), Stomoxys calcitrans Linnaeus (autumn stingers)); field flies, stingers, meat flies, Chrysomya spp.; Phormia spp. and other pest flies of the superfamily Muscoidea, horseflies Tabanus spp.; nasopharyngeal gadflies Gastrophilus spp.; Oestrus spp.; bull gadflies Hypoderma spp.; variegated Chrysops spp.; Melophagus ovinus Linnaeus (sheep bloodsuckers) and other Brachycera, Aedes spp. mosquitoes; Anopheles spp.; Culex spp.; midges Prosimulium spp.; Simulium spp.; midges, mosquitoes, sciarids and other Nematocera.

As insects of interest, adults and nymph-stage insects of the orders Hemiptera and Homoptera are included, such as, but not limited to, Hermes of the family Adelgidae, horseflies of the family Miridae, cicadas of the family Cicadidae, leafhoppers, Empoasca spp.; from the family Cicadellidae, insects of the superfamily Fulgoroidea from the families Cixiidae, Flatidae, Fulgoroidea, Issidae and Delphacidae, humpbacks from the family Membracidae, psyllids from the family Psyllidae, whiteflies from the family Aleyrodidae, aphids from the family Aphididae, phylloxera from the family Phylloxeridae, mealybugs from the family Pseudococcidae, mealybugs from the families Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae and Margarodidae, laceworms from the family Tingidae, bugs from the family Pentatomidae, turtle bugs, Blissus spp.; and other terrestrial bugs from the Lygaeidae family, penny bugs from the Cercopidae family, shorebirds from the Coreidae family, as well as red bugs and cotton bugs from the Pyrrhocoridae family.

Agriculturally important members of the order Homoptera further include, without limitation: Acyrthisiphon pisum Harris (pea aphid); Aphis craccivora Koch (aphid

- 58 040502 alfalfa); A. fabae Scopoli (beet aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (citrus green aphid); Aulacorthum solani Kaltenbach (common potato aphid); Chaetosiphon fragaefolii Cockerell (American strawberry aphid); Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantaginea Paaserini (pink apple aphid); Eriosoma lanigerum Hausmann (apple blood aphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopterus pruni Geoffroy (plum pollinated aphid); Lipaphis erysimi Kaltenbach (false cabbage aphid); Metopolophium dirrhodum Walker (roseweed aphid); Macrosiphum euphorbiae Thomas (large potato aphid); Myzus persicae Sulzer (peach aphid, greenhouse aphid); Nasonovia ribisnigri Mosley (lettuce green aphid); Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch (varietal aphid); R. padi Linnaeus (common bird cherry aphid); Schizaphis graminum Rondani (common grass aphid); Sipha flava Forbes (yellow sugar cane aphid); Sitobion avenae Fabricius (leaf aphid); Therioaphis maculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer de Fonscolombe (orange aphid) and T. citricida Kirkaldy (citrus aphid); Adelges spp. (hermes); Phylloxera devastatrix Pergande (hickory phylloxera); Bemisia tabaci Gennadius (tobacco whitefly, cotton whitefly); B. argentifolii Bellows and Perring (magnolia whitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus (striped-winged whitefly) and T. vaporariorum Westwood (hothouse whitefly); Empoasca fabae Harris (potato leafhopper); Laodelphax striatellus Fallen (dark leafhopper); Macrolestes quadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal (rice leafhopper); Nilaparvata lugens Stal (brown rice leafhopper); Peregrinus maidis Ashmead (corn leafhopper); Sogatella furcifera Horvath (white-backed leafhopper); Sogatodes orizicola Muir (rice plantworm); Typhlocyba pomaria McAtee (apple leafhopper); Erythroneoura spp. (vine leafhoppers); Magicicada septendecim Linnaeus (periodic cicada); Icerya purchasi Maskell (Australian grooved mealybug); Quadraspidiotus perniciosus Comstock (California scale insect); Planococcus citri Risso (grape mealybug); Pseudococcus spp. (other mealybugs); Cacopsylla pyricola Foerster (pear psyllid); Trioza diospyri Ashmead (persimmon psyllid).

Species of agricultural interest in the order Hemiptera include, without limitation: Acrosternum hilare Say (green stinkweed); Anasa tristis De Geer (sad bug); Biissus leucopterus leucopterus Say (turtle bug); Corythuca gossypii Fabricius (cotton lace); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellus Herrich-Schaffer (cotton red bug); Euschistus servus Say (brown stink bug); E. variolarius Palisot de Beauvois (single-spotted shield beetle); Graptostethus spp. (complex of ground bugs); Leptoglossus corculus Say (pine edge bug); Lygus lineolaris Palisot de Beauvois (meadow bug); L. hesperus Knight (western matted horsefly); L. pratensis Linnaeus (field bug); L. rugulipennis Poppius (European grass bug); Lygocoris pabulinus Linnaeus (green horsefly); Nezara viridula Linnaeus (green vegetable bug); Oebalus pugnax Fabricius (rice shield bug); Oncopeltus fasciatus Dallas (large euphorbia bug); Pseudatomoscelis seriatus Reuter (cotton bug).

In addition, embodiments may be effective against Hemiptera such as Calocoris norvegicus Gmelin (potato bug); Orthops campestris Linnaeus; Plesiocoris rugicollis Fallen (northern apple bug); Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant (horse bug); Spanagonicus albofasciatus Reuter (white-spotted horsefly); Diaphnocoris chlorionis Say (bed bug of honey locust); Labopidicola allii Knight (onion horsefly); Pseudatomoscelis seriatus Reuter (cotton bug); Adelphocoris rapidus Say (quick bug); Poecilocapsus lineatus Fabricius (four-lined horsefly); Nysius ericae Schilling (heather nisius); Nysius raphanus Howard (false turtle); Nezara viridula Linnaeus (green vegetable bug); Eurygaster spp.; Coreidae spp.; Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp. and Cimicidae spp.

Also included are adults and larvae from the order Acari (mites), such as Aceria tosichella Keifer (wheat gall mite); Petrobia latens Muller (polyphagous petrobia); spider mites and red mites of the family Tetranychidae, Panonychus ulmi Koch (red fruit mites); Tetranychus urticae Koch (common spider mite); (T. mcdanieli McGregor (McDaniel's tick); T. cinnabarinus Boisduval (red spider mite); T. turkestani Ugarov & Nikolski (Turkestan spider mite); flat mites of the family Tenuipalpidae, Brevipalpus lewisi McGregor (orange mite); rust and bud mites of the family Eriophyidae and other leaf-eating mites, as well as mites important to human and animal health, i.e. dust mites of the family Epidermoptidae, iron mites of the family Demodicidae, grain mites of the family Glycyphagidae, ixodid mites of the order Ixodidae Ixodes scapularis Say (black-legged tick); I. holocyclus Neumann (Australian paralytic tick), Dermacentor variabilis Say (Ixodes dog tick), Amblyomma americanum Linnaeus (Amblyomma Ixodes tick), and horse and scabies mites of the families Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of interest from the order Thysanura are, for example, Lepisma saccharina Linnaeus (silverfish); Thermobia domestica Packard (thermobia).

Additional arthropod pests covered include: Araneae spiders such as Loxosceles reclusa Gertsch and Mulaik (brown recluse) and Latrodectus mactans Fabricius (black

- 59 040502 widow), as well as centipedes from the order Scutigeromorpha, such as Scutigera coleoptrata Linnaeus (common flycatcher).

The pest of interest includes the superfamily of stink bugs and other related insects, including without exception species belonging to the family Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorus guildini, Euschistus servus, Acrosternum hilare, Euschistus heros, Euschistus tristigmus, Acrosternum hilare , Dichelops furcatus, Dichelops melacanthus, and Bagrada hilaris (the bug of the genus Bagrada)), the family Plataspidae (Megacopta cribraria hemispherical bug) and the family Cydnidae (Scaptocoris castanea - brown bug digger), as well as species of Lepidoptera, including without limitation: cabbage moth, for example Helicoverpa zea Boddie; soybean cutworm such as Pseudoplusia includens Walker; and velvet bean cutworm such as Anticarsia gemmatalis Hubner.

Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang, (1990) J. Econ. Entomol. 83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone, et al., (1985) J. of Economic Entomology 78:290-293 and US Pat. No. 5,743,477, all of which are incorporated herein by reference in their entirety. Typically, the protein is mixed and used in nutritional analyses. See, for example, Marrone, et al., (1985) J. of Economic Entomology 78:290293. Such assays may include bringing the plants into contact with one or more pests and determining the plant's ability to survive and/or kill the pests.

Nematodes include parasitic nematodes such as root-knot, cyst-forming and wounding nematodes, including Heterodera spp., Meloidogyne spp. and Globodera spp.; in particular representatives of cyst nematodes, including without limitation Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode), as well as Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Wounding nematodes include Pratylenchus spp.

Seed treatment agent.

For protection and to increase yields and improve trait improvement technologies, additional seed treatments can provide additional crop adaptability and cost-effective control of insects, weeds and diseases. The seed material can be treated, usually surface treated, with a composition containing combinations of chemical or biological herbicides, herbicide safeners, insecticides, fungicides, germination inhibitors and enhancers, nutrients, plant growth regulators and activators, bactericides, nematicides, avicides and/or molluscicides. These compounds are usually formulated with additional carriers, surfactants or application aids conventionally used in the formulation art. Coatings can be applied by impregnating the propagation material with a liquid formulation or by coating with a combination of wet or dry formulation. Examples of various types of compounds that can be used as seed treatments are provided in The Pesticide Manual: A World Compendium, C.D.S. Tomlin Ed., published by the British Crop Production Council, which is hereby incorporated by reference herein.

Some seed treatments that can be applied to crop seeds include, without limitation, one or more of abscisic acid, acibenzolar-S-methyl, avermectin, amitrol, azaconazole, azospirillum, azadirachtin, azoxystrobin, Bacillus spp. (including one or more of the species cereus, firmus, megaterium, pumilis, sphaericus, subtilis and/or thuringiensis), bradyrhizobium spp. (including one or more of betae, canariense, elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/or yuanmingense), captane, carboxin, chitosan, clothianidin, copper, cyazipyr, difenoconazole, ethidiazole, fipronil, fludioxonil, fluoxastrobin, fluquinconazole , flurazol, fluxofenim, harpin protein, imazalil, imidacloprid, ipconazole, isoflavenoids, lipochitooligosaccharide, mancozeb, manganese, maneb, mefenoxam, metalaxyl, metconazole, myclobutanil, PCNB, penflufen, pinecillum, pentiopyrad, permethrhinazoline, pyroxystrobin, pyroxystrobin, pyroxystrobin, S-metolachlor, saponin, sedaxan, TSMTB, tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram, tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin, triticonazole and/or zinc. The PCNB seed coating, EPA registration number 00293500419, contains quintozene and terrasol. TCMTB stands for 2-(thiocyanomethylthio)benzothiazole.

Seed varieties and seeds with specific transgenic traits can be tested to determine what additional seed treatments and application rates can complement such varieties and transgenic traits to increase yield. For example, a cultivar with good yield potential but susceptibility to loose smut may benefit from a seed treatment that provides protection against loose smut, a cultivar with good yield potential but susceptibility to cyst nematodes may benefit from a seed treatment which provides protection against cyst nematodes, etc. Similarly, a cultivar comprising a transgenic trait that confers insect resistance may benefit from a second mechanism of action conferred by a seed treatment, a cultivar comprising a transgenic trait that confers herbicide resistance may benefit from a seed treatment with an antidote that increases the resistance of plants to such a herbicide, etc. In addition, good establishment and early emergence that result from the correct application of a seed treatment can result in more efficient nitrogen utilization, better drought tolerance, and an overall increase in potential yield of a variety or varieties containing a particular trait when combined with a seed treatment. seeds.

Methods for the destruction of an insect pest and control of insect populations.

In some embodiments, methods are provided for controlling an insect pest, comprising bringing the insect pest, either simultaneously or sequentially, into contact with an insecticidally effective amount of a recombinant IPD090 polypeptide or a chimeric IPD090 polypeptide of the present invention. In some embodiments, methods are provided for killing an insect pest, comprising bringing the insect pest into contact with an insecticidally effective amount of a recombinant pesticide protein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, SEQ ID NO: 384 or variants thereof.

In some embodiments, methods are provided to control an insect pest population, comprising contacting the insect population, either simultaneously or sequentially, with an insecticidally effective amount of a recombinant IPD090 polypeptide or a chimeric IPD090 polypeptide of the present invention. In some embodiments, methods are provided for controlling an insect pest population, comprising contacting the insect population with an insecticidally effective amount of a recombinant IPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ, SEQ ID NO: 379, SEQ ID NO: 384 or a variant thereof. The terms pest control or pest control used in this document refer to any effect on a pest that results in limiting the harm caused by the pest. Pest control includes, without limitation, killing the pest, suppressing the development of the pest, changing the fertility or growth of the pest so that the pest causes less harm to the plant, reducing the number of offspring produced, making pests less fit, making pests more susceptible to predation, or keeping pests away. from eating the plant.

In some embodiments, methods are provided to control a population of a pesticidal protein-resistant insect pest comprising contacting the pest population, either simultaneously or sequentially, with an insecticidally effective amount of a recombinant IPD090 polypeptide or a chimeric IPD090 polypeptide of the present invention. In some embodiments, methods are provided to control a population of a pesticidal protein resistant insect pest comprising contacting the pest population with an insecticidally effective amount of a recombinant IPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 , SEQ ID NO: 379, SEQ ID NO: 384, or variants thereof.

In some embodiments, methods are provided for protecting a plant from an insect pest, comprising causing expression in the plant or cell of at least one recombinant polynucleotide encoding an IPD090 polypeptide or a chimeric IPD090 polypeptide. In some embodiments, methods are provided for protecting a plant from an insect pest, comprising causing expression in the plant or its cell of a recombinant polynucleotide encoding an IPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, SEQ ID NO: 384 or variants thereof.

In some embodiments, methods are provided for protecting a plant from an insect pest, comprising causing expression in the plant or its cell of a recombinant polynucleotide encoding a polypeptide of SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, or its options. In some embodiments, methods are provided for protecting a plant from an insect pest, comprising causing the recombinant polynucleotide of SEQ ID NO: 381, SEQ ID NO: 382, or SEQ ID NO: 383 to be expressed in the plant or its cell.

Insect resistance management (IRM) strategies.

Expression of B. thuringiensis δ-endotoxins in transgenic maize has been shown to be an effective means of controlling agriculturally important insect pests (Perlak, et al., 1990; 1993). However, insects have emerged that are resistant to B. thuringiensis δ-endotoxins expressed in transgenic plants. Such resistance, if it becomes widespread, would clearly limit the commercial value of germplasm containing genes encoding such B. thuringiensis δ-endotoxins.

One way to increase the effectiveness of transgenic insecticides against target pests while reducing the development of insecticide-resistant pests is to use derived non-transgenic (i.e., with a non-insecticide protein) refugia (non-insecticide crop/maize section) to apply transgenic crops,

- 61 040502 producing one insecticidal protein active against target pests. The United States Environmental Protection Agency (ep.gov/oppbppdh/biopesticides/pips/bt_corn_refUge_2006.htm, available with the prefix www) publishes requirements for the use of transgenic crops that produce a single Bt protein active against target pests. In addition, the National Corn Growers Association, on its website: (ncga.com/insect-resistance-management-factsheet-bt-corn, which can be accessed using the prefix www) also offers similar guidance regarding refugee requirements. Due to losses caused by insects within the refugia zone, larger refugia can reduce overall yield.

Another way to increase the effectiveness of transgenic insecticides against target pests and at the same time reduce the development of insecticide-resistant pests would be to store insecticidal genes that are effective against groups of insect pests and that exert their effects through different mechanisms of action.

Expression in a plant of two or more insecticidal compositions that are toxic to the same insect species, with each insecticide being expressed at effective levels, would be another way to achieve resistance development control. This is based on the principle that the evolution of resistance to two separate mechanisms of action is much less likely than to only one. For example, Roush describes a two-toxin strategy, also called pyramiding or bundling, for managing insecticidal transgenic crops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998) 353:1777-1786). Bundling or pyramiding of two different proteins, each of which is effective against the target pests, and with little or no cross-resistance, may allow less refugia to be applied. The US EPA requires significantly less (typically 5%) structured refugia for planting non-Bt corn than for single trait products (typically 20%). There are various ways to achieve the effects of IRM refugia, including various geometric planting patterns in the fields and seed-in-bag mixes, as discussed further by Roush.

In some embodiments, the IPD090 polypeptides of the present invention are useful as an insect resistance management strategy in combination (i.e., pyramided) with other pesticidal proteins, including but not limited to Bt toxins, Xenorhabdus sp. insecticidal proteins. or Photorhabdus sp., other insecticidally active proteins, and the like.

Methods are provided for controlling infestation(s) by insects from the order Lepidoptera and/or Coleoptera of a transgenic plant, which provide management of insect resistance, providing for the expression in the plant of at least two different insecticidal proteins characterized by different mechanisms of action.

In some embodiments, methods for controlling infestation by insects from the order Lepidoptera and/or Coleoptera of a transgenic plant and assisting in the management of insect resistance include providing at least one of the insecticidal IPD090 protein polypeptides to insects from the order Lepidoptera and/or Coleoptera.

In some embodiments, methods for controlling infestation by insects from the order Lepidoptera and/or Coleoptera of a transgenic plant and assisting in the management of insect resistance comprise providing at least one of the IPD090 polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 , SEQ ID NO: 379, SEQ ID NO: 384 or their variants, insecticidal against insects from the order Lepidoptera and/or Coleoptera.

In some embodiments, methods for controlling infestation by insects from the order Lepidoptera and/or Coleoptera of a transgenic plant and assisting in the management of insect resistance include providing expression in the transgenic plant of an IPD090 polypeptide and a Cry protein or other protein that is insecticidal against insects from the order of Lepidoptera and/or Coleoptera, however, they are characterized by different mechanisms of action.

In some embodiments, methods for controlling infestation by insects from the order Lepidoptera and/or Coleoptera of a transgenic plant and assisting in the management of insect resistance include providing expression in the transgenic plant of the IPD090 polypeptide of SEQ ID NO: 2, SEQ ID nO: 4, SEQ ID NO: 6, SEQ ID NO: 379, SEQ ID NO: 384 or variants thereof, and the Cry protein or other insecticidal protein against insects from the order Lepidoptera and/or Coleoptera, wherein the IPD090 polypeptide and the Cry protein have different mechanisms of action.

Also provided are methods for reducing the likelihood of resistance in insects from the order Lepidoptera and/or Coleoptera to transgenic plants expressing insecticidal proteins in plants for controlling insect species, providing for the expression of an IPD090 polypeptide, insecticidal against insect species, in combination with a second protein, insecticidal in species of insects, while they are characterized by different mechanisms of action.

Means are also provided for the effective management of the resistance of insects from the order Lepidoptera and/or Coleoptera to transgenic plants, providing for the provision of joint

- 62 040502 expression at high levels in plants of two or more insecticidal proteins toxic to insects from the order Lepidoptera and/or Coleoptera, but each exhibits a different mechanism for carrying out its activity in relation to destruction, where two or more insecticidal proteins provide an IPD090 polypeptide and Cry protein. Means are also provided for effectively managing the resistance of insects of the order Lepidoptera and/or Coleoptera to transgenic plants, providing for co-expression at high levels in plants of two or more insecticidal proteins that are toxic to insects of the order Lepidoptera and/or Coleoptera, but each exhibits a different mechanism for carrying out its activity in relation to destruction, where two or more insecticidal proteins are an IPD090 polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, SEQ ID NO: 384 or its variant and the Cry protein or other isecticidally active protein.

Also provided are methods of obtaining regulatory approval for growing or commercializing plants expressing proteins that are insecticidal against insects from the order Lepidoptera and/or Coleoptera, comprising the step of requesting, providing, or linking to data from insect protein binding assays demonstrating that the IPD090 polypeptide does not compete with binding sites for Cry proteins in such insects. Also provided are methods of obtaining regulatory approval for the cultivation or commercialization of plants expressing proteins that are insecticidal against insects of the order Lepidoptera and/or Coleoptera, comprising the step of requesting, providing, or linking to data from insect protein binding assays showing that the IPD090 polypeptide with SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 379, SEQ ID NO: 384 or variants thereof do not compete with binding sites for Cry proteins in such insects.

Ways to increase plant productivity.

There are ways to increase productivity. The methods include obtaining a plant or plant cell expressing a polynucleotide encoding a pesticidal polypeptide sequence disclosed herein, and growing the plant or its seed in a field infested with a pest against which the polypeptide has pesticidal activity. In some embodiments, the polypeptide has pesticidal activity against a Lepidoptera, Coleoptera, Diptera, Hemiptera, or Nematode pest and the field is infested with a Lepidoptera, Hemiptera, Coleoptera, Diptera, or Nematode pest.

As defined herein, plant yield refers to the quality and/or amount of biomass produced by a plant. The term biomass as used herein refers to any measured plant product. An increase in biomass production is any improvement in the yield of a measured plant product. Increasing the yield of a plant has several commercial applications. For example, an increase in the biomass of the leaves of a plant can lead to an increase in the yield of leafy vegetables for human or animal consumption. In addition, the increase in leaf biomass can be used to increase the production of pharmaceutical or industrial plant products. An increase in yield may include any statistically significant increase, including, but not limited to, at least 1% increase, at least 3% increase, at least 5% increase, at least 10% increase, at least 20% increase, by at least 30% increase, at least 50% increase, at least 70% increase, at least 100% or more increase in yield compared to a plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improved pest resistance of a plant expressing the IPD090 polypeptide disclosed herein. Expression of the IPD090 polypeptide results in a reduced ability of the pest to infect or feed on the plant, thus improving plant yield.

Processing methods.

Additionally provided are methods for processing a plant, plant part, or seed to produce a food or feed product from a plant, plant part, or seed containing an IPD090 polynucleotide. The plants, plant parts, or seeds provided herein can be processed to produce oil, protein products, and/or by-products that are processed derivatives that are of commercial value. Non-limiting examples include transgenic seeds containing a nucleic acid molecule encoding an IPD090 polypeptide that can be processed to produce soybean oil, soy products and/or soy by-products.

Processing refers to any physical and chemical methods used to produce any soy product and includes, without limitation, heat conditioning, rolling and milling, extrusion, solvent extraction or soaking in water, and extraction of whole or crushed seeds.

The following examples are provided for illustration and not limitation.

- 63 040502

Experimental part.

Example 1 Identification of an insecticidal protein active against the western corn bug (Diabrotica virgifera virgifera LeConte - WCRW) from strain JH34071-1.

The insecticidal protein, designated IPD090Aa, was identified by protein purification, N-terminal amino acid sequencing, and PCR cloning from Pseudomonas sp. strain JH34071-1. in the following way. Insecticidal activity against WCRW was observed with cell lysate JH34071-1 grown in tryptic soy broth (TSB, casein peptone 15 g/l; soy peptone 5.0 g/l; NaCl 5.0 g/l) and cultured for 1 day at 28°C with shaking at 200 rpm. This insecticidal activity was characterized by sensitivity to heat and protease, which indicated a proteinaceous nature.

Biological assays with WCRW were performed using cell lysate samples mixed with Diabrotica diet (Frontier Agricultural Sciences, Newark, Delaware) in a 96-well format. WCRW neonates were placed in each well of a 96-well plate. The analysis was carried out for four days at 25° C. and then insect mortality and insect stunting were assessed. Scores were dead (3), severely stunted (2) (little or no growth, but alive), stunted (1) (growth to second larval stage, but not equivalent to controls) or no activity (0) . Samples demonstrating mortality or severe arrest were studied further.

The genomic DNA of the isolated strain JH34071-1 was prepared according to the library construction protocol developed by Illumina and sequenced using the Illumina® Genome Analyzer IIx (Illumina Inc., 9885 Towne Center Drive, San Diego, CA 92121). The nucleic acid contig sequences were assembled and open reading frames were obtained. The 16S ribosomal DNA sequence of strain JH34071-1 was searched with BLAST™ against the NCBI database, identifying strain JH34071-1 as belonging to a species of the genus Pseudomonas.

The JH34071-1 cell pellet was homogenized at ~30,000 psi after resuspension in 20 mM Tris buffer, pH 8, with a complete, EDTA-free protease inhibitor mixture (Roche, Indianapolis, Ind.). The crude lysate was clarified by centrifugation and brought to 75% saturation with ammonium sulfate. The 75% ammonium sulfate solution was then centrifuged and the supernatant was discarded. A portion of the pellet was suspended in 20 mM Tris, pH 8.0 and then adjusted to 1.5 M ammonium sulfate by adding 2 M ammonium sulfate, 20 mM Tris, pH 8.0. This solution was clarified and loaded onto a TSKgel™ Phenyl-5PW column (Tosoh Bioscience, Tokyo, Japan) equilibrated in 20 mM Tris, pH 8.0, 1.5 M ammonium sulfate. Insecticidal activity was eluted with a gradient to 20 mM Tris, pH 8. Active fractions were pooled, concentrated on centrifuge concentrators with a molecular weight limit of 10 kDa (Sartorius Stedim, Göttingen, Germany) and desalted in 20 mM piperazine, pH 9.5, using Sephadex G25 columns (GE Healthcare, Piscataway, NJ). The desalted pool was loaded onto a Mono Q™ column (GE Healthcare, Piscataway, NJ) equilibrated in 20 mM piperazine, pH 9.5, and eluted with a 0-0.4 M NaCl gradient. Active fractions were pooled and loaded onto a Superdex™ 200 column (GE Healthcare), equilibrated in phosphate buffered saline (PBS). In SDS-PAGE analysis of the fractions, it was noted that the activity against WCRW coincided with a pronounced band after staining with GelCode™ blue staining reagent (Thermo Fisher Scientific®). The protein strip was excised, digested with trypsin, and analyzed by nano-liquid chromatography/tandem mass spectrometry (nano-LC/ESI-MS/MS) on a Thermo Q Exactive™ Orbitrap™ mass spectrometer (Thermo Fisher Scientific®, 81 Wyman Street , Waltham, MA, 02454) coupled to an Eksigent™ NanoLC™ 1-D Plus nano-lc system (AB Sciex™, 500 Old Connecticut Path, Framingham, MA, 01701). Protein identification was performed by internal database search using Mascot® (Matrix Science, 10 Perrins Lane, London, NW3 1QY, UK), whereby the IPD090Aa polypeptide (SEQ ID NO: 2) encoded by the polynucleotide of SEQ ID NO: 1. The insecticidal activity of the IPD090Aa polypeptide (SEQ ID NO: 2) against WCRW was confirmed by cloning and recombinant expression.

Example 2 Identification of IPD090Aa homologues.

Gene identity can be determined by performing BLAST™ searches (Basic Local Alignment Finder 20; Altschul, et al. (1993) J. Molec. Biol. 215: 403-410; see also ncbi.nlm.nih.gov/BLAST/ , which can be accessed using the prefix www) with standard sequence similarity parameters contained in the public BLAST database (containing all non-redundant CDS translations from GenBank, sequences derived from the 3-dimensional structure of the Brookhaven and DDBJ databases. In addition to public databases, DuPont Pioneer databases were searched IPD090Aa (SEQ ID NO: 2) showed distant homology to proteins that have Pfam ID no. : en.wikipedia.org/wiki/Pfam, which can be accessed using the www prefix) Several identified homologues of the IPD090 polypeptide Aa (SEQ ID NO: 2), characterized by different percentage identity, are shown in table. 1. In the table. 2 shows a matrix table of pairwise identity relationships for IPD090 global homologue alignments based on the ClustalW algorithm implemented using the ALIGNX® module of the Vector NTI® software package (Invitrogen Corporation, Carlsbad, CA) with all standard parameters.

Table 1

Protein identification number Identity with IPD090Aa Strain identification number View Polynucleus OTID Polypep TID IPD090Aa JH34071-1, SSP342A9-1 Pseudomonas sp. SEQ ID NO: 1 SEQ ID NO: 2 IPD090A6 99.8% SS342A7-1 Pseudomonas monteilii SEQ ID NO: 3 SEQ ID NO: 4 IPD090Ca 79.3% JH23589-1, JH23611-2, JH23959-1, JH59556-2, JH61488-2, JH62159-2, JH62167-1, JH62246-2, JH62258-1, JH62270-2, and JH62417-2 Pseudomonas entomophila SEQ ID NO: 5 SEQ ID NO: 6 IPD090Fa 49.4% Number access to GenBank No. WP_019961352 woodsholea maritima SEQ ID NO: 7 SEQ ID NO: 8 IPD090AC 89.1% SSP1049E7- Pseudomonas monteilii SEQ ID NO: 380 SEQ ID NO: 384 IPD090Ga 38.9 Number access to GenBank No. WP_012039071 Clavibacter michiganens is SEQ ID NO:381 SEQ ID NO: 385 IPD090Gb 35.6 Number access to GenBank No. WP_012145116 Serratia proteamaculans SEQ ID NO: 382 SEQ ID NO: 386 IPD090GC 37.9 Number access to GenBank No. WP_046018755 . 1 Marinomonas sp. SEQ ID NO: 383 SEQ ID NO:387 IPD090Gd 36.2 Number access to GenBank No. WP_073439185 Serratia plymuthica SEQ ID NO: 388

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table 2

IPD090Ab SEQ ID NO: 4 IPD090AC SEQ ID NO: IPD090Ca SEQ ID NO: 6 IPD090Cd SEQ ID NO: 379 IPD090Fa SEQ ID NO: 8 IPD090Ga SEQ ID NO: 385 IPD090Gb SEQ ID NO: 386 IPD090GC SEQ ID NO: 387 IPD090Gd SEQ ID NO: 388 IPD090Aa SEQ ID NO: 2 99.8 89.1 79.3 79.8 50.8 38.9 35.6 37.9 36.2 IPD090Ab SEQ ID NO: 4 88.9 79.1 79.5 50.8 38.7 35.4 37.7 36.0 IPD090AC SEQ ID NO: 384 — — 76.8 77.6 48.9 36.8 35.1 36.6 35.4 IPD090Ca SEQ ID NO: 6 80.3 48.5 36.2 35.5 36.9 36.9 IPD090Cd SEQ ID NO: 379 47.2 37.7 36, 3 37.7 38.7 IPD090Fa SEQ ID NO: 8 — — — — — 34.5 34.3 39.4 35.3 IPD090Ga SEQ ID NO: 385 34.3 32.7 33.5 IPD090Gb SEQ ID NO: 386 33.5 82.8 IPD090GC SEQ ID NO: 387 32.9

Using genome sequencing of a pool of bacterial strains, a polynucleotide with SEQ ID NO: 378 was identified, encoding an IPD090 homologue, IPD090Cd (SEQ ID NO: 379), characterized by ~80% amino acid sequence identity with IPD090Ca (SEQ ID NO: 6).

Example 3 Expression of IPD090Aa in E. coli.

Peptide fragments from the MS analysis were used to position the coding sequence for IPD090Aa (SEQ ID NO: 1) in the JH34071-1 contig. In addition, N-terminal sequencing was used to confirm the predicted site of initiation. The coding sequence was used to construct primers CTB54-FOR (SEQ ID NO: 354) and CTB55-REV (SEQ ID NO: 355) to clone the coding sequence IPD090Aa (SEQ ID NO: 1) (with native TAG stop codon) into pET- 24a (Novagen®) for unlabeled translation and pET-14b (Novagen®) for N-terminal translation of the 6X-His tag using NdeI/XhoI sites. In addition, the coding sequence was used to design primers CTB54-FOR (SEQ ID NO: 354) and CTB56-REV (SEQ ID NO: 356) to clone the coding sequence IPD090Aa (SEQ ID NO: 1) (without stop codon) into pET-24a (Novagen®) for C-terminal translation of the 6X-His tag using NdeI/XhoI sites. KOD Hot Start Master Mix (EMD Biosciences, San Diego, CA) was used for PCR amplification of the IPD090Aa gene in a Bio-Rad™ C1000 Touch™ thermal cycler. Cycle parameters were as follows: 1 cycle at 95° C. for 2 minutes; 35 cycles at 95°C for 20 s, 60°C for 10 s and 70°C for 15 s; 1 cycle at 70°C for 2 min. Amplicons were gel purified, ligated (T4 DNA ligase, New England BioLabs, Ipswich, MA) into expression vectors (as described above), transformed into high efficiency chemically competent E. coli One Shot® TOP10 cells (Invitrogen™ - Thermo Fisher Scientific, 81 Wyman Street, Waltham, MA) and clones were verified by sequencing.

Expression constructs IPD090Aa with N-terminal 6X His (SEQ ID NO: 346) and IPD090Aa with C-terminal 6X-His (SEQ ID NO: 348) were transformed into E. coli BL21 cells (DE3, Agilent, Santa Clara, CA) for expression. . Cultures in one liter of Luria broth (containing the appropriate antibiotic) were grown to reach an OD ₆₀₀ of about 0.6 and then cultures were induced with 0.3 mM isopropyl-v-D-1-thiogalactopyranoside (IPTG) and allowed to grow for additional 18 hours at 16°C, 100 rpm. Cultures were centrifuged at 5000 rcf for 15 min to pellet the cells. The cell pellet was lysed with 1/4 B-PER™ II reagent (Thermo Scientific), 20 mM Tris pH 8.0, OmniCleave™ endonuclease (Epicentre), ReadyLyse™ lysozyme (Epicentre), and HALT™ protease inhibitors (Life Technologies) in for 30 min with rocking at room

- 66 040502 temperature. Lysates were clarified by centrifugation at 13,000 rcf for 10 min and supernatants were transferred to 10 mM imidazole and then loaded onto separate 2.5 ml Ni-NTA columns (QIAGEN® Inc., Valencia, CA 91355) equilibrated with PBS, 10 mM imidazole. The columns were washed with 5 ml of 20, 40 and 80 mm imidazole in PBS. Recombinantly expressed N-terminal 6X-His IPD090Aa polypeptide (SEQ ID NO: 347) and C-terminal 6XHis IPD090Aa polypeptide (SEQ ID NO: 349) were eluted from the columns with 2.5 ml of 150 mM imidazole in PBS. Both 2.5 ml eluates were applied to separate PD10 desalting columns (GE Healthcare) and proteins were eluted with 3.5 ml PBS. Purified and desalted N-terminal IPD090Aa polypeptide (SEQ ID NO: 347) and IPD090Aa polypeptide with C-terminal 6X-His tag (SEQ ID NO: 349) were bioassayed for WCRW and were active (Table 3) . In addition, a pure IPD090Aa polypeptide lysate (SEQ ID NO: 2) from 50 ml of induction solution was clarified by FPLC and bioassayed for WCRW and was active (see Example 4 below).

Example 4 Purification of the recombinant IPD090Aa polypeptide.

A cell pellet from a 1 L culture of E. coli expressing the IPD090Aa polypeptide (SEQ ID NO: 2) was suspended in 100 ml of 20 mM Tris, pH 8.0+1:100 HALT™ proteinase inhibitor mixture (Life Technologies). Cells were lysed at 30,000 PSI and the lysate was centrifuged at 30,000 g for 30 minutes. Ammonium sulfate was added to the supernatant to a final concentration of 1.5 M and the solution was equilibrated overnight. After clarification, the supernatant was loaded onto a Phenyl-5PW column (GE Healthcare, Piscataway, NJ) equilibrated in 1.5 M ammonium sulfate, 20 mM Tris, pH 8.0. The column was washed with 4× column volume (CV) and fractions containing the IPD090Aa polypeptide (SEQ ID NO: 2) were eluted with a 10 CV gradient to 20 mM Tris, pH 8.0. The IPD090Aa polypeptide eluate (SEQ ID NO: 2) was concentrated and further purified by size exclusion chromatography on an S200 column (GE Healthcare, Piscataway, NJ) equilibrated in PBS. Fractions with purified IPD090Aa polypeptide (SEQ ID NO: 2) were pooled based on SDS-PAGE.

Example 5 Purified IPD090Aa protein assays using Coleoptera.

Biological assays screening for insecticidal activity were performed with purified recombinant IPD090Aa polypeptide (SEQ ID NO: 2), as well as N-terminal His-tagged IPD090Aa polypeptide (SEQ ID NO: 347) and C-terminal His-tagged IPD090Aa polypeptide (SEQ ID NO : 349) to assess the effects of an insecticidal protein on the larvae of a variety of Coleoptera, including the western corn beetle (Diabrotica virgifera) - WCRW and the northern corn beetle (Diabrotica barberi) - NCRW, with Coleoptera nutritional analyzes performed on an artificial diet containing the insecticidal protein . The insecticidal proteins were included in an artificial beetle-specific diet (Frontier Agricultural Sciences, Newark, Delaware). Proteins were analyzed in a series of dilutions from 188 ppm to 6 ppm. One newborn larva was placed in each well for feeding ad libitum for 4 days. Each biological assay was performed in eight replicates at each dose. The results were expressed as positive for larval reactions such as developmental arrest and/or mortality. The results were expressed as negative if the larvae were similar to the negative control, which was fed a diet that used only the above buffer. Average WCRW for 8 replicate dilution series for IPD090Aa polypeptide (SEQ ID NO: 2), IPD090Aa polypeptide with N-terminal 6X His (SEQ ID NO: 347), and IPD090Aa polypeptide with C-terminal 6X-His (SEQ ID NO : 349) are shown in Table. 3.

Table 3

Purified polypeptide D Polyp ptide concentration (ppm) Avg. WCRW indicator Purified polypeptide Conc. ptida polype (ppm) Avg. WCRW indicator Purified polypeptide Conc. ptida polype (ppm) Avg. WCRW indicator IPD090Aa 188 2.4 IPD090 188 2.5 IPD090 188 2.0 SEQ ID 131 2.0 Aa s 131 2.0 Aa s 131 2.0

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NO: 2 94 2.0 N- END . 6X-His SEQ ID NO: 347 94 2.3 With- END . 6X-His SEQ ID NO: 349 94 1.8 66 2.0 bb 2.0 66 1.8 47 2.0 47 2.0 47 1.3 33 18 33 1.8 33 1.4 23 1.4 23 2.0 23 1.5 16 1.3 16 1.0 16 1.0 12 0.3 12 1.1 12 0.4 8 0 8 0.5 8 0.3 6 0 6 0.1 6 0.0 PBS buffer control 0 0 Control l in form buffer PBS 0 0 Control l in form buffer PBS 0 0

Example 6 E. coli expression and insecticidal activity of an N-terminally truncated IPD090Aa polypeptide.

Using biological assays for WCRW with the trypsinized IPD090Aa polypeptide (SEQ ID NO: 2), the truncated IPD090Aa product was noted to be insecticidal. Using N-terminal sequencing of a fragment of the trypsinized IPD090Aa polypeptide, it was shown that a polypeptide product was formed starting from alanine 25 SEQ ID NO: 2. A polynucleotide (SEQ ID NO: 9) encoding the IPD090Aa(TR1) polypeptide (SEQ ID NO: 10) was synthesized , by amplifying the IPD090Aa (SEQ ID NO: 1) gene using primers CTB142-FOR (SEQ ID NO: 357) and CTB55-REV (SEQ ID NO: 355) to clone the IPD090Aa (TR1) coding sequence (with native stop- codon TAG) in pET-24a (Novagen) for unlabeled translation. The KOD hot start mastermix (EMD Biosciences, San Diego, CA) was used for PCR amplification of the IPD090Aa (TR1) gene in a Bio-Rad® C1000 Touch™ thermal cycler. Cycle parameters were as follows: 1 cycle at 95° C. for 2 minutes; 35 cycles at 95°C for 20 s, 60°C for 10 s and 70°C for 15 s; 1 cycle at 70°C for 2 min. Amplicons were gel purified, ligated (T4 DNA ligase, New England BioLabs, Ipswich, MA) into expression vectors (as described above), transformed into high efficiency E. coli One Shot® TOP10 cells (Invitrogen) and clones confirmed by sequencing.

Confirmed clones expressing the IPD090Aa (TRI) polypeptide (SEQ ID NO: 10) were transformed into BL21-Gold cells for expression for 1 L inductions. Induction pellets were lysed in 30 ml lysis buffer (20 mM Tris pH 8, 1/4X B-PER™ II, Omni-Cleave™, Ready-Lyse™ and HALT™ (Life Technologies)) with shaking at room temperature for 1 h. The lysate was centrifuged at 30,000 g for 30 minutes. Ammonium sulfate was added to the supernatant to a final concentration of 1.5 M and the solution was equilibrated overnight. After clarification, the supernatant was loaded onto a Phenyl-5PW column (GE Healthcare, Piscataway, NJ) equilibrated in 1.5 M ammonium sulfate, 20 mM Tris, pH 8.0. The column was washed with 4x column volume (CV) and fractions containing the IPD090Aa polypeptide (TRI) (SEQ ID NO: 10) were eluted with a 10x column volume gradient to 20 mM Tris, pH 8.0. Eluate fractions containing the IPD090Aa (TRI) polypeptide (SEQ ID NO: 10) were concentrated and desalted in PBS buffer using a Sephadex™ G-25 column (GE Healthcare) and subjected to bioassay for WCRW. Average WCRW values for the 8 replicate dilution series for the IPD090Aa (TRI) polypeptide (SEQ ID NO: 10) are shown in Table 1. 4.

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Example 7 Expression of the 1PP090Ca polypeptide in E. coli.

The sequence encoding the IPD090Ca polypeptide (SEQ ID NO: 6) was isolated from strain JH23959-1 using primers CTB60-FOR (SEQ ID NO: 358) and CTB63-REV (SEQ ID NO: 359) to amplify the gene from strain JH23959- 1 and cloning the coding sequence for IPD090Ca (SEQ ID NO: 5) (with native stop codon (TAA)) into pET-24a (Novagen) for translation and pET14b (Novagen) for N-terminal translation of the bX-His tag using NdeI sites /BamHI. KOD Hot Start Master Mix (EMD Biosciences, San Diego, CA) was used for PCR amplification of the IPD090Ca gene in a Bio-Rad™ C1000 Touch™ thermal cycler. Cycle parameters were as follows: 1 cycle at 95° C. for 2 minutes; 35 cycles at 95°C for 20 s, 60°C for 10 s and 70°C for 15 s; 1 cycle at 70°C for 2 min. Amplicons were gel purified, ligated (T4 DNA ligase, New England BioLabs, Ipswich, MA) into expression vectors (as described above), transformed into high efficiency E. coli One Shot® TOP10 chemically competent cells (Invitrogen) and clones verified with sequencing.

Confirmed clones expressing IPD090Ca (SEQ ID NO: 5) in pET-24a /BL21, 50 ml LBCARB and KAN culture were plated with 500 μl overnight culture and incubated at 37°C, 200 rpm until OD ₆₀₀ ~0.8. Cultures were induced with 0.3 mM IPTG and incubated at 16° C., 100 rpm overnight (~20 hours). After induction, the cultures were centrifuged at 5000 rcf for 15 min to pellet the cells. The cell pellet was stored at -80° C. overnight until lysis. After freeze/thaw, the cell pellet was lysed with 3 ml lysis buffer (20 mM Tris pH 8, 1/4X B-PER™ II, Omni-Cleave™, Ready-Lyse™ and Halt™ protease inhibitors) with rocking at room temperature within 1 h. Cell lysates were clarified by centrifugation at 13,000 rcf for 10 min. 2.5 ml of each clarified lysate was applied to a PD10 desalting column (GE Healthcare, Piscataway, NJ) equilibrated with PBS. The IPD090Ca polypeptide (SEQ ID NO: 6) was eluted from the PD10 columns with 3.5 ml PBS and the lysate was bioassayed for WCRW. The mean WCRW for the 8 replicate dilution series for the IPD090Ca polypeptide (SEQ ID NO: 6) is shown in Table 1. 5.

Example 8 Expression of the IPD090Fa polypeptide in E. coli.

The amino acid sequence of IPD090Fa (SEQ ID NO: 8) was identified by a BLAST™ search on the NCBI public non-redundant protein sequence database (NCBI reference sequence: WP_019961352.1).

The corresponding coding sequence optimized for E. coli (SEQ ID NO: 7) was generated as two overlapping synthetic DNA fragments (IDT, Coralville, Iowa) with 30 nucleotide ends homologous to pET-24a (Novagen) at the NdeI sites. /XhoI. The expression vector IPD090Fa with C-terminal 6X-His (SEQ ID NO: 350) was generated using NEBuilder™ (New England Biolabs, Ipswich, MA).

Positive clones were confirmed by DNA sequencing.

The IPD090Fa expression construct with C-terminal 6X-His (SEQ ID NO: 350) was transformed into E. coli BL21 cells (DE3, Agilent, Santa Clara, CA) for expression. Cultures in 250 ml of Luria broth (containing kanamycin) were grown at 37°C until an OD600 of about 0.6 was reached, and then the cultures were induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and allowed to grow for additional 18 hours at 16°C, 250 rpm. Cultures were centrifuged at 5000 rcf for 15 min to pellet the cells. The cell pellet was lysed with 1/4 B-PER™ II reagent (Thermo Scientific), 20 mM Tris pH 8.0, OmniCleave™ endonuclease (Epicentre), lysocys

- 69 040502 mA ReadyLyse™ (Epicentre) and HALT™ V protease inhibitor mixture (Millipore) with rocking for 120 minutes at 30°C. Lysates were clarified by centrifugation at 13,000 rcf for 10 min and supernatants were transferred to 10 mM imidazole and then loaded onto individual 1 ml Ni-NTA columns (QIAGEN® Inc., Valencia, CA 91355) equilibrated with Tris buffered saline. solution (TBS), 10 mM imidazole. The columns were washed twice with 5 ml of 10 mM imidazole in TBS. Recombinantly expressed C-terminal 6X-His IPD090Fa polypeptide (SEQ ID NO: 351) was eluted from the columns with 1.2 ml of 300 mM imidazole in TBS. 1.2 ml of the eluate was applied to a Zeba Spin desalting column (Thermo) and the buffer was changed to TBS. Purified and desalted 6X-His C-terminal tagged IPD0 90Fa polypeptide (SEQ ID NO: 351) was bioassayed for WCRW and was active (Table 6).

A cell pellet from a 1 L culture of E. coli expressing the IPD090Fa polypeptide (SEQ ID NO: 8) was suspended in 5X volume (volume by weight) of 20 mM Tris pH 8.0+1:100 HALT™ proteinase inhibitor mixture (Thermo). Cells were lysed at 25,000 PSI and the lysate was centrifuged at 30,000 g for 30 minutes. An equal volume of 3 M ammonium sulfate was added dropwise to the supernatant with stirring to a final concentration of 1.5 M, and the solution was stirred for at least 30 minutes. After clarification, the supernatant was loaded onto a Phenyl-5PW column (Tosoh Bioscience, King of Prussia, PA) equilibrated in 1.5 M ammonium sulfate, 20 mM Tris, pH 8.0. The column was washed with 4× column volume (CV) and fractions containing the IPD090Fa polypeptide (SEQ ID NO: 8) were eluted with a 15 CV gradient to 20 mM Tris, pH 8.0. The IPD090Fa polypeptide eluate (SEQ ID NO: 8) was loaded onto a Mono Q™ column (GE Healthcare, Piscataway, NJ) equilibrated in 20 mM Tris buffer pH 8.0 and fractions containing IPD090Fa were eluted with a 40 CV gradient to 0.5 M NaCl, 20 mM Tris pH 8.0 was concentrated and further purified by size exclusion chromatography on a Superdex™ 200 column (GE Healthcare, Piscataway, NJ) equilibrated in PBS. Fractions with purified IPD090Fa polypeptide (SEQ ID NO: 8) were pooled based on SDS-PAGE.

Example 9 Diet based biological assays using corn bug to determine LC50 and IC50.

Biological assays using a standardized corn bug diet were used to test the activity of the IPD090Aa polypeptide (SEQ ID NO: 2) against WCRW. The corn bug diet was prepared according to the manufacturer's recommendation for the Diabrotica diet (Frontier, Newark, Delaware). The test included six different doses of the IPD090Aa polypeptide (SEQ ID NO: 2) plus a buffer control with 32 observations for each dose in each bioassay. Neonates were plated in 96-well plates containing a mixture of IPD090Aa polypeptide (SEQ ID NO: 2) (5 µl/well) and diet (25 µl/well), with each well containing approximately 5-8 larvae (<24 h. after hatching). One day later, one larva was transferred to each well of a second 96-well plate containing a mixture of IPD090Aa polypeptide (SEQ ID NO: 2) (20 µl/well) and diet (100 µl/well) at the same concentration as the treatment, to which the insect was subjected on the first day. The plates were incubated at 27°C, 65% RH in the dark for 6 days. The 50% lethal concentration for the polypeptides in the biological assay was calculated using the Excel add-in program to determine the dose effect of the Dose Response Add-In for Excel based on probit analysis. Mortality and the number of serious developmental arrests were evaluated and combined as an overall response to calculate the suppression of 50% of individuals using the same method. The LC50 and IC50 for WCRW (Diabrotica virgifera virgifera) were 16.3 ppm and 7.4 ppm, respectively, and for NCRW (Diabrotica barberi) they were 35.6 ppm and 13 ppm, respectively. For Diabrotica speciosa, the LC50 was >400 ppm and the IC50=320 ppm. The same assay protocol was used to evaluate the toxicity of the IPD090Aa C-terminal 6X-His polypeptide (SEQ ID NO: 349) and the IPD090Fa C-terminal 6X-His polypeptide (SEQ ID NO: 351) to WCRW and NCRW. The results are shown in table. 6.

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Table 6

Naseko my Sample LC/IC ppm Below 95% CL Higher 95%CL Incline N WCRW IPD090Aa with C-conc. 6xHis (SEQ ID NO: 349) LC50 42.0 31, 8 63.9 2.1 128.0 IC50 17.6 14.2 21, 9 2.6 158.0 IPD090Fa c C-conc. 6xHis (SEQ ID NO: 351) LC50 9.0 7.0 11.3 2.4 186.0 IC50 5.7 4.4 7.0 2.7 154.0 NCRW IPD090Ac C-conc. 6xHis (SEQ ID NO: 349) LC50 100.2 69.7 122.6 7.9 47.0 IC50 54.4 36, 4 81.7 2.6 79.0 IPD090Fa c C-conc. 6xHis (SEQ ID NO: 351) LC50 13.9 10.5 18.2 3.4 79.0 IC50 9.2 6.5 12.0 4.0 63.0

Example 10 Cross-resistance testing in mCry3A-selected WCRW.

LC50 was also determined for the IPD090Aa polypeptide (SEQ ID NO: 2) against mCry3A resistant WCRW and compared to susceptible WCRW using the same method as the diet based bioassays for WCRW described in Example 8 above. The mCry3A resistant WCRW line was developed through selections on transgenic maize plants with high mCry3A expression (>10,000 ng/mg total soluble protein in T0 roots) and high WCRW potency. The mCry3A resistance ratio (RR) of the colony was >92-fold higher based on LC50 in a diet based analysis (Patent Application Publication No. US 20140033361). RR was calculated as follows: RR=(LC50 resistant WCRW)/(LC50 susceptible WCRW). In table. 7 shows that the mCry3A resistant WCRW line did not cross-resistance (CC=1.4-fold) to the IPD090Aa polypeptide (SEQ ID NO: 2).

Table 7

The colony WCRW n IPD090Aa (SEQ ID NO: 2), (µg/ml) 95%CL Tilt (SE) Stability ratio (RR) Control 230 25, 4 19, 3- 34.5 1.8 (0.3) 1.0 mCryZAres* 240 35, 3 26, 5- 46.5 2.3 (0.4) 1.4

Example 11 Chimeras between IPD090 homologues.

To obtain active variants of the IPD090Aa (SEQ ID NO: 2) polypeptide with diversified sequences, chimeric genes between IPD090Aa (SEQ ID NO: 1) and IPD090Ca (SEQ ID NO: 5) were generated by assembling multiple overlapping PCR fragments. For this purpose, the nucleotide sequence of IPD090Ca was codon-harmonized with the sequence of IPD090Aa, which increases DNA homology to allow for family shuffling and chimera construction. The codon-modified IPD090Ca coding sequence contains the nucleic acid sequences of SEQ ID NO: 345. A total of seven IPD090Aa/IPD090Ca chimeric polynucleotides were constructed and cloned into pET24a. The constructs were transformed into BL21 DE3 and cultured in 48-well protein expression plates. Cell lysates were prepared using B-PER® Protein Extraction Reagent from Thermo Scientific (3747 N. Meridian Rd., Rockford, IL, USA 61101) and screened for insecticidal activity against WCRW. In table. 8 shows the chimeric protein boundaries and percent sequence identity with the IPD090Aa polypeptide (SEQ ID NO: 2).

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Table 8

Designation chimeras PolynuCL eotid IPD090Aa (SEQ ID NO: 2) N- END . fragment IPD090Ca (SEQ ID NO: 6) C-conc. fragment identity ate with IPD090Aa Activity in a relationship WCRW Chimera 1 SEQID NO: 12 SEQ ID NO:11 M1-A239 R241- K4 8 3 88.6 Yes Chimera 2 SEQID NO: 14 SEQ ID NO:13 M1-V296 P297- K4 8 3 90.9 Yes Chimera 3 SEQID NO: 16 SEQ ID NO:15 M1-G348 D349- K4 8 3 93.8 Yes Chimera 4 SEQID NO: 18 SEQ ID NO:17 M1-Q382 A383- K4 8 3 94.8 Yes Chimera 5 SEQID NO: 20 SEQ ID NO:19 M1-G422 A423- K4 8 3 97.7 Yes Chimera 6 SEQID NO: 22 SEQ ID NO: 21 M1-K442 1443- K4 8 3 98.8 Yes Chimera 7 SEQID NO: 24 SEQ ID NO: 2 3 M1-Q144 S146- K4 8 3 86.5 Yes

Example 12 Multiple amino acid substitution variants of IPD090Aa.

To generate variants of the IPD090Aa (SEQ ID NO: 2) polypeptide with multiple amino acid changes, libraries of variants were generated by family shuffling (Chia-Chun J. Chang et al, 1999, Nature Biotechnology 17, 793-797) of polynucleotides encoding IPD090Aa (SEQ ID NO: 2) and IPD090Ca (SEQ ID NO: 6).

Three libraries were designed to generate IPD090Aa variants. In the first library, the polynucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 5 were used as library sources. In the second library, the polynucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 5 were amplified in seven fragments with overlapping homology. The primers used to amplify the fragments are summarized in Table 1. 9. Overlapping fragments were pooled and collected.

Table 9

primer Subsequence 90Aa Fragl straight GAA GGA GAT ATA CAT ATG GAA MAC RTA GAC TTG CCA CAR GGA CTT GTA AAS TTT TCC (SEQ ID NO: 360) 90Ca Fragl straight GAA GGA GAT ATA CAT ATG GAA MAC RTC GAC CTG CCG ACR GGA CTC GTC AAA TTT TCC (SEQ ID NO: 361) 90-2 straight TC GTR CCS GAG ATC GTC GAC GTS CAR CAG AAY GAC AGC GCM ASC TAG ACC AAC (SEQ ID NO: 362) 90-2 RC GTT GGT GTA GST KGC GCT GTC RTT CTG YTG SAC GTC GAC GAT CTC (SEQ ID NO: 363) 90-3 straight AAC GAG TTC CAC YCG YAT YCA GCA ATC GAT CAA CCT CTG GTC G (SEQ ID NO: 364) 90-3RC ACC GAA GGC ARG CGC ARC GAC CAG AGG TTG ATC GAT TGC TG (SEQ ID NO: 365) 90-4 straight ACC GGC ATC GTR ATG GGY GGM CGR GCC ATM CTC GCC KCC TCG GAC CAA C (SEQ ID NO: 366) 90-4RC GTT GGT CCG AGG MGG CGA GKA TGG CYC GKC CRC CCA TYA CGA TGC CGG T (SEQ ID NO: 367) 90-5 straight TTC CAG GCC TGG GTM GAC AGY GTG RGC RCC TCG CCS GAY TTC GTC GAY TTC GTY CCC ACC ATC CC (SEQ ID NO: 368) 90-5RC GGG ATG GTG GGR ACG AAR TCG ACG AAR TCS GGC GAG

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GYG CYC ACR CTG TSK ACC GAG GCC TGG AA (SEQ ID NO: 369) 90-6 straight TAC GAG CTC AAT GCC GG (SEQ ID NO: 370) 90-6RC CCG GCA TTG AGG TCG TA (SEQ ID NO: 371) 90-7 straight TAC AAC ACC GAY ACC GCR ATC AAC AAG (SEQ ID NO: 372) 90-7 RC CTT GTT GAT YGC GGT RTC GGT GTT GTA (SEQ ID NO: 372) 90Aa Frag 8 reverse CTC AGT GGT GGT GGT GGT GGT GCT CGA GCT ACT TGC STA CGA AGG TAC AGG CAT AGA TG (SEQ ID NO: 374) 90Ca Frag 8 Reverse CTC AGT GGT GGT GGT GGT GGT GCT CGA GTT ACT TGC CGA CGA AAG TGC AGG CAT AGA TG (SEQ ID NO: 375)

In the third library, a native polynucleotide sequence (SEQ ID NO: 1) encoding an IPD090Aa polypeptide (SEQ ID NO: 2) and a codon-optimized E. coli polynucleotide sequence (SEQ ID NO: 345) encoding an IPD090Ca polypeptide (SEQ ID NO: 6) were used as library sources.

After transformation with the E. coli cell library variants, colonies were harvested and cultured in 48-well protein expression plates. Cell lysates were prepared with B-PER® protein extraction reagent from Thermo Scientific (3747 N Meridian Rd, Rockford, IL, USA 61101) and screened for insecticidal activity against WCRW. Active variants were sequenced and amino acid substitutions identified. In Library 1, 144 variants were screened and the sequence of 11 active unique variants was identified. Library 2 screened 96 variants and identified the sequence of 10 active unique variants. Library 3 screened 168 variants and identified the sequence of 64 active unique variants.

Percent sequence identity of the active IPD090Aa variants compared to the IPD090Aa polypeptide (SEQ ID NO: 2) was calculated using the Needleman-Wunsch algorithm implemented in the Needle program (EMBOSS toolkit). Percent identity compared to the IPD090Aa polypeptide (SEQ ID NO: 2), variant designation, nucleotide sequences, and amino acid sequences of the resulting active variants of the IPD090Aa polypeptide are shown in Table 1. 10. In the table. 11 summarizes the percent identity of active variants compared to the IPD090Aa polypeptide (SEQ ID NO: 2), the number of variants with each percent identity, and the identification of the variant.

Table 10

% identity c IPD090Aa (SEQ ID NO: 2) Option Polynucleotide Polypeptide 90.5 S04515584 SEQ ID NO: 25 SEQ ID NO: 114 84.1 S04515608 SEQ ID NO: 26 SEQ ID NO: 115 89.9 S04515618 SEQ ID NO: 27 SEQ ID NO: 116 80.2 S04515626 SEQ ID NO: 28 SEQ ID NO: 117 81.6 S04515631 SEQ ID NO: 29 SEQ ID NO: 118 82.2 S04515638 SEQ ID NO: 30 SEQ ID NO: 119 79.8 S04515642 SEQ ID NO: 31 SEQ ID NO: 120 81.8 S04515648 SEQ ID NO: 32 SEQ ID NO: 121 94.2 S04515711 SEQ ID NO: 33 SEQ ID NO: 122 80, 8 S04515723 SEQ ID NO: 34 SEQ ID NO: 123 80 S04515724 SEQ ID NO: 35 SEQ ID NO: 124 92.1 S04519420 SEQ ID NO: 36 SEQ ID NO: 125 84.1 S04519434 SEQ ID NO: 37 SEQ ID NO: 126 83, 9 S04519435 SEQ ID NO: 38 SEQ ID NO: 127 93.4 S04519439 SEQ ID NO: 39 SEQ ID NO: 128 87.8 S04519446 SEQ ID NO: 40 SEQ ID NO: 129 83, 9 S04519447 SEQ ID NO: 41 SEQ ID NO: 130 89 S04519473 SEQ ID NO: 42 SEQ ID NO: 131 96.7 S04519475 SEQ ID NO: 43 SEQ ID NO: 132 94.2 S04519477 SEQ ID NO: 44 SEQ ID NO: 133 83.3 S04519504 SEQ ID NO: 45 SEQ ID NO: 134 96.9 S04529311 SEQ ID NO: 46 SEQ ID NO: 135

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90.7 S04529312 SEQ ID NO: 47 SEQ ID NO: 136 89.6 S04529313 SEQ ID NO: 48 SEQ ID NO: 137 88 S04529314 SEQ ID NO: 49 SEQ ID NO: 138 88.4 S04529317 SEQ ID NO: 50 SEQ ID NO: 139 89.9 S04529318 SEQ ID NO: 51 SEQ ID NO: 140 93 S04529319 SEQ ID NO: 52 SEQ ID NO: 141 92.4 304529320 SEQ ID NO: 53 SEQ ID NO: 142 96.1 S04529325 SEQ ID NO: 54 SEQ ID NO: 143 93.6 S04529326 SEQ ID NO: 55 SEQ ID NO: 144 91.7 304529329 SEQ ID NO: 56 SEQ ID NO: 145 93.6 S04529331 SEQ ID NO: 57 SEQ ID NO: 146 95.4 304529338 SEQ ID NO: 58 SEQ ID NO: 147 96.3 S04529347 SEQ ID NO: 59 SEQ ID NO: 148 94.8 304529348 SEQ ID NO: 60 SEQ ID NO: 149 89.9 S04529351 SEQ ID NO: 61 SEQ ID NO: 150 89.2 S04529352 SEQ ID NO: 62 SEQ ID NO: 151 86 304529353 SEQ ID NO: 63 SEQ ID NO: 152 97.3 S04529355 SEQ ID NO: 64 SEQ ID NO: 153 86.2 S04529359 SEQ ID NO: 65 SEQ ID NO: 154 88.8 304529361 SEQ ID NO: 66 SEQ ID NO: 155 95.9 S04529363 SEQ ID NO: 67 SEQ ID NO: 156 86.8 S04529365 SEQ ID NO: 68 SEQ ID NO: 157 88 S04529371 SEQ ID NO: 69 SEQ ID NO: 158 90.5 304529372 SEQ ID NO: 70 SEQ ID NO: 159 97.3 S04529374 SEQ ID NO: 71 SEQ ID NO: 160 96.5 S04529375 SEQ ID NO: 72 SEQ ID NO: 161 83, 9 304529376 SEQ ID NO: 73 SEQ ID NO: 162 95.2 S04529377 SEQ ID NO: 74 SEQ ID NO: 163 96.5 304529378 SEQ ID NO: 75 SEQ ID NO: 164 85.1 S04529380 SEQ ID NO: 76 SEQ ID NO: 165 94.2 S04529383 SEQ ID NO: 77 SEQ ID NO: 166 92.1 304529386 SEQ ID NO: 78 SEQ ID NO: 167 94 304529390 SEQ ID NO: 79 SEQ ID NO: 168 86 S04529393 SEQ ID NO: 80 SEQ ID NO: 169

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86.2 S04529396 SEQ ID NO: 81 SEQ ID NO: 170 92.8 S04529397 SEQ ID NO: 82 SEQ ID NO: 171 94.2 S04529401 SEQ ID NO: 83 SEQ ID NO: 172 90.3 S04529402 SEQ ID NO: 84 SEQ ID NO: 173 92.3 S04529404 SEQ ID NO: 85 SEQ ID NO: 174 90.3 S04529407 SEQ ID NO: 86 SEQ ID NO: 175 95 S04529410 SEQ ID NO: 87 SEQ ID NO: 176 97.5 S04529419 SEQ ID NO: 88 SEQ ID NO: 177 99.4 S04529422 SEQ ID NO: 89 SEQ ID NO: 178 95.2 S04529423 SEQ ID NO: 90 SEQ ID NO: 179 95.2 S04529426 SEQ ID NO: 91 SEQ ID NO: 180 90.3 S04529432 SEQ ID NO: 92 SEQ ID NO: 181 91.1 S04529434 SEQ ID NO: 93 SEQ ID NO: 182 93.6 S04529436 SEQ ID NO: 94 SEQ ID NO: 183 91.3 S04529437 SEQ ID NO: 95 SEQ ID NO: 184 93.6 S04529443 SEQ ID NO: 96 SEQ ID NO: 185 98.3 S04529446 SEQ ID NO: 97 SEQ ID NO: 186 89.9 S04529447 SEQ ID NO: 98 SEQ ID NO: 187 96.5 S04529455 SEQ ID NO: 99 SEQ ID NO: 188 97.7 S04529458 SEQ ID NO: 100 SEQ ID NO: 189 92.1 S04529460 SEQ ID NO: 101 SEQ ID NO: 190 91.3 S04529461 SEQ ID NO: 102 SEQ ID NO: 191 93.8 S04529462 SEQ ID NO: 103 SEQ ID NO: 192 88 S04529463 SEQ ID NO: 104 SEQ ID NO: 193 93.6 S04529469 SEQ ID NO: 105 SEQ ID NO: 194 87.4 S04529471 SEQ ID NO: 106 SEQ ID NO: 195 87.4 S04529479 SEQ ID NO: 107 SEQ ID NO: 196 91.1 S04529481 SEQ ID NO: 108 SEQ ID NO: 197 89.4 S04529483 SEQ ID NO: 109 SEQ ID NO: 198 87 S04529486 SEQ ID NO: 110 SEQ ID NO: 199 92.1 S04529493 SEQ ID NO: 111 SEQ ID NO: 200 95.2 S04529495 SEQ ID NO: 112 SEQ ID NO: 201 87.4 304529498 SEQ ID NO: 113 SEQ ID NO: 202

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Table 11

% ident. With IPD090Aa (SEQ ID NO: 2) Number of options Options 99 1 S04529422 98 1 S04529446 97 4 S04529458, S04529419, S04529355, S04529374 96 7 S04529311, S04519475, S04529375, S04529378, S04529455, S04529347, S04529325 95 7 S04529363, S04529338, S04529377, S04529423, S04529426, S04529495, S04529410 94 6 S04529348, S04515711, S04519477, S04529383, S04529401, S04529390 93 8 S04529462, S04529326, S04529331, S04529436, S04529443, S04529469, S04519439, S04529319 92 7 S04529397, S04529320, S04529404, S04519420, S04529386, S04529460, S04529493 91 5 S04529437, S04529481, S04529329, S04529434, S04529461 90 6 S04529312, S04515584, S04529372, S04529402, S04529407, S04529432 89 8 S04515618, S04529318, S04529351, S04529447, S04529313, S04529483, S04529352, S04519473 88 5 S04529361, S04529317, S04529314, 304529371, 304529463 87 5 S04519446, S04529471, S04529479, S04529498, S04529486 86 5 304529365, 304529359, 304529396, S04529353, S04529393 85 1 S04529380 84 2 S04515608, S04519434 83 4 S04519435, S04519447, 304529376, S04519504 82 1 S04515638 81 2 S04515648, S04515631 80 3 S04515723, S04515626, S04515724 79 1 S04515642

Example 13 Variants of IPD090Aa with modified physical properties.

A number of variants of the IPD090Aa polypeptide (SEQ ID NO: 2) with modified physical properties were generated by mutagenesis methods using the QuikChange™ multisite site-directed mutagenesis kit (Agilent).

Oligonucleotides were designed and combined to introduce conservative amino acid changes I to L and Y to F at selected positions within the IPD090Aa polypeptide (SEQ ID NO: 2). The library was expressed in E. coli and 204 isolates were screened as purified lysates for insecticidal activity against WCRW. 71 unique active WCRW clones were identified and summarized in Table 1. 12.

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Table 12

Option Polynucleotide Polypeptide Amino acid substitutions relative to IPD090Aa (SEQ ID NO: 2) 304509867 SEQ ID NO: 203 SEQ ID NO: 274 I038L S04509903 SEQ ID NO: 204 SEQ ID NO: 275 I004L, I038L, I375L S04509914 SEQ ID NO: 205 SEQ ID NO: 276 I340L S04509946 SEQ ID NO: 2 0 6 SEQ ID NO: 277 I375L 304513757 SEQ ID NO: 207 SEQ ID NO: 278 I080L S04537215 SEQ ID SEQ ID NO: 279 I080L, Y321F, Y333F, NO: 208 Y434F, I446L, I453L S04537217 SEQ ID NO: 209 SEQ ID NO: 280 I080L, I099L, Y321F, Y333F, I353L, Y434F, I453L S04537221 SEQ ID NO: 210 SEQ ID NO: 281 I080L, Y091F, I099L, I353L, I440L, Y457F S04537226 SEQ ID NO: 211 SEQ ID NO: 282 I080L, Y333F, I340L, I446L, Y457F 304537227 SEQ ID NO: 212 SEQ ID NO: 283 I080L, I099L, Y333F, Y434F S04537230 SEQ ID NO: 213 SEQ ID NO: 284 I080L, Y091F, Y339F, I353L, I440L S04537235 SEQ ID NO: 214 SEQ ID NO: 285 I080L, Y091F, Y321F, Y333F, I340L, I346L, I362L, Y434F, I440L S04537237 SEQ ID NO: 215 SEQ ID NO: 286 I080L, Y333F, Y434F S04537243 SEQ ID NO: 216 SEQ ID NO: 287 I080L, I099L, Y339F, Y457F S04537244 SEQ ID NO: 217 SEQ ID NO: 288 I080L, Y091F, Y333F, I362L, I446L S04537246 SEQID NO: 218 SEQ ID NO: 289 I080L, Y333F, I362L S04537249 SEQ ID NO: 219 SEQ ID NO: 290 I080L, Y091F, Y333F, I440L S04537256 SEQ ID NO: 220 SEQ ID NO: 291 I080L, Y091F, I099L, Y321F, Y333F S04537260 SEQ ID NO: 221 SEQ ID NO: 292 I080L, I099L, E331K, K332V, Y333P, R334G, V335Q, K336G, A337, N338, Y339, 1340, D341, Q342, L343, V344, V345, 1346, T347, G348, G349, S350, S351, T352,

-77040502

1353, E354, P355 P356, V357, G358 Y359, S360, K361 1362, E363, Y364 D365, L366, N367 A368, G369, A370 G371, G372, D373 F374, 1375, Y376 L377, C378, Y379 H380, E381, Q382 T383, w384, Q385 A386, D387, R388 P389, K390, D391 A392, V393, T394 D395, 1396 R397 1398, 1399 F400 N401, K402, E403 P404, T405, P406 P407, G408, Y409 T410, K411, L412 P413, Q414, D415 L416, N417, K418 G419, A420, G421 G422, D423, D424 V425, F426, L427 C428, Y429, K430 T431, E432, A433 Y434, N435, T436 D437, T438, A439 1440, N441, K442 V443, T444, V445 1446 G447, G448 N449, N450, A451 D452, 1453, N454 A455, P456, Y457

- 78 040502

G458, Y459, l460, K461, V4 62, P463, G464, D465, L466, N467, R468, G469, A470, G471, G472, N473, F474, 1475, Y476, A477, C478, T479, F480, V481, G482 S04537262 SEQ ID NO: 222 SEQ ID NO: 293 I362L, I440L 304537263 SEQ ID NO: 223 SEQ ID NO: 294 I080L, I362L Y091F, Y333F, S04537266 SEQ ID NO: 224 SEQ ID NO: 295 Y091F, Y321F, I440L S04537271 SEQ ID NO: 225 SEQ ID NO: 296 I080L, Y434F, Y333F, I440L I346L, S04537273 SEQID NO: 226 SEQ ID NO: 297 I080L, Y333F, I099L, I362L, Y321F, I440L S04537275 SEQID NO: 227 SEQ ID NO: 298 I080L, Y339F S04537281 SEQID NO: 228 SEQ ID NO: 299 I080L, I353L, I099L, Y434F, Y333F, I446L S04537282 SEQID NO: 229 SEQ ID NO: 300 I080L, Y333F, I440L S04537285 SEQID NO: 230 SEQ ID NO: 301 I080L, I346L, Y091F, I440L, Y333F, Y457F 304537286 SEQID NO: 231 SEQ ID NO: 302 I080L, Y434F I340L, I362L, 304537292 SEQID NO: 232 SEQ ID NO: 303 I080L, I362L, I340L, Y434F I346L, S04537293 SEQID NO: 233 SEQ ID NO: 304 I080L, I353L I099L, Y333F, S04537294 SEQID NO: 234 SEQ ID NO: 305 I080L, I362L, I099L, I440L I346L,

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I080L,

Y321F, S350I, I353S, P356R, Y359T, I362S, D365T, A368P, G371A, F374S, L377C, H380T, T383P, A386P, P389L,

V393, 1396, 1399,

K402, T405,

G408,

K411,

Q414,

N417,

A420,

D423, F426,

Y429,

E432,

N435, T438,

N441, T444,

G447,

Y091F,

Y333F,

S351Q,

E354N,

V357S,

S360A,

E363S,

L366S,

G369V,

G372V,

i375s,

C378A,

E381N,

W384G,

D387T,

D391M,

T394,

R397,

F400,

E403,

P406,

Y409,

L412,

D415,

K418,

G421,

D424,

L427,

K430,

A433,

T436,

A439,

K442,

V445,

G448,

I099L, I340L, T352P, P355H, G358A, K361R, Y364T, N367M, A370P, D373T, Y376T, Y379I, Q382K, Q385R, R388G, A392L,

D395, 1398, N401, P404, P407, T410,

P413, L416, G419, G422, V425, C428,

T431,

Y434,

D437, 1440, V443, 1446, N449,

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N450, A451, D452 1453, N454, A455 P456, Y457, G458 Y459, l460, K461 V4 62, P463, G464 D465, L466, N467 R468, G469, A470 G471, G472, N473 F474, 1475, Y476 A477, C478, T479 F480, V481, G482

S04537298 SEQID NO: 236 SEQ ID NO: 307 I080L, I340L, I362L, D437A, T438P, A439H, I440S, N441T, K442R, V443S, T444R, V445S, I446S, G447A, G448A, N449T, N450M, A451R, D452I, I453S, N454T, A455L, Y457L, G458V, Y459I, L460, K461, V462, P463, G464, D465, L466, N467, R468, G469, A470, G471, G472, N473, F474, 1475, Y476, A477, 0478, T479, F480, V481, G482 304537300 SEQID NO: 237 SEQ ID NO: 308 Y333F, I340L, I362L S04537301 SEQID NO: 238 SEQ ID NO: 309 I080L, Y091F, I340L S04537303 SEQID NO: 239 SEQ ID NO: 310 Y091F, Y333F, I446L S04537304 SEQID NO: 240 SEQ ID NO: 311 I080L, Y091F, Y333F, Y434F

- 81 040502

SEQID NO: S04537305 SEQ ID NO: 312 Y091F, Y333F 241 I080L, Y091F, I099L, SEQID NO: S04537309 SEQ ID NO: 313 Y339F, I346L, I353L, 242 Y434F, I440L, Y457F I080L, Y091F, K319S, H320I, Y321S, D322M, D323T, V324S, W325G, A326R, P327R, A328R, Q329N, S330R, E331K, K332S, Y333S, R334G, V335S, K336R, A337L, N338T, Y339T, I340S, D341T, Q342N, L343W, V344W, V345S, I346S, T347P, G348A, G349V, S350V, S351Q, T352P, I353S, E354N, P355H, P356R, V357S, G358A, SEQID NO: Y359T, S360A, K361S, S04537312 SEQ ID NO: 314 243 I362S, E363S, Y364T, D365T, L366S, N367M, A368P, G369V, A370P, G371A, G372V, D373T, F374S, I375S, Y376T, L377C, C378A, Y379I, H380T, E381N, Q382K, T383P, W384G, Q385R, A386P, D387T, R388G, P389L, D391M, A392L, V393, T394, D395, 1396, R397, 1398, 1399, F400, N401, K402, E403, P404, T405, P406, P407,

- 82 040502

G408, Y409, T410, K411, L412, P413, Q414, D415, L416, N417, K418, G419, A420, G421, G422, D423, D424, V425, F426, L427, C428, Y429, K430, T431, E432, A433, Y434, N435, T436, D437, T438, A439, 1440, N441, K442, V443, T444, V445, 1446, G447, G448, N449, N450, A451, D452, 1453, N454, A455, P456, Y457, G458, Y459, L460, K461, V462, P463, G464, D465, L466, N467, R468, G469, A470, G471, G472, N473, F474, 1475, Y476, A477, C478, T479, F480, V481, G482 S04537314 SEQID NO: 244 SEQ ID NO: 315 I080L, I099L, I346L, I440L S04537315 SEQID NO: 245 SEQ ID NO: 316 I080L, Y091F, Y321F, Y333F S04537319 SEQID NO: 246 SEQ ID NO: 317 I080L, I099L, Y333F, I362L, I453L S04537321 SEQID NO: 247 SEQ ID NO: 318 Y091F, Y333F, I440L, Y457F S04537322 SEQID NO: SEQ ID NO: 319 I080L, Y091F, I099L,

- 83 040502

- 84 040502

- 85 040502

S04537337 SEQ ID 255 NO: SEQ ID NO: 326 I080L, Y091F, Y333F S04537339 SEQ ID 256 NO: SEQ ID NO: 327 Y091F, Y333F S04537347 SEQ ID 257 NO: SEQ ID NO: 328 I080L, I440L, Y321F, Y333F, Y457F S04537349 SEQ ID 258 NO: SEQ ID NO: 329 I080L, Y333F, I446L S04537350 SEQ ID 259 NO: SEQ ID NO: 330 I080L, I446L Y091F, Y333F, S04537351 SEQ ID 260 NO: SEQ ID NO: 331 I080L, I340L, I440L S04537352 SEQ ID 261 NO: SEQ ID NO: 332 Y091F, Y434F, Y333F, I346L, Y457F S04537359 SEQ ID 262 NO: SEQ ID NO: 333 I080L, I346L, Y091F, Y339F, Y434F S04537360 SEQ ID 263 NO: SEQ ID NO: 334 I080L, I362L, I099L, Y333F, Y434F S04537367 SEQ ID 264 NO: SEQ ID NO: 335 I080L, Y091F, Y333F S04537369 SEQ ID 265 NO: SEQ ID NO: 336 I080L, Y333F, I353L, Y091F, I099L, Y339F, I346L, I362L, I440L S04537371 SEQ ID 266 NO: SEQ ID NO: 337 I080L, Y321F, I346L, Y091F, I099L, Y333F, Y339F, Y434F, I453L S04537373 SEQ ID 267 NO: SEQ ID NO: 338 I080L, Y434F, Y321F, Y333F, I446L S04537377 SEQ ID 268 NO: SEQ ID NO: 339 I080L, Y339F, Y091F, A239T, I453L S04537385 SEQ ID 269 NO: SEQ ID NO: 340 I080L, Y457F Y333F, Y434F, S04537389 SEQ ID 270 NO: SEQ ID NO: 341 I080L, I453L Y321F, Y333F, S04537400 SEQ ID NO: SEQ ID NO: 342 I080L, Y091F, Y333F, 271 I353L S04537401 SEQ ID 272 NO: SEQ ID NO: 343 I080L, I353L, I099L, Y333F, I440L S04537402 SEQ ID 273 NO: SEQ ID NO: 344 I080L, I446L Y091F, Y333F,

Example 14. Mechanism of action.

The biological activity of the purified recombinant protein included in the artificial diet showed the toxicity of the IPD090Aa polypeptide (SEQ ID NO: 2) against WCRW larvae. To understand the mechanism of toxicity of the IPD090Aa polypeptide (SEQ ID NO: 2), the specific binding of the purified protein to WCRW midgut tissues was assessed by in vitro competition assays. The midgut was harvested from third instar WCRW larvae to obtain brush border membrane vesicles (BBMV) following the method modified by Wolfersberger et al. (Comp Bioch Physiol 86A: 301-308, 1987) using aminopeptidase activity to track enrichment. BBMVs are a component of the apical membrane of the epithelial cells lining insect midgut tissue and thus serve as a model system for how insecticidal proteins interact within the gut after ingestion.

The IPD090Aa polypeptide (SEQ ID NO: 2) was repurified by anion exchange chromatography using an AKTA™ Purifier 10 (GE Life Sciences) with a Frac-950 fraction collector. An aliquot of the purified IPD090Aa polypeptide (SEQ ID NO: 2) from Example 4 was taken from storage at -80° C. and dialyzed for 1 hour at 4° C. against 20 mM CAPS pH 9.6 ('eluent A') and loaded into a 1 ml HiTrap™ Q FF column (GE Life Sciences) equilibrated in eluent A. A 30x column volume of a gradient of 0-50% eluent B (20 mM CAPS pH 9.6 + 1 M NaCl) was applied at 1 ml/min. . Fractions near the top of the elution peak were pooled and dialyzed in binding buffer (50 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM sodium monohydrogen phosphate and 1.47 mM potassium dihydrogen phosphate, pH

7.5).

The purified IPD090Aa polypeptide (SEQ ID NO: 2) was labeled with Alexa-Fluor® 488 (Life Technologies) and the unincorporated fluorophore was separated from the labeled protein with a buffer exchange resin (Life Technologies, A30006) according to the manufacturer's recommendations. Prior to binding experiments, the amount of proteins was assessed by gel absorbance measurement followed by Simply Blue® (Thermo Scientific) staining of SDS-PAGE separated samples that included BSA as a standard.

To demonstrate specific binding and assess affinity, BBMV (5 µg) was incubated with 6.3 nM Alexa labeled IPD090Aa polypeptide (SEQ ID NO: 2) in 100 µl binding buffer for 1 hour at RT in the absence and presence of 13 µM unlabeled polypeptide IPD090Aa (SEQ ID NO: 2). Centrifugation at 20000xg was used to precipitate the BBMV to separate the unbound IPD090Aa polypeptide (SEQ ID NO: 2) remaining in solution. The BBMV pellet was then washed twice with binding buffer to remove the remaining unbound IPD090Aa polypeptide (SEQ ID NO: 2). The final pellet of BBMV (with bound fluorescent protein) was solubilized in Laemmli's reconstitution sample buffer, heated to 100° C. for 5 min, and subjected to SDSPAGE using 4-12% Bis-Tris polyacrylamide gels (Life Technologies). The amount of Alexa labeled IPD090Aa polypeptide (SEQ ID NO: 2) in the gel from each sample was measured by a digital fluorescence imaging system (ImageQuant™ LAS4000 - GE Healthcare). The digitized images were analyzed with optical density measurement software (Phoretix™ ID, TotalLab, Ltd.). In FIG. 2 shows that the IPD090Aa polypeptide (SEQ ID NO: 2) binds specifically to 5 μg of BBMV WCRW.

Example 15 Vector constructs for the expression of IPD090Aa polypeptides in plants.

Plant expression vectors were constructed to include a transgene cassette containing two different gene constructs encoding the IPD090 polypeptide of SEQ ID NO: 377 and one gene construct encoding the IPD090 polypeptide of SEQ ID NO: 10 under the control of the maize ubiquitin promoter (Christensen, et al. al., 1992, Christensen and Quail 1996) and associated with the PINII terminator (Keil et al., 1986, Nucleic Acids Research 14: 5641-5650; An et al., 1989, The Plant Cell 1: 115-122). The resulting constructs, PHP73234, PHP73237, for the IPD090 polypeptide of SEQ ID NO: 377 and PHP77372 for the IPD090 polypeptide of SEQ ID NO: 10, were used to generate maize transgenic events for testing efficacy against the corn bug when these polypeptides are expressed.

Example 16 Agrobacterium mediated transformation of maize and regeneration of transgenic plants.

Agrobacterium-mediated transformation of maize with the expression vectors PHP73234, PHP73237 and PHP77372 used the Zhao method (US Patent No. 5,981,840 and PCT Patent Application Publication No. WO 1998/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos were isolated from maize and the embryos were brought into contact with a suspension of Agrobacterium under conditions where the bacteria were able to transfer the vectors PHP73234, PHP73237 and PHP77372 into at least one cell of at least one of the immature embryos (step 1: infection step ). At this stage, immature embryos were immersed in Agrobacterium suspension to initiate inoculation. The embryos were co-cultured with Agrobacterium for some time (step 2: co-cultivation step). Immature embryos were cultured on solid medium after the infection stage. After a period of co-cultivation, an optional dormant stage was assumed. At this dormancy stage, the embryos were incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective plant transformation agent (stage 3: dormancy stage). Immature embryos were cultured on solid medium with antibiotic but no selective agent to eliminate Agrobacterium and during the dormant phase for infected cells. Then, the inoculated embryos were cultured on a medium containing a selective agent, and the growing transformed callus was isolated (step 4: selection step). Immature embryos were cultured on a solid medium with a selective agent, which led to the selective growth of the transformed cells. Then, the callus was regenerated to obtain plants (step 5: regeneration step), and the calli grown on the selective medium were cultured on the solid plant regeneration medium.

To detect IPD090Aa polypeptide (SEQ ID NO: 2) and IPD090Aa polypeptide (TR1) (SEQ ID NO: 10) in leaf tissue, 4 lyophilized leaf punches/samples were ground into powder and resuspended in 100 µl of PBS containing 0.1% Tween 20 (PBST), 1% beta-mercaptoethanol containing 1 tablet/7 ml Proteinase Inhibitor Complete Mini (Roche 1183615301). The suspension was sonicated for 2 min and then centrifuged at 4°C, 20,000 g for 15 min. To the supernatant was added a 1/3 volume aliquot of 3X NuPAGE® LDS Sample Buffer (Invitrogen™ (California, USA), 1% beta-mercaptoethanol containing 1 tablet/7 mL of Complete Mini proteinase inhibitor. The mixture was heated at 80°C for 10 min and then centrifuged.A sample of the supernatant was loaded onto 4-12% Bis-Tris Midi gels with MES running buffer according to the manufacturer's instructions (Invitrogen™) and transferred to a nitrocellulose membrane using the iBlot® device ( Invitrogen™) The nitrocellulose membrane was incubated in PBST containing 5% skimmed milk powder for 2 hours prior to overnight incubation with affinity purified rabbit polyclonal anti-IPD090Aa (SEQ ID NO: 2) in PBST overnight. The membrane was washed three times with PBST and then incubated in PBST for 15 min, and then twice for 5 min before incubation for 2 h in PBST with HRP-conjugated goat anti-rabbit Ig for 3 h. Detected proteins were visualized using ECL western blotting reagents (GE Healthcare cat. no. RPN2106) and visualized using a luminescent image analyzer (ImageQuant LAS 4000, GE Healthcare). To detect IPD090Aa polypeptide (SEQ ID NO: 2) and IPD090Aa polypeptide (TR1) (SEQ ID NO: 10) in roots, roots were lyophilized and 2 mg of powder per sample were resuspended in LDS, 1% beta-mercaptoethanol containing 1 tablet/7 ml of proteinase inhibitor Complete Mini. The mixture was heated at 80°C for 10 min and then centrifuged at 4°C, 20,000 g for 15 min. The supernatant sample was loaded onto 4-12% Bis-Tris Midi gels with MES running buffer according to the manufacturer's instructions (Invitrogen™) and transferred to a nitrocellulose membrane using an iBlot® device (Invitrogen™). The nitrocellulose membrane was incubated in PBST containing 5% skimmed milk powder for 2 hours before overnight incubation with affinity purified rabbit polyclonal antibody to IPD090Aa in PBST overnight. The membrane was washed three times with PBST and then incubated in PBST for 15 min and then two times for 5 min before incubation for 2 h in PBST with HRP-conjugated goat anti-rabbit Ig for 3 h. insecticidal proteins were detected using ECL™ Western Blotting Reagents (GE Healthcare, Cat # RPN2106) and visualized using a Luminescent Image Analyzer (ImageQuant™ LAS 4000, GE Healthcare). Transgenic maize plants positive for the expression of insecticidal proteins were tested for pesticidal activity using standard biological assays known in the art. Such methods included, for example, root excision bioassays and whole plant bioassays. See, for example, US Patent Application Publication No. US 2003/0120054 and International Publication No. wO 2003/018810.

Example 17 Efficiency of IPD090 polypeptide objects in a greenhouse.

The greenhouse efficiency results for T0 for objects derived from the designs PHP73234, PHP73237, and PHP77372 are shown in FIG. 3. Efficacy for objects derived from all 3 constructs was observed against negative control objects (blank), which was assessed by root protection against western corn bug. Root protection was assessed based on the number of affected root nodes (CRWNIS = Cornworm Node Infestation Index) using the method developed by Oleson, et al. (2005) [J. Econ Entomol. 98(1):1-8]. The root lesion score was scored from 0 to 3, with 0 indicating no visible root lesion, 1 indicating 1 root lesion node, 2 indicating 2 root lesion nodes, and 3 indicating a maximum score of 3 root lesion nodes. Mean scores (eg, 1.5) indicate an additional proportion of damaged nodes (eg, one and half affected node). In FIG. 3 shows that most of the events from PHP73234, PHP73237 and PHP77372 performed better than the negative control and had corn bug scores <1.0.

Example 18 Three-dimensional structure of IPD090Aa determined by X-ray crystallography.

IPD090Aa 1167 variant crystals were grown by hanging drop method by vapor diffusion at 25°C. Crystals were obtained by mixing 2 μl of a 10 mg/ml protein solution and 2 μl of crystallization solution containing 0.2 M MgCl ₂ hexahydrate, 0.1 M HEPES, pH 7.5, and 30% PEG 400. Crystals were set to 0. 5 mM loop and provided cryoprotection by adding ~20% glycerol to the crystallization solution. They were flash frozen in liquid N ₂ and mounted on a Rigaku Micromax-007 HF X-ray source at the Iowa State University Macromolecular X-ray Crystallography Center. 2.1A, data were collected using a R-Axis IV++ signal plate detector at a distance of 165.0 mM. 60° data were collected at a working field width of 0.5°. Diffraction data were indexed and integrated with iMOSFILM (CCP4 GNU License) (Battye, TGG, et al. (2011) Acta Cryst. D67, 271-281) (Steller, I et al. (1997) J. Appl. Cryst. 30 , 1036-1040) and scaled using SCALA (Kabsch, W. 1998) J. Appl. Cryst. 21, 916-924. The structure was determined using the PhaserMR molecular displacement program (McCoy, AJ et al (2007) J. Appl. Cryst. 40, 658-674). The structure of the MACPF/perforin-like protein from Photorhabdus luminescens (PDB ID 2QP2) (Rosado, CJ et al. (2007) Science 317, 1548-1551) was used as a search model. A suitable solution for the rotation and translation functions was identified. The sequence of the IPD090Aa 1167 variant was then inserted into electron density with

- 88 040502 using WinCoot© (Emsley P, et al. (2010) ASTA CRYSTALLOGRAPHICA SECTION DBIOLOGICAL CRYSTALLOGRAPHY 66, 486-501). The model was refined using Refmac5 (Murshudov, G. et al. (1996) in the Refinement of Protein structures, Proceedings of Daresbury Study Weekend;

Murshudov, G.N. et al. (1997) Acta Cryst. D53, 240-255) to R-factor=0.236 and R-free=0.267 with >96% amino acids in the eligible areas of the Ramachandran map. In table. 13 shows the collected data and updated statistics.

Table 13

Collected data statistics

Spatial β 90 Υ 90 group Permission Cell dimensions Р4ХХ 2.13 a b 127.61 127.61 with a 116.12 90 Reflections 244629 merge 10.40% Completion (%) 99.5 I / SigmaI 10, 8 Plurality 4, 6 comparisons Refined statistical characteristics Resolution (A) 2.13 No. reflections 51601 Rwork/Rf gay 21.45/24.54 number of atoms Protein 3731 Water 184 ligand 1 B factors (A ² ) 32.84 R.M.S. deviations Length values communications (A) 0.019 Angle values 1.943 connection (°)

Ramachandran Map

Favorable 95.62%

Allowed 3.55%

dropdown

0.84% value

General Procheck

G-factor -0.1

The general structure of IPD090Aa resembles that of other proteins containing the membrane attack complex/porphyrin (MACPF). Its N-terminal domain consists of the MACPF domain, while the C-terminal domain contains a β-prismatic domain (Fig. 4). Secondary structures were labeled according to Rosado et al (2007 Science 317, 1548-1551). The Mg+ atom is shown as a sphere at the bottom of the β-prismatic domain. The two clusters of helices (CH1 and CH2) are structurally similar to the transmembrane helices (TMH1 and TMH2) of toxins from the cholesterol-dependent cytolysin (CDC) family. The overall shape of the N-terminal MACPF domain is somewhat box-shaped (~42A x 44A x 24A) with a central L-shaped 4-strand antiparallel β-layer and 2 a-helix clusters. The 17 N-terminal amino acids in the MACPF domain form the 5th member of the central L-shaped β-layer, but are parallel to strand 4 (FIG. 5). The MACPF domain from P. luminescens has an a-helical N-terminus. The C-terminal β-prismatic domain is located in the lower part and under the central β-layer. It is connected to the MACPF domain via a five amino acid linker that adopts a β-strand-like extended conformation. The β-prismatic domain consists of three 3-strand anti-parallel β-layers with a 3-dimensional axis running through the center of the domain (Fig. 6). The Mg ⁺² ion is located on this 3-dimensional axis and is coordinated with the carbonyl atoms of the backbone L365, L415, L465 and the carbonyl atoms of the side

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Claims (5)

chains N366, N416 and N466. Although the role of Mg ⁺² in insecticidal activity has not been observed, the Mg ⁺² ion fills an anionic gap at this location in the molecule and helps maintain the alignment of the 3 antiparallel β-sheets around the 3D axis. The above description of the various illustrated embodiments of the present invention is not intended to be exhaustive or intended to limit the scope of the exact form disclosed. While specific embodiments and examples are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the present invention as will be appreciated by those skilled in the art. The ideas presented herein may be applied to purposes other than the examples described above. Numerous modifications and variations are possible in light of the foregoing ideas, and are therefore within the scope of the appended claims. These and other changes can be made in light of the foregoing detailed description. In general, in the following claims, the terms used should not be construed as limiting the scope of the present invention to the specific embodiments disclosed in the specification and claims. The full disclosures of each document cited (including patents, patent applications, journal publications, abstracts, manuals, books, or other disclosures) in the Background, Detailed Description, and Examples sections are incorporated herein by reference in their entirety. Attempts have been made to ensure accuracy with respect to the numbers used (eg amounts, temperatures, concentrations, etc.), but some experimental errors and deviations must be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight; temperature is given in degrees Celsius and pressure is atmospheric or substantially atmospheric. CLAIM 1. Recombinant insecticidal polypeptide with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 384, or its fragments having insecticidal activity, where the fragment : (a) contains amino acids 1-315, amino acids 1-330, amino acids 1-349, amino acids 1-450, amino acids 25-315, amino acids 25-330, amino acids 25-349, amino acids 25-450 or amino acids 25-483 from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 384; or (b) is an N-terminal and/or C-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 amino acids from the N-terminus and / or C-terminus relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 384. 2. The recombinant insecticidal polypeptide of claim 1(a), wherein the polypeptide comprises amino acids 25-483 of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. 3. The recombinant insecticidal polypeptide of claim 1, wherein the insecticidal polypeptide is linked to a heterologous signal sequence or transit sequence. 4. Insecticidal chimeric polypeptide containing the N-terminal region of the first polypeptide with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; and the C-terminal region of a second different polypeptide with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, having insecticidal activity, where the N-terminal region contains: (a) an amino acid sequence with at least 90% sequence identity to the amino acid sequence of amino acids 1-144, amino acids 1-239, amino acids 1296, amino acids 1-348, amino acids 1-382, amino acids 1-422, amino acids 1-442, amino acids 25144, amino acids 25-239, amino acids 25-296, amino acids 25-348, amino acids 25-382, amino acids 25-422, amino acids 25-442 of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 ; and the C-terminal region contains an amino acid sequence with at least 90% sequence identity to the amino acid sequence of amino acids 146-483, amino acids 241-483, amino acids 297-483, amino acids 349-483, amino acids 383-483, amino acids 423-483, or amino acids 443-483 of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; or (b) amino acids 1-144, amino acids 1-239, amino acids 1-296, amino acids 1-348, amino acids 1-382, amino acids 1-422, amino acids 1-442 of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; and the C-terminal region contains amino acids 146-483, amino acids 241-483, amino acids 297-483, amino acids 349-483, amino acids 383-483, amino acids 423-483 or amino acids 443483 from SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. 5. An insecticidal composition comprising at least one recombinant insecticidal polypeptide according to claim 1, 2 or 3, or a chimeric polypeptide according to claim 4. -