Insecticidal proteins from plants and methods of use thereof

Abstract

The present invention provides compositions and methods for controlling harmful organisms. The methods involve transforming organisms with nucleic acid sequences encoding insecticidal proteins. In particular, the nucleic acid sequences can be used to prepare plants and microorganisms with insecticidal activity. Therefore, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. The compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences can be used to construct expression vectors that are subsequently transformed into target organisms, including plants, as probes for isolating other homologous (or partially homologous) genes. The insecticidal proteins can be used to control Lepidoptera, Coleoptera, Diptera, Fungi, Hemiptera and nematode pest populations, inhibit their growth or kill them, and can be used to produce compositions with insecticidal activity.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 521,084 (filed June 16, 2017), which is incorporated herein by reference in its entirety.

[0003] References to sequence lists submitted electronically

[0004] An official copy of this sequence list was submitted electronically via EFS-Web as an ASCII format sequence list, filenamed 5296WOPCT_SequenceListing, created on March 13, 2018, and is 186 kilobytes in size, and was submitted with this specification. The sequence list contained in the file in the ASCII format is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0005] This disclosure relates to the field of molecular biology. Novel genes encoding pest-killing proteins are provided. These pest-killing proteins and the nucleic acid sequences encoding them can be used to prepare pest-killing agents and produce pest-resistant transgenic plants. Background Technology

[0006] The use of microbial agents (such as fungi, bacteria, or other insect species) for the biological control of agriculturally significant insect pests offers an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Biopesticides pose a lower risk of pollution and environmental harm and offer greater target specificity than traditional broad-spectrum chemical insecticides. Furthermore, biopesticides are often less expensive to produce, thus increasing the economic yield of various crops.

[0007] Certain species of Bacillus microorganisms are known to possess pest-killing activity against a range of insect pests, including those in the orders Lepidoptera, Diptera, Coleoptera, and Hemiptera. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity is also attributed to strains of Bacillus larvae, Bacillus lentimorbus, Bacillus sphaericus, and Bacillus cereus. Microbial insecticides, particularly those derived from Bacillus strains, play an important role in agriculture as an alternative to chemical pest control.

[0008] Insect-enhanced crop plants have been developed by genetically engineering them to produce pest-killing proteins from Bacillus species. For example, maize and cotton plants have been genetically engineered to produce pest-killing proteins isolated from Bacillus thuringiensis strains. These genetically engineered crops are now widely used in agriculture, providing farmers with an environmentally friendly alternative to traditional insect control methods. While they have proven commercially successful, these genetically engineered insect-resistant crop plants only provide resistance to a narrow range of economically important insect pests. In some cases, insects can develop resistance to different insecticidal compounds, necessitating the identification of alternative biocontrol agents for pest control.

[0009] Therefore, there remains a need for novel insecticidal proteins with varying ranges of insecticidal activity against insect pests, such as insecticidal proteins active against a variety of insects in the Lepidoptera and Coleoptera orders, including but not limited to insect pests that have developed resistance to existing insecticides. Summary of the Invention

[0010] In one aspect, compositions and methods are provided for conferring pest-killing activity upon bacteria, plants, plant cells, tissues, and seeds. The compositions include nucleic acid molecule coding sequences of pest-killing and insecticidal polypeptides, a vector containing those nucleic acid molecules, and a host cell containing said vector. The compositions also include the pest-killing polypeptide sequence and antibodies against those polypeptides. The compositions further comprise transformed bacteria, plants, plant cells, tissues, and seeds.

[0011] On the other hand, isolated or recombinant nucleic acid molecules encoding the IPD059 polypeptide are provided, the polypeptide including amino acid substitutions, deletions, insertions, and fragments thereof. An isolated or recombinant nucleic acid molecule is provided, which encodes SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 6 ... The IPD059 polypeptides of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 78, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Nucleic acid sequences complementary to or hybridizing with the nucleic acid sequences of the embodiments are also included. These nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in a variety of organisms, including but not limited to microorganisms and plants. The nucleotide or amino acid sequences can be synthetic sequences designed for expression in organisms including, but not limited to, microorganisms or plants.

[0012] On the other hand, it includes the IPD059 peptide. Isolated or recombinant SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ IPD059 polypeptides of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 78, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0013] On the other hand, isolated or recombinant nucleic acid molecules encoding the IPD098 polypeptide are provided, said polypeptide comprising amino acid substitutions, deletions, insertions, and fragments thereof. Isolated or recombinant nucleic acid molecules are provided capable of encoding the IPD098 polypeptides of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Nucleic acid sequences complementary to or hybridizing with the sequences of the embodiments are also covered. The nucleic acid sequence can be used in DNA constructs or expression cassettes for transformation and expression in a variety of organisms, including microorganisms and plants. The nucleotide or amino acid sequence can be a synthetic sequence designed for expression in organisms, including but not limited to microorganisms or plants.

[0014] On the other hand, the IPD098 peptide is included. Also provided are isolated or recombinant IPD098 peptides of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0015] On the other hand, isolated or recombinant nucleic acid molecules encoding the IPD108 polypeptide are provided, the polypeptide comprising amino acid substitutions, deletions, insertions, and fragments thereof. Isolated or recombinant nucleic acid molecules are provided capable of encoding the IPD108 polypeptides of SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID NO: 136, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Nucleic acid sequences complementary to or hybridizing with the nucleic acid sequences of the embodiments are also covered. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in a variety of organisms, including but not limited to microorganisms and plants. The nucleotide or amino acid sequences can be synthetic sequences designed for expression in organisms including, but not limited to, microorganisms or plants.

[0016] On the other hand, the IPD108 peptide is included. Isolated or recombinant IPD108 peptides of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, and SEQ ID NO: 136 are also provided, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0017] On the other hand, isolated or recombinant nucleic acid molecules encoding the IPD109 polypeptide are provided, the polypeptide comprising amino acid substitutions, deletions, insertions, and fragments thereof. Isolated or recombinant nucleic acid molecules are provided capable of encoding the IPD109 polypeptide of SEQ ID NO: 138, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof. Nucleic acid sequences complementary to or hybridizing with the nucleic acid sequences of the embodiments are also covered. The nucleic acid sequences can be used in DNA constructs or expression cassettes for transformation and expression in a variety of organisms, including but not limited to microorganisms and plants. The nucleotide or amino acid sequences can be synthetic sequences designed for expression in organisms including, but not limited to, microorganisms or plants.

[0018] On the other hand, the IPD109 polypeptide is covered. Isolated or recombinant IPD109 polypeptide of SEQ ID NO: 138 is also provided, as well as amino acid substitutions, deletions, insertions, fragments thereof, and combinations thereof.

[0019] On the other hand, methods are provided for producing polypeptides and using those polypeptides to control or kill Lepidoptera, Coleoptera, nematodes, fungi, and / or Diptera pests. The transgenic plants of the embodiments express one or more of the pest-killing sequences disclosed herein. In various embodiments, the transgenic plants further comprise one or more additional insect-resistant genes, for example, one or more additional genes for controlling Coleoptera, Lepidoptera, Hemiptera, or nematode pests. Those skilled in the art will understand that the transgenic plants may contain any genes conferring the desired agronomic trait.

[0020] On the other hand, methods for detecting the nucleic acids and peptides of the embodiments in a sample are also included. Kits are provided for detecting the presence of peptides of this disclosure or the presence of polynucleotides encoding peptides of this disclosure in a sample. The kits may be provided with all reagents and control samples required to perform the intended reagent detection method, as well as instructions for use.

[0021] In another aspect, the compositions and methods of the embodiments can be used to produce organisms with enhanced resistance or tolerance to pests. These organisms, and compositions comprising said organisms, are desirable for agricultural purposes. The compositions of the embodiments can also be used to produce proteins with altered or improved pest-killing activity, or to detect the presence of IPD059, IPD098, IPD108, or IPD109 peptides. Attached Figure Description

[0022] Figure 1 Display using Vector kit The phylogenetic tree of the following IPD059 homologs generated by the adjacency-joining method in the module is as follows: IPD059Aa (SEQ ID NO: 39), IPD059Ab (SEQ ID NO: 40), IPD059Ac (SEQ ID NO: 41), IPD059Ad (SEQ ID NO: 42), IPD059Ae (SEQ ID NO: 43), IPD059Af (SEQ ID NO: 44), IPD059Ag (SEQ ID NO: 45), IPD059Ah (SEQ ID NO: 46), IPD059Ca (SEQ ID NO: 47), IPD059Cb (SEQ ID NO: 48), IPD059Cc (SEQ ID NO: 49), IPD059Da (SEQ ID NO: 50), IPD059Db (SEQ ID NO: 51), IPD059Ea (SEQ ID NO: 52), IPD059Eb (SEQ ID NO: 53). NO: 53), IPD059Ec (SEQ ID NO: 54), IPD059Ed (SEQ ID NO: 55), IPD059Ee (SEQ ID NO: 56), IPD059Ef (SEQ ID NO: 57), IPD059Eg (SEQ ID NO: 58), IPD059Eh (SEQ ID NO: 59), IPD059Ei (SEQ ID NO: 60), IPD059Ej (SEQ ID NO: 61), IPD059Ek (SEQ ID NO: 62), IPD059El (SEQ ID NO: 63), IPD059Em (SEQ ID NO: 64), IPD059En (SEQ ID NO: 65), IPD059Eo (SEQ ID NO: 66), IPD059Ep (SEQ ID NO: 67), IPD059Eq (SEQ ID NO: 68), IPD059Er (SEQ ID NO: 69), IPD059Es (SEQ ID NO: 70), IPD059Et (SEQ ID NO: 71), IPD059Eu (SEQ ID NO: 72), IPD059Ev (SEQ ID NO: 73), IPD059Ew (SEQ ID NO: 74), and IPD059Ex (SEQ ID NO: 75).

[0023] Figure 2A-2B Display using Vector kit The module compares the amino acid sequences of the following IPD059 homologs: IPD059Aa (SEQ ID NO: 39), IPD059Ab (SEQ ID NO: 40), IPD059Ac (SEQ ID NO: 41), IPD059Ad (SEQ ID NO: 42), IPD059Ae (SEQ ID NO: 43), IPD059Af (SEQ ID NO: 44), IPD059Ag (SEQ ID NO: 45), IPD059Ah (SEQ ID NO: 46), IPD059Ca (SEQ ID NO: 47), IPD059Cb (SEQ ID NO: 48), and IPD059Cc (SEQ ID NO: 49). The amino acid sequence diversity among these sequences is highlighted. Above the alignment, conserved cysteine ​​residues are indicated by… Symbol representation. The starting point of the deletion variant IPD059AaTR1 (SEQ ID NO: 78) is represented by a "·" symbol above residue 29 (aspartic acid) of IPD059Aa (SEQ ID NO: 39).

[0024] Figure 3 Display using Vector kit The module, with the following amino acid sequences aligned to IPD059 homologs: IPD059Da (SEQ ID NO: 50), IPD059Db (SEQ ID NO: 51), IPD059Ea (SEQ ID NO: 52), IPD059Eb (SEQ ID NO: 53), IPD059Ec (SEQ ID NO: 54), IPD059Ed (SEQ ID NO: 55), IPD059Ee (SEQ ID NO: 56), IPD059Ef (SEQ ID NO: 57), and IPD059Eg (SEQ ID NO: 58). The amino acid sequence diversity among these sequences is highlighted. Above the alignment, conserved cysteine ​​residues are indicated by… Symbolic representation.

[0025] Figure 4 Display using Vector kit The module, with the following amino acid sequences aligned to IPD059 homologs: IPD059Em (SEQ ID NO: 64), IPD059En (SEQ ID NO: 65), IPD059Eo (SEQ ID NO: 66), IPD059Ep (SEQ ID NO: 67), IPD059Eq (SEQ ID NO: 68), IPD059Er (SEQ ID NO: 69), IPD059Es (SEQ ID NO: 70), and IPD059Et (SEQ ID NO: 71). The amino acid sequence diversity among these sequences is highlighted. Above the alignment, conserved cysteine ​​residues are indicated by… Symbolic representation.

[0026] Figure 5 Display using Vector kit The module, with the following amino acid sequences aligned to IPD098 homologs: IPD098Aa (SEQ ID NO: 102), IPD098Ab (SEQ ID NO: 103), IPD098Ac (SEQ ID NO: 104), IPD098Ba (SEQ ID NO: 105), IPD098Bb (SEQ ID NO: 106), IPD098Bc (SEQ ID NO: 107), IPD098Bd (SEQ ID NO: 108), IPD098Be (SEQ ID NO: 109), IPD098Bf (SEQ ID NO: 110), IPD098Bg (SEQ ID NO: 111), IPD098Bh (SEQ ID NO: 112), and IPD098Bi (SEQ ID NO: 113). The amino acid sequence diversity among these sequences is highlighted. Above the alignment, conserved cysteine ​​residues are indicated by… Symbolic representation.

[0027] Figure 6 Display using Vector kit The module provides amino acid sequence alignments of the following IPD098 homologs: IPD098Fa (SEQ ID NO: 118), IPD098Ga (SEQ ID NO: 119), IPD098Gb (SEQ ID NO: 120), IPD098Gc (SEQ ID NO: 121), IPD098Gd (SEQ ID NO: 122), and IPD098Ge (SEQ ID NO: 123). The amino acid sequence diversity among these sequences is highlighted. Above the alignment, conserved cysteine ​​residues are indicated by… Symbolic representation.

[0028] Figures 7A-7B Display using Vector kit The module, with the following amino acid sequences aligned to IPD108 homologs: IPD108Aa (SEQ ID NO: 131), IPD108Da (SEQ ID NO: 132), IPD108Dd (SEQ ID NO: 133), IPD108Df (SEQ ID NO: 134), IPD108Dj (SEQ ID NO: 135), and IPD108Eb (SEQ ID NO: 136). The amino acid sequence diversity among these sequences is highlighted. Above the alignment, conserved cysteine ​​residues are indicated by… Symbolic representation.

[0029] Figure 8 The graph shows the density measurements of the intragel fluorescence from the SDS-PAGE gel of Example 12, for homologous competition between 40 nM Alexa-labeled IPD098Aa peptide (SEQ ID NO: 102) and unlabeled IPD098Aa peptide (SEQ ID NO: 102), as the percentage of total binding to WCRW BBMV (in the absence of competitors) relative to the concentration of unlabeled IPD098Aa peptide (SEQ ID NO: 102). Detailed Implementation

[0030] It should be understood that this disclosure is not limited to the specific methods, protocols, cell lines, genera, and reagents described, and therefore variations are possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure.

[0031] As used herein, the singular forms “a / an” and “the” include a plural indicator unless the context clearly indicates otherwise. Thus, for example, reference to “cell” includes a plurality of such cells, and reference to “protein” includes one or more proteins known to those skilled in the art and their equivalents, and so on. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains, unless otherwise expressly stated.

[0032] This disclosure relates to compositions and methods for controlling pests. The methods involve transforming organisms with nucleic acid sequences encoding polypeptides of the present disclosure. The nucleic acid sequences of the embodiments can be used to prepare plants and microorganisms with pest-killing activity. Thus, transformed bacteria, plants, plant cells, plant tissues, and seeds are provided. The compositions comprise pest-killing nucleic acids and proteins of bacterial species. The nucleic acid sequences can be used to construct expression vectors subsequently transformed into target organisms, as probes for isolating other homologous (or partially homologous) genes, and for producing altered IPD059, IPD098, IPD108, or IPD109 polypeptides by methods known in the art, such as directed mutagenesis, domain exchange, or DNA shuffling. The IPD059, IPD098, IPD108, or IPD109 polypeptides can be used to control or kill populations of Lepidoptera, Coleoptera, Diptera, fungi, Hemiptera, and nematodes, and can be used to produce compositions with pest-killing activity. The target insect pests include, but are not limited to: Lepidoptera species, including but not limited to: Corn Earworm (CEW) (Helicoverpa zea); European Corn Borer (ECB) (Ostrinia nubialis); Diamondback Moth, such as Corn Earworm; Soybean Looper, such as Soybean Includers Walker; and Velvet Bean Caterpillar, such as Pseudoplusia gemmatalis Hübner; and Coleoptera species, including but not limited to: Western Corn Rootworm (Diabrotica virgifera) - WCRW, Southern Corn Rootworm (Diabrotica undecimpunctata) howardi))-SCRW, and Northern corn rootworm (Diabrotica barberi)-NCRW.

[0033] As used herein, "biotoxic toxin" or "biotoxic protein" refers to a toxin or a protein homologous to it, which has toxic activity against one or more of the following pests, including but not limited to members of the orders Lepidoptera, Diptera, Hemiptera, Coleoptera, or Nematoda. Biotoxic proteins have been isolated from organisms including, for example, species of the genera *Bacillus*, *Pseudomonas*, *Photorhabdus*, *Xenorhabdus*, *Clostridium bifermentans*, and *Paenibacillus popilliae*. Insecticidal proteins include, but are not limited to: insecticidal proteins from Pseudomonas species, such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens, 7: 1-13); insecticidal proteins from Pseudomonas protegens strains CHA0 and Pf-5 (formerly fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology, 10: 2368-2386; Genebank accession number EU400157); insecticidal proteins from Pseudomonas taiwanensis (Liu et al., (2010) J. Agric. Food Chem., 58: 12343-12349); and Pseudomonas pseudoalkaloid. Insecticidal proteins from *Pseudoalcaligenes* (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 *Proteobacterium* and *Bacillus* species (Hinchliffe et al., (2010) The Open Toxicology Journal, 3:101-118 and Morgan et al., (2001) Applied and Envir. Micro. 67:2062-2069); insecticidal proteins from US Patent Nos. 6,048,838 and 6,379,946; and the PIP-1 polypeptide of US 9,688,730.AfIP-1A and / or AfIP-1B peptides in US 9,475,847; PIP-47 peptide in US Publication No. US 20160186204; IPD045, IPD064, IPD074, IPD075, and IPD077 peptides in PCT Publication No. WO 2016 / 114973; IPD080 peptide in PCT Serial No. PCT / US17 / 56517; IPD078, IPD084, IPD085, IPD086, IPD087, IPD088, and IPD089 peptides in PCT / US17 / 54160; PIP-72 peptide in US Patent Publication No. US 20160366891; US ​​Publication No. US PtIP-50 and PtIP-65 peptides (US Serial No. 20170166921); IPD098, IPD059, IPD108, and IPD109 peptides (US Serial No. 62 / 521084); PtIP-83 peptide (US Publication No. 20160347799); PtIP-96 peptide (US Publication No. 20170233440); IPD079 peptide (PCT Publication No. WO 2017 / 23486); PCT Publication No. WO IPD082 peptide (serial number 2017 / 105987), IPD090 peptide (serial number PCT / US17 / 30602), IPD093 peptide (serial number 62 / 434020), IPD103 peptide (serial number PCT / US17 / 39376), IPD101 peptide (serial number 62 / 438179), and IPD121 peptide (serial number US62 / 508,514).And delta-endotoxins, including but not limited to 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, as well as the Bacillus thuringiensis cell-lysing cyt1 and cyt2 genes. Members of these classes of Bacillus thuringiensis insecticidal proteins are well known to those skilled in the art (see, Crickmore et al., “Bacillus thuringiensis toxin nomenclature” (2011), lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / , which can be accessed on the World Wide Web with the “www” prefix).

[0034] Examples of delta-endotoxins include, but are not limited to: Cry1A protein of U.S. Patent Nos. 5,880,275, 7,858,849, and 8,878,007; Cry1Ac mutant of US 9,512,187; DIG-3 or DIG-11 toxins (N-terminal deletion of α-helix 1 and / or α-helix 2 variants of cry proteins (such as Cry1A, Cry3A)) of U.S. Patent Nos. 8,304,604, 8,304,605, and 8,476,226; Cry1B of U.S. Patent Application Serial No. 10 / 525,318, U.S. Patent Application Publication No. US 20160194364, and U.S. Patent Nos. 9,404,121, and 8,772,577; and PCT Publication No. WO. Variants of Cry1B (serial number PCT / US17 / 27160, 2016 / 61197); Cry1C (US Patent No. 6,033,874); Cry1D protein (US 20170233759); Cry1E protein (PCT serial number PCT / US17 / 53178); Cry1F protein (US Patent Nos. 5,188,960 and 6,218,188); Cry1A / F chimeras (US Patent Nos. 7,070,982, 6,962,705 and 6,713,063); Cry1I protein (PCT Publication No. WO 2017 / 0233759); Cry1J variant (US Publication No. 20170240603); Cry2 proteins such as Cry2Ab protein (US Patent No. 7,064,249) and US... Cry2A.127 protein of US Patent No. 7208474; Cry3A protein, including but not limited to engineered hybrid insecticidal protein (eHIP) produced by a unique combination of variable and conserved regions of at least two different Cry proteins (US Patent Application Publication No. 2010 / 0017914); Cry4 protein; Cry5 protein; Cry6 protein; Cry8 protein of US Patent Nos. 7,329,736, 7,449,552, 7,803,943, 7,476,781, 7,105,332, 7,339,092, 7,378,499, 7,462,760, and 9,593,345; Cry9 protein, such as Cry9A, Cry9B, Cry9C, Cry9D, etc.Members of the Cry9E and Cry9F families, including the Cry9 protein (US Patents 9,000,261 and 8,802,933 and US Serial No. WO 2017 / 132188); the Cry15 protein, described in the following references: Naimov et al., (2008) Applied and Environmental Microbiology, 74: 7145-7151; the Cry14 protein (US Patent No. US 8,933,299); the Cry22 and Cry34Ab1 proteins (US Patent Nos. 6,127,180, 6,624,145 and 6,340,593); and the Cry9 protein (US Patent No. US 8,933,299). The truncated Cry34 protein of US Patent Nos. 8,816,157; ​​CryET33 and CryET34 proteins of US Patent Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; CryET33 and CryET34 homologs of US Patent Publications Nos. 2006 / 0191034, 2012 / 0278954 and PCT Publication No. WO 2012 / 139004; Cry35Ab1 protein of US Patent Nos. 6,083,499, 6,548,291 and 6,340,593; Cry46 protein and CryET34 homolog of US Patent No. 9,403,881. 51 protein, Cry binary toxin; TIC901 or related toxins; TIC807 of US Patent Application Publication No. 2008 / 0295207; TIC853 of US Patent 8,513,493; ET29, ET37, TIC809, TIC810, TIC812, TIC127, and TIC128 of PCT US 2006 / 033867; engineered hemipteran toxin proteins of US Patent Application Publication No. 20160150795; AXMI-027, AXMI-036, and AXMI-038 of US Patent No. 8,236,757; AXMI-031, AXMI-039, AXMI-040, and AXMI-049 of US Patent No. 7,923,602; and AXMI-018 of WO 2006 / 083891.AXMI-020 and AXMI-021; AXMI-010 of WO 2005 / 038032; AXMI-003 of WO 2005 / 021585; AXMI-008 of U.S. Patent Application Publication No. 2004 / 0250311; AXMI-006 of U.S. Patent Application Publication No. 2004 / 0216186; AXMI-007 of U.S. Patent Application Publication No. 2004 / 0210965; AXMI-009 of U.S. Patent Application Publication No. 2004 / 0210964; AXMI-014 of U.S. Patent Application Publication No. 2004 / 0197917; AXMI-004 of U.S. Patent Application Publication No. 2004 / 0197916; AXMI-028 and AXMI-029 of WO 2006 / 119457; WO AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014, and AXMI-004 of U.S. Patent No. 2004 / 074462; AXMI-150 of U.S. Patent No. 8,084,416; AXMI-205 of U.S. Patent Application Publication No. 2011 / 0023184; AXMI-011, AXMI-012, AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063, and AXMI-064 of U.S. Patent Application Publication No. 2011 / 0263488; U.S. Patent No. 8461421 and U.S. Patent No. 8461421. 8,461,422 of AXMI046, AXMI048, AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMI055, AXMI056, AXMI057, AXMI058, AXMI 059, AXMI060, AXMI061, AXMI067, AXMI069, AXMI071, AXMI072, AXMI073, AXMI074, AXMI075, AXMI087, AXMI088, AXMI093, AXM I070,AXMI080,AXMI081,AXMI082,AXMI091,AXMI092,AXMI096,AXMI097,AXMI098,AXMI099,AXMI100,AXMI101,AXMI102,A XMI103, 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; AXMI-R1 and related proteins of U.S. Patent Application Publication No. 2010 / 0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z, and AXMI225z of WO 2011 / 103248: WO AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230, and AXMI231 of U.S. Patent No. 2011 / 103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163, and AXMI-184 of U.S. Patent No. 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035, and AXMI-045 of U.S. Patent Application Publication No. 2010 / 0298211; AXMI-066 and AXMI-076 of U.S. Patent Application Publication No. 2009 / 0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, and AXMI231 of U.S. Patent No. 8,318,900. MI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI 169. AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189; U.S. patent US AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109 of 8461421AXMI110, dsAXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137; AXMI192 of U.S. Patent No. US 8,461,415; AXMI281 of U.S. Patent Application Publication No. US20160177332; U.S. Patent No. US AXMI422 (US Patent No. 8,252,872); Cry proteins with modified proteolytic sites, such as Cry1A and Cry3A, in US Patent No. 8,319,019; Cry1Ac, Cry2Aa, and Cry1Ca toxin proteins from Bacillus thuringiensis strain VBTS 2528 in US Patent Application Publication No. 2011 / 0064710; and Cry proteins MP032, MP049, MP051, MP066, MP068, MP070, MP091S, MP109S, MP114, MP121, MP134S, MP183S, MP185S, MP186S, MP195S, MP197S, MP208S, MP209S, MP212S, and MP214S, in US Serial No. 62 / 607372. MP217S, MP222S, MP234S, MP235S, MP237S, MP242S, MP243, MP248, MP249S, MP251M, MP252S, M P253, MP259S, MP287S, MP288S, MP295S, MP296S, MP297S, MP300S, MP304S, MP306S, MP310S, M P312S, 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, MP575 S, 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, MP802 S, 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. The insecticidal activity of Cry proteins is well known to those skilled in the art (see van Frannkenhuyzen, (2009) J. Invert. Path. [Journal of Invertebrate Pathology] 101: 1-16 for a review). The use of Cry proteins as a trait in transgenic plants is well known to those skilled in the art, and Cry-transgenic plants (including, but not limited to, plants expressing the following: Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A) are known to be transgenic plants.Cry9c and CBI-Bt have received regulatory approval (see Sanahuja, (2011) Plant Biotech Journal 9: 283-300 and CERA (2010) Center for Environmental Risk Assessment of Transgenic Crops (CERA), ILSI Research Foundation, Washington, D.C., at cera-gmc.org / index.php?action=gm_crop_database, which can be accessed on the World Wide Web with the prefix "www"). It is well known to those skilled in the art that several pest-killing proteins can be expressed in plants, such as Vip3Ab and Cry1Fa (US 2012 / 0317682); Cry1BE and Cry1F (US 2012 / 0311746); Cry1CA and Cry1AB (US 2012 / 0311745); Cry1F and CryCa (US 2012 / 0317681); Cry1DA and Cry1BE (US 2012 / 0331590); Cry1DA and Cry1Fa (US 2012 / 0331589); Cry1AB and Cry1BE (US 2012 / 0324606); Cry1Fa and Cry2Aa; and Cry1I and Cry1E (US 2012 / 0324606). 2012 / 0324605); Cry34Ab / 35Ab&Cry6Aa (US 20130167269); Cry34Ab / VCry35Ab&Cry3Aa (US 20130167268); Cry1Da&Cry1Ca (US 9796982); Cry3Aa&Cry6Aa (US 9798963); and Cry3A&Cry1Ab or Vip3Aa (US 9,045,766). Harm-killing proteins also include insecticidal lipases, including the lipid acyl hydrolase of US Patent No. 7,491,869, and cholesterol oxidases, such as those from Streptomyces (Purcell et al., (1993) Biochem Biophys Res Commun [Biochemistry and Biophysics Research Communications] 15: 1406-1413). Harmful organism-killing proteins also include US patents 5,877,012, 6,107,279, 6,137,033, and 7,244,820.Vital toxins, such as those in 7,615,686 and 8,237,020, are examples. Other VIP proteins are well known to those skilled in the art (see lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / vip.html, accessible via the World Wide Web with the prefix "www"). Vital toxins also include Cyt proteins (including the Cyt1A variant) of PCT sequence number PCT / US2017 / 000510; vital toxins also include toxin complex (TC) proteins, which are available from organisms such as pathogenic bacteria, luminescent bacteria, and spore-forming bacteria (see U.S. Patents 7,491,698 and 8,084,418). Some TC proteins have "independent" insecticidal activity, and others enhance the activity of stand-alone toxins produced by the same given organism. The toxicity of "independent" TC proteins (e.g., from *Proteobacterium*, *Pathobacterium*, or *Bacillus* genera) can be enhanced by one or more "synergists" derived from organisms of different genera. There are three main types of TC proteins. As mentioned herein, type A proteins ("protein A") are independent toxins. Type B proteins ("protein B") and type C proteins ("protein C") enhance the toxicity of type A proteins. Examples of type A proteins are TcbA, TcdA, XptA1, and XptA2. Examples of type B proteins are TcaC, TcdB, XptB1Xb, and XptC1Wi. Examples of type C proteins are TccC, XptC1Xb, and XptB1Wi. Harm-killing proteins also include spider, snake, and scorpion venom proteins. Examples of spider venom peptides include, but are not limited to, lecotoxin-1 peptide and its mutants (US Patent No. 8,334,366). The resulting combinations may also include multiple copies of any of the target polynucleotides.

[0035] In some embodiments, the IPD059, IPD098, IPD108, or IPD109 polypeptide comprises an amino acid sequence deduced from the full-length nucleic acid sequence disclosed herein, and an amino acid sequence shorter than the full-length sequence due to the use of an alternative downstream start site or due to processing to produce a shorter protein with necrotic activity. Processing can occur in an organism expressing the protein or in a necrotic organism that has ingested the protein.

[0036] Therefore, this article provides novel isolated or recombinant nucleic acid sequences conferring pest-killing activity. The amino acid sequences of IPD059, IPD098, IPD108, or IPD109 polypeptides are also provided. Proteins translated from these IPD059, IPD098, IPD108, or IPD109 genes allow cells to control or kill certain pests that take up said proteins.

[0037] IPD059 protein and its variants and fragments

[0038] This disclosure covers the IPD059 polypeptide. As used interchangeably herein, “IPD059 polypeptide” and “IPD059 protein” refer to a polypeptide having insecticidal activity (including, but not limited to, insecticidal activity against one or more insect pests of the orders Lepidoptera and / or Coleoptera) and being sufficiently homologous to the IPD059Aa polypeptide of SEQ ID NO: 39. Various IPD059 polypeptides are considered. Sources of IPD059 polypeptides or related proteins include ferns or other primitive plant species selected from, but not limited to, species of the genera *Polypodium*, *Colysis*, *Asplenium*, *Polystichium*, or *Phyllitis*.

[0039] In some embodiments, the IPD059 polypeptide is derived from fern species belonging to the order Polypodiaceae and the genus Polypodiaceae. In some embodiments, the IPD059 polypeptide is derived from fern species of the genus *Polypodium*, selected from, but not limited to: *Polypodium absidatum*, *Polypodium acutifolium*, *Polypodium adiantiforme*, *Polypodium aequale*, *Polypodium affine*, *Polypodium albidopaleatum*, *Polypodium alcicorne*, *Polypodium alfarii*, *Polypodium alfredii*, *Polypodium alfredii var. curtii*, *Polypodium allosuroides*, *Polypodium alsophilicola*, *Polypodium amamianum*, *Polypodium amoenum*, *Polypodium amorphum*, and *Polypodium*. anetioides, twisted water caltrop (Polypodium anfractuosum), snake-shaped water caltrop (Polypodium anguinum), narrow-leaved water caltrop (Polypodium angustifolium f. remotifolia), Polypodium angustifolium var. amphostenon, narrow-leaved water caltrop heterobud variety (Polypodium angustifolium var. heterolepis), narrow-leaved water caltrop monstrosa variety (Polypodium angustifolium var. monstrosa), Polypodium angustipaleatum, linear-leaved water caltrop (Polypodium angustissimum), non-heterogeneous water caltrop anisomeron var.Polypodium pectinatum, Polypodium antioquianum, Polypodium aoristisorum, Polypodium aagolepis, apical keel (Polypodium apicidens), pointed keel (Polypodium apiculatum), Polypodium apoense, Appalachian keel (Polypodium appalachianum), appressum, sandy keel (Polypodium arenarium), Argentinum, argutum, armatum, aromatic keel (Polypodium aromaticum), aspergillum, assurgens, atrum, auriculatum, balaonense, Polypodium balliviani, Polypodium bamleri, Polypodium bangii, Polypodium bartlettii, Polypodium basale, Polypodium bernoullii, Polypodium biauritum, Polypodium bifrons, Polypodiumblepharodes, Polypodium bolivari, Polypodium bolivianum, Polypodium bolobense, Polypodium bombycinum, Polypodium bombycinum var.insularum), Polypodium bradeorum, Polypodium bryophilum, Polypodium bryopodum, Polypodium buchtienii, Polypodium buesii, Polypodium bulbotrichum, Polypodiumcaceresii, Polypodium californicum f.brauscombii, Polypodium californicumf.parsonsiae, Polypodium californicum, Polypodiumcalophlebium, Polypodium calvum, Polypodium camptophyllarium var.abbreviatum, Polypodium capitellatum, Polypodium carpinterae, Polypodium chachapoyense, Polypodium chartaceum, Polypodium chimantense, Polypodium chiricanum,Polypodiumchoquetangense,Polypodium christensenii, Polypodium christii, Polypodium chrysotrichum, Polypodium ciliolepis, Polypodium cinerascens, Polypodium collinsii, Polypodium colysoides, Polypodium confluens, Polypodium conforme, Polypodium Confusum, Polypodium congregatifolium, Polypodium connellii, Polypodium consimile var. bourgaeanum, Polypodium consimile var.The following are various species of water caltrop: Polypodium conterminans, Polypodium contiguum, Polypodium cookii, Polypodium coriaceum, Polypodium coronans, Polypodium costaricense, Polypodium costatum, Polypodium crassifolium f. angustissimum, Polypodium crassifolium var. longipes, Polypodium crassulum, Polypodium craterisorum, Polypodium cryptum, Polypodium crystalloneuron, and Polypodium cucullatum var.Polypodium planum, Polypodium cuencanum, Polypodium cumingianum, Polypodium cupreolepis, Polypodium curranii, Polypodium curvans, Polypodium cyathicola, Polypodium cyathisorum, Polypodium cyclocolpon, Polypodium daguense, Polypodium damunense, Polypodium dareiformioides, Polypodium dasypleura, Polypodium decipiens, Polypodium decorum, Polypodium delicatulum, Polypodium deltoideum, Polypodium demeraranum, Polypodium denticulatum, Polypodium Polypodium dilatatum, Polypodium dispersum, Polypodium dissectum, Polypodium dissimulans, Polypodium dolichosorum, Polypodium dolorense, Polypodium donnell-smithii, Polypodium drymoglossoides, Polypodium ebeninum, Polypodium eggersii, Polypodium elmeri, Polypodium elongatum, Polypodium enterosoroides, Polypodium erubescens, Polypodium erythrolepis, Polypodium erythrotrichum, Polypodium eurybasis, Polypodium eurybasis var.Polypodium villosum, Polypodium exornans, Polypodium falcoideum, Polypodium fallacissimum, Polypodium farinosum, Polypodium faucium, Polypodium feei, Polypodium ferrugineum, Polypodium feuillei, Polypodium firmulum, Polypodium firmum, Polypodium flaccidum, Polypodium flagellare, Polypodium flexuosum, Polypodium flexuosum var. ekmanii, Polypodium forbesii, Taiwan Polypodium (non-), Polypodium fraxinifolium subsp. articulatum, Polypodium fraxinifolium subsp.Polypodium luridum, Polypodium fructuosum, Polypodium fucoides, Polypodium fulvescens, Polypodium galeottii, Polypodium glaucum, Polypodium glycyrrhiza, Polypodium gracillimum, Polypodium gramineum, Polypodium grandifolium, Polypodium gratum, Polypodium graveolens, Polypodium griseo-nigrum, Polypodium griseum, Polypodium guttatum, Polypodium haalilioanum, Polypodium hammatisorum, Polypodium han's water dragon (Polypodium hammatisorum) hancockii), Polypodium haplophlebicum, Harris water porpoise (Polypodium harrisii), simple variety of spear-shaped water porpoise (Polypodium hastatum var. simpleplex), Hawaiian water porpoise (Polypodium hawaiiense), Polypodium heanophyllum, Helleri water porpoise (Polypodium helleri), Polypodium hemmionitidium, Henry water porpoise (Polypodium henryi), Southern tree water porpoise (Polypodium herzogii), Western water porpoise (Polypodium hesperium), Polypodium hessii, Polypodium hombersleyi, Hostmann water porpoise (Polypodium hostmannii), Dwarf water porpoise (Polypodium humile), Colorless water porpoise (Polypodium hyalinum), Polypodium iboense, Polypodium induens var. subdentatum, Cunning water porpoise (Polypodium Polypodium insidiosum, Polypodium insigne, Polypodium intermedium subsp.*Polypodium masafueranum* var. *obtuseserratum*, *Polypodium intramarginale*, *Polypodium involutum*, *Polypodium itatiayense*, *Polypodium javanicum*, *Polypodium juglandifolium*, *Polypodium kaniense*, *Polypodium knowltoniorum*, *Polypodium kyimbilense*, *Polypodium l′herminieri var. *costaricense*, *Polypodium lachniferum f. incurvata*, *Polypodium lachniferum var. *glabrescens*, *Polypodium lachnopus*, *Polypodium lanceolatum var. *complanatum*, *Polypodium lanceolatum* var. *lanceolatum* Polypodium var. trichophorum), Polypodium latevagans, Polypodium laxifrons, Polypodium laxifrons var. lividum, Polypodium lehmannianum, Polypodium leiorhizum, Polypodium leptopodon, Polypodium leuconeuron var. angustifolia, Polypodium leuconeuron var. latiifolium, Polypodium leucosticta, Polypodium limulum, Polypodium lindigii, Polypodium lineatum, Polypodium lomarioides, Polypodium longifrons, Polypodium loretense, Polypodium loriceum var.Polypodium umbraticum, Polypodium loriforme, Polypodium loxogramme f. gigas, Polypodium ludens, Polypodium luzonicum, Polypodium lycopodioides f. obtusum, Polypodium lycopodioides L., Polypodium macrolepis, Polypodium macrophyllum, Polypodium macrosorum, Polypodium macrosphaerum, Polypodium maculosum, Polypodium madrense, Polypodium manmeiense, Polypodium margaritiferum, Polypodium maritimum, Polypodium maritimum Polypodium martensii, Polypodium mayoris, Polypodium megalolepis, Polypodium melanotrichum, Polypodium menisciifolium var. pubescens, Polypodium meniscioides, Polypodium merrillii, Polypodium mettenii, Polypodium mexiae, Polypodium microsorum, Polypodium militare, Polypodium minimum, Polypodium minusculum, Polypodium mixtum, Polypodium mollendense, Polypodium mollissimum, Polypodium moniliforme var.minus), Polypodium monooides, Polypodium monticola, Polypodium montigenum, Polypodium morizianum, Polypodium moultonii, Polypodium multicaudatum, Polypodium multilineatum, Polypodium multisorum, Polypodium munchii, Polypodium muscoides, Polypodiummyriolepis, Polypodium myriophyllum, Polypodium myriotrichum, Polypodium nematorhizon, Polypodium nemorale, Polypodium nesioticum, Polypodium nigrescentium, Polypodium nigripes, Polypodium nigrocinctum, Polypodium Polypodium nimbatum, Polypodium nitidissimum, Polypodium nitidissimum var. latior, Polypodium nubrigenum, Polypodium oligolepis, Polypodium oligosorum, Polypodium oligosorum, Polypodium olivaceum, Polypodium olivaceum var.Polypodium oodes, Polypodium oosphaerum, Polypodium oreophilum, Polypodium ornatissimum, Polypodium ornatum, Polypodium ovatum, Polypodium oxylobum, Polypodium oxypholis, Polypodium pakkaense, Polypodium pallidum, Polypodium palmatopedatum, Polypodium palmeri, Polypodium panamense, Polypodium parvum, Polypodium patagonicum, Polypodium paucisorum, Polypodium pavonianum, Polypodium pectinatum (warm-up variant) var. caliense), Polypodium pectinatum var. hispidum, Polypodium pellucidum, Polypodium pendulum var. boliviense, Polypodium percrassum, Polypodium perpusillum, Polypodium peruvianum var. subgibbosum, Polypodium phyllitidis var. elongatum, Polypodium pichinchense, Polypodium pilosissimum, Polypodium pilosissimum var. glabriusculum, Polypodium pilossimum var. tunguraquensis, Polypodium pityrolepis, Polypodium platyphyllum, Polypodium brie's var. playfairii), and the common water pod Cooper variety (Polypodium plebeium var.).(The remaining text appears to be a list of names and related terms, possibly related to Polypodium, ... Polypodium propinguum, Polypodium proteus, Polypodium pruinatum, Polypodium pseudocapillare, Polypodium pseudofraternum, Polypodium pseudonutans, Polypodium pseudoserratum, Polypodium pulcherrimum, Polypodium pulogense, Polypodium pungens, Polypodium purpusii, Polypodium radicale, Polypodium randallii, Polypodium ratiborii, Polypodium reclinatum, Polypodium recreense, and Polypodium repensvar.Polypodium abruptum, Polypodium revolvens, Polypodium rhachipterygium, Polypodium rhomboideum, Polypodium rigens, Polypodium robustum, Polypodium roraimense, Polypodium rraimense, Polypodium rosei, Polypodium rosenstockii, Polypodium rubidum, Polypodium rudimentum, Polypodium rusbyi, Polypodium sablanianum, Polypodium sarmentosum, Polypodium saxicola, Polypodium schenckii schenckii), Narrow-leaved water dragon (Polypodium schlechteri), Centipede water dragon (Polypodium scolopendria), Centipede water dragon (Polypodium scolopendria), Brown-scaled water dragon (Polypodium scolopendrium), Polypodium scouleri, Polypodium scutulatum, Segregatum, Semihirsutum, Polypodium semihirsutum var. fuscosetosum, Senile var. minor, Sericeolanatum, Serraeforme, Serricula, Sesquipedala, Sessilifolium, Bald variety of the linear-scaled water dragon (Polypodium setosum) var.calvum), bristly kelp (Polypodium setulosum), Polypodium shaferi, Polypodium sibomense, dry kelp (Polypodium siccum), Polypodium simacense, mimicking kelp (Polypodium simulans), singer kelp (Polypodium singeri), Chinese kelp (Polypodium sinicum), Polypodium sintenisii, Polypodium skutchii, sloanei (Polypodium sloanei), Polypodium sodiroi, filthy kelp (Polypodium sordidulum), despicable kelp (Polypodium sordidum), spherical kelp (Polypodium sphaeropteroides), wedge-shaped kelp (Polypodium sphenodes), neat kelp (Polypodium sprucei), Polypodium sprucei var. fructativenosa, Polypodium steirolepis, Polypodium stenobasis, Polypodium stenolepis, Polypodium stenopterum, Polypodium subcapillare, Polypodium subflabelliforme, Polypodium subhemionitidium, Polypodium subinaequale, Polypodium subintegrum, Polypodium subspathulatum, Polypodium subtile, Polypodium subvestitum, Polypodium subviride, Polypodium superficiale var. attenuatum, Polypodium superficiale vair.chinensis), Polypodium sursumcurrens, Polypodium tablazianum, Polypodium taenifolium, Polypodium tamandarei, Polypodium tatei, Polypodium tenuiculum var. acrosora, Polypodium tenuiculum var. brasiliense, Polypodium tenuilore, Polypodium tenuinerve, Polypodium tepuiense, Polypodium teresae, Polypodium tetragonum var. incompletum, Polypodium thysanolepis var. bipinnatifidum, Polypodium tepuiense thyssanolepis, var. thyssanolepis), Polypodium thyssanolepsi, Polypodium tobagense, Polypodium trichophyllum, Polypodium tridactylum, Polypodium tridentatum, Polypodium trifurcatum var. brevipes, Polypodium triglossum, Polypodium truncatulum, Polypodium truncicola var. major, Polypodium truncicola var.minor), nodular water stalk (Polypodium tuberosum), Polypodium tunguraguae, turquinum, turrialbae, ursipes, vagans, valdealatum, versteegii, villagranii, cambroideum f. cambroideum, peraferens f. peraferens, vittarioides, vulgare, vulgare L., and ore-loving subspecies of Polypodium. Polypodium vulgare subsp. oreophilum, Polypodium vulgare var. acuminatum, Polypodium vulpinum, Polypodium williamsii, Polypodium wobbense, Polypodium x fallacissimum-guttatum, Polypodium xantholepis, Polypodium xiphopteris, Polypodium diunyarumalense, Polypodium yungense, and Polypodium zosteriforme.

[0040] In some embodiments, the IPD059 polypeptide is derived from fern species belonging to the order Polypodiaceae, family Polypodiaceae, or genus Adiantum. In some embodiments, the IPD059 polypeptide is derived from fern species in the genus *Colysis*, selected from, but not limited to: *Colysis ampla*, *Colysis digitata*, *Colysis elegans*, *Colysis elliptica*, *Colysis flexiloba*, *Colysis hemionitidea*, *Colysis hemitoma*, *Colysis henryi*, *Colysis insignis*, *Colysis intermedia*, *Colysis leveillei*, *Colysis longipes*, *Colysis pentaphylla*, *Colysis pothifolia*, *Colysis pteropus*, *Colysis shintenensis*, *Colysis simplicifrons*, *Colysis triphylla*, and *Colysis wrightii*.

[0041] In some embodiments, the IPD059 polypeptide is derived from fern species belonging to the order Polypodales, family Acernaceae, and genus Acernaceae. In some embodiments, the IPD059 polypeptide is derived from fern species of the genus *Asplenium*, selected from, but not limited to: *Asplenium abscissum*, *Asplenium actinopteroides*, *Asplenium adiantum-nigrum*, *Asplenium aegaeum*, *Asplenium ethiopicum*, *Asplenium aff.hallbergii*, *Asplenium aff.heterochroum*, *Asplenium affine*, *Asplenium alatum*, *Asplenium amboinense*, *Asplenium anceps*, *Asplenium angustum*, *Asplenium anisophyllum*, *Asplenium anogrammoides*, *Asplenium anogrammoides x Asplenium*, *Asplenium antiquum*, *Asplenium antrophyoides*, and *Asplenium*. Asplenium apogamum, Asplenium appendiculatum, Asplenium aureum, Asplenium auriculatum, Asplenium auritum, Asplenium australiscum, Asplenium azoricum, Asplenium barclayanum, Asplenium billotii, Asplenium bipartitum, Asplenium blepharodes, Asplenium boltonii, Asplenium boreale, Asplenium bourgaei, Asplenium bulbiferum, Asplenium bulbiferum x Asplenium bulbiferum subsp. gracillimum, Asplenium bulbiferumAsplenium bullatum, Asplenium capillipes, Asplenium castaneum, Asplenium caudatum, Asplenium ceterach, Asplenium chathamense, Asplenium christii, Asplenium cimmeriorum, Asplenium coenobiale, Asplenium compressum, Asplenium contiguum, Asplenium cordatum, Asplenium crinicaule, Asplenium cristataum, Asplenium cuneatiforme, Asplenium cuneifolium, Asplenium currorii, Asplenium Asplenium cuspidatum, Asplenium cymbifolium, Asplenium cyprium, Asplenium dalhousiae, Asplenium dareoides, Asplenium daucifolium, Asplenium decompositum, Asplenium delicatulum, Asplenium dielerectum, Asplenium difforme, Asplenium dimorphum, Asplenium dissectum, Asplenium dregeanum, Asplenium elliottii, Asplenium emarginatum, Asplenium ensiforme, Asplenium erectum, Asplenium exiguum, Asplenium feei, fibrous asplenium, aspleniumAsplenium filipes, Asplenium finlaysonianum, Asplenium fissum, Asplenium flabellifolium, Asplenium flaccidum, Asplenium fontanum, Asplenium foresiense, Asplenium formosum, Asplenium fragile, Asplenium friesiorum, Asplenium gemmiferum, Asplenium griffithianum, Asplenium gulingense, Asplenium hallbergii, Asplenium harpeodes, Asplenium hastatum, Asplenium haughtonii, Asplenium haurakiense, Asplenium hemionitis. Asplenium hemitomum, Asplenium heterochpoum, Asplenium heteroresiliens, Asplenium hispanicum, Asplenium hobdyi, Asplenium hookerianum, Asplenium hostmannii, Asplenium hybridum, Asplenium incisum, Asplenium interjectum, Asplenium jahandiezii, Asplenium juglandifolium, Asplenium kukkonenii, Asplenium laciniatum, Asplenium laetum, Asplenium lamprophyllum, Asplenium laserpitiifolium, Asplenium lividum, Asplenium lolegnamense, Asplenium loriceum), Aspleniumloxoscaphoides, transparent asp (Asplenium lucidum), crescent asp (Asplenium lunulatum), Lushan asp (Asplenium lushanense), alpine asp (Asplenium lyallii), asplenium majoricum, large asp (Asplenium majus), small-flowered asp (Asplenium mannii), marine asp (Asplenium marinum), asplenium mauritiensis, asplenium micantifrons, red-tailed asp (Asplenium milnei), single-sac asp (Asplenium monanthes), mountain asp (Asplenium montanum), multileaved asp (Asplenium myriophyllum), wrinkled-leaved bird's nest fern (Asplenium nidus), mountain wax plum asp (Asplenium nitens), hanging asp (Asplenium normale), asplenium northlandicum, Asplenium obliquissimum, Asplenium obliquum, Asplenium oblongifolium, Asplenium obovatum, Asplenium obtusatum, Asplenium octoploideum, Asplenium oligophlebium, Asplenium onopteris, Asplenium palmeri, Asplenium papaverifolium, Asplenium parvifolium, Asplenium paucivenosum, Asplenium pauperequitum, Asplenium pekinense, Asplenium pellucidum, Asplenium petiolulatum, Asplenium petrarchae, Asplenium phillipsianum, narrow-leaved asplenium phyllitidis, oblique-leaved aspleniumAsplenium planicaule, Asplenium platyneuron, Asplenium polyodon, Asplenium polyphyllum, Asplenium praegracile, Asplenium praemorsum, Asplenium preussii, Asplenium prolongatum, Asplenium protensum, Asplenium pseudolaserpitiifolium, Asplenium pseudowilfordii, Asplenium pteridoides, Asplenium pteropus, Asplenium punjabense, Asplenium radicans, Asplenium resiliens, Asplenium rhizophyllum, Asplenium richardii, Asplenium Asplenium ritoense, Asplenium ruprechtii, Asplenium rutamuraria, Asplenium rutifolium, Asplenium sagittatum, Asplenium salicifolium, Asplenium sandersonii, Asplenium sarelii, Asplenium scalare, Asplenium scleropium, Asplenium scolopendrium, Asplenium seelosii, Asplenium septentrionale, Asplenium serratum, Asplenium setoi, Asplenium shimurae, Asplenium shuttleworthianum, Asplenium simplicifrons, Asplenium smedsii, Aspleniumsoleirolioides, Asplenium sphenotomum, Asplenium squamulatum, Asplenium stoloniferum, Asplenium subglandulosus, Asplenium sulcatum, Asplenium surrogatum, Asplenium tenerum, Asplenium tenuicaule, Asplenium theciferum, Asplenium thunbergii, Asplenium tricholepis, Asplenium trichomanes, Asplenium trigonopterum, Asplenium tripteropus, Asplenium variabile, Asplenium varians, Asplenium Asplenium viride, Asplenium volkensii, Asplenium vulcanicum, Asplenium wilfordii, Asplenium wrightii, Asplenium wrightioides, Asplenium x adulterinum, Asplenium x chasmophilum, Asplenium xlessinense, Asplenium x mantoniae, Asplenium x protomajoricum, Asplenium xtenuivarians, Asplenium yoshinagae, and Asplenium yunnanense.

[0042] In some embodiments, the IPD059 polypeptide is derived from fern species belonging to the order Polypodales, family Dryopteridaceae, and genus Aristolochia. In some embodiments, the IPD059 polypeptide is derived from fern species of the genus *Polystichum*, selected from, but not limited to: *Polystichum acanthophyllum*, *Polystichum aculeatum*, *Polystichum acutidens*, *Polystichum acutipinnulum*, *Polystichum adungense*, *Polystichum alcicorne*, *Polystichum altum*, *Polystichum anomalum*, *Polystichum mariticticulatipilosum*, *Polystichum assurgentipinnum*, *Polystichum atkinsonii*, *Polystichum attenuatum*, *Polystichum auriculum*, and *Polystichum auriculum*. The following are a list of fern species: bakerianum, baoxingense, biaristatum, bifidum, bifidum, bissectum, bomiense, brachypterum, braunii, capillipes, castaneum, chingiae, christii, chunii, consimile, costularisorum, and others.craspedosorum), *Polystichum crassinervium*, *Polystichum cringerum*, *Polystichum cuneatiforme*, *Polystichum cyclolobum*, *Polystichum daguanense*, *Polystichum dangii*, *Polystichum delavayi*, *Polystichum deltodon*, *Polystichum dielsii*, *Polystichum diiffundens*, *Polystichum discretum*, *Polystichum disjunctum*, *Polystichum duthiei*, *Polystichum elevatovenusum*, *Polystichum erosum*, *Polystichum erosum* exauriforme, Polystichum excellens, Polystichum excelsius, Polystichum fimbriatum, Polystichum formosanum, Polystichum frigidicola, Polystichum fugongense, Polystichum gongboense, Polystichum grandifrons, Polystichum guangxiense, Polystichum gymnocarpium, Polystichum habaense, Polystichum hancockii, Polystichum hecatopteron, Polystichum herbaceum, Polystichum houchangense, Polystichum houchangense, Polystichum huae), Yichang ear fern (Polystichum ichangense), small ear fern (Polystichuminaense), deep-fiss ear fern (Polystichum*Polystichum incisopinnulum*, *Polystichum integrilimbum*, *Polystichum integrilobum*, *Polystichum jinfoshaense*, *Polystichum jiulaodongense*, *Polystichum jizhushanense*, *Polystichum kangdingense*, *Polystichum kungianum*, *Polystichum kwangtungense*, *Polystichum lachenense*, *Polystichum lanceolatum*, *Polystichum langchungense*, *Polystichum latilepis*, *Polystichum lentum*, *Polystichum leveillei*, *Polystichum liui*, *Polystichum lanceolate* lonchitis, Polystichum longiaristatum, Polystichum longidens, Polystichum longipaleatum, Polystichum longipes, Polystichum longipinnulum, Polystichum longispinosum, Polystichum longissimum, Polystichum macrochlaenum, Polystichum makinoi, Polystichum manmeiense, Polystichum martinii, Polystichum mayebarae, Polystichum medogense, Polystichum mehrae, Polystichum meiguense, Polystichum melanostipes), Polystichum mollissimum, Polystichummorii), Muping ear fern (Polystichum moupinense), accompanied moss ear fern (Polystichum muscicola), rock-living ear fern (Polystichum nayongense), new Zhengyu ear fern (Polystichum neoliuii), leather-leaved ear fern (Polystichum neolobatum), soft-boned ear fern (Polystichum nepalense), dark-scaled ear fern (Polystichum nigrum), Ningshan ear fern (Polystichum ningshenense), naked-fruit ear fern (Polystichum nudisorudm), Zhiben ear fern (Polystichum obliquum), Zhenkang ear fern (Polystichum oblongum), sparse-fruit ear fern (Polystichum oligocarpum), Emei ear fern (Polystichum omeiense), false half-grown ear fern (Polystichum oreodoxa), Tibetan eastern ear fern (Polystichum orientalitibeticum), alpine ear fern (Polystichum otophorum, Polystichum ovato-paleaceum, Polystichum paramoupinense, Polystichum parvifoliolatum, Polystichum parvipinnulum, Polystichum pianmaense, Polystichum piceo-paleaceum, Polystichum polyblepharum, Polystichum prescottianum, Polystichum prionolepis, Polystichum pseudocastaneum, Polystichum pseudolanceolatum, Polystichum pseudomakinoi, Polystichum pseudorhomboideum, Polystichum pseudosetosum, Polystichum pseudoxiphophyllum, Polystichumpunctiferum), Polystichum puteicola, Polystichum pycnopterum, Polystichum qamdoense, Polystichum retrosopaleaceum, Polystichum revolutum, Polystichum rhombiforme, Polystichum rigens, Polystichum robustum, Polystichum rufopaleaceum, Polystichum saxicola, Polystichum semifertile, Polystichum setillosum, Polystichum shandongense, Polystichum shensiense, Polystichum shimurae, Polystichum simplicipinnum), Chinese ear fern (Polystichum sinense), Chinese ear fern (Polystichum sinotsus-simense), grass ear fern (Polystichum sozanense), cave ear fern (Polystichum speluncicola), dense-scaled ear fern (Polystichum squarrosum), bud ear fern (Polystichum stenophyllum), cat-ear ear fern (Polystichum stimulans), multi-feathered ear fern (Polystichum subacutidens), coarse-toothed ear fern (Polystichum subdeltodon), pseudo-fringe ear fern (Polystichum subfimbriatum), near-marginal ear fern (Polystichum submarginale), Qinling ear fern (Polystichum submite), drill-scaled ear fern (Polystichum subulatum), South Asian ear fern (Polystichum tacticopterum), Taichung ear fern (Polystichum *Polystichum tangmaiense*, *Polystichum taizhongense*, *Polystichum tangmaiense*, *Polystichum taizhongense*The species include *Polystichum thomsonii*, *Polystichum tibeticum*, *Polystichum tonkinense*, *Polystichum tripteron*, *Polystichum tsingkanshanense*, *Polystichum tsus-simense*, *Polystichum wattii*, *Polystichum xiphophyllum*, *Polystichum yadongense*, *Polystichum yuanum*, *Polystichum yunnanense*, and *Polystichum zayuense*.

[0043] As used herein, “sufficient homology” refers to an amino acid sequence that, using standard parameters and one of the alignment procedures described herein, has at least about 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 with a reference sequence. In some embodiments, the sequence homology refers to the full-length sequence of the IPD059 peptide.

[0044] In some embodiments, the IPD059 polypeptide is identical to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID Compared with SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 78, it has at least about 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 used in conjunction with the percentage of sequence identity, the term “about” means / -0.5%. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of proteins, taking into account amino acid similarity, etc.

[0045] In some embodiments, relative to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID IPD059 polypeptides of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 78, wherein the IPD059 polypeptide has 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, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more amino acids from the N-terminus.

[0046] In some embodiments, relative to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID IPD059 polypeptides of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 78, wherein the IPD059 polypeptide has 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, or 28 N-terminal truncations.

[0047] IPD098 protein and its variants and fragments

[0048] This disclosure covers the IPD098 polypeptide. As used interchangeably herein, “IPD098 polypeptide” and “IPD098 protein” refer to a polypeptide having insecticidal activity (including, but not limited to, insecticidal activity against one or more insect pests of the orders Lepidoptera and / or Coleoptera) and being sufficiently homologous to the IPD098Aa polypeptide of SEQ ID NO: 102. Various IPD098 polypeptides are considered. Sources of IPD098 polypeptides or related proteins include ferns or other primitive plant species selected from, but not limited to, species of the genera *Platycerium*, *Aspergillus*, or *Selaginella*.

[0049] In some embodiments, the IPD098 polypeptide is derived from fern species belonging to the order Polypodales, family Acernaceae, and genus Acernaceae. In some embodiments, the IPD098 polypeptide is derived from fern species of the genus *Asplenium*, selected from, but not limited to: *Asplenium abscissum*, *Asplenium actinopteroides*, *Asplenium adiantum-nigrum*, *Asplenium aegaeum*, *Asplenium ethiopicum*, *Asplenium aff.hallbergii*, *Asplenium aff.heterochroum*, *Asplenium affine*, *Asplenium alatum*, *Asplenium amboinense*, *Asplenium anceps*, *Asplenium angustum*, *Asplenium anisophyllum*, *Asplenium anogrammoides*, *Asplenium anogrammoides x Asplenium*, *Asplenium antiquum*, *Asplenium antrophyoides*, and *Asplenium*. Asplenium apogamum, Asplenium appendiculatum, Asplenium aureum, Asplenium auriculatum, Asplenium auritum, Asplenium australiscum, Asplenium azoricum, Asplenium barclayanum, Asplenium billotii, Asplenium bipartitum, Asplenium blepharodes, Asplenium boltonii, Asplenium boreale, Asplenium bourgaei, Asplenium bulbiferum, Asplenium bulbiferum x Asplenium bulbiferum subsp. gracillimum, Asplenium bulbiferumAsplenium bullatum, Asplenium capillipes, Asplenium castaneum, Asplenium caudatum, Asplenium ceterach, Asplenium chathamense, Asplenium christii, Asplenium cimmeriorum, Asplenium coenobiale, Asplenium compressum, Asplenium contiguum, Asplenium cordatum, Asplenium crinicaule, Asplenium cristataum, Asplenium cuneatiforme, Asplenium cuneifolium, Asplenium currorii, Asplenium Asplenium cuspidatum, Asplenium cymbifolium, Asplenium cyprium, Asplenium dalhousiae, Asplenium dareoides, Asplenium daucifolium, Asplenium decompositum, Asplenium delicatulum, Asplenium dielerectum, Asplenium difforme, Asplenium dimorphum, Asplenium dissectum, Asplenium dregeanum, Asplenium elliottii, Asplenium emarginatum, Asplenium ensiforme, Asplenium erectum, Asplenium exiguum, Asplenium feei, fibrous asplenium, aspleniumAsplenium filipes, Asplenium finlaysonianum, Asplenium fissum, Asplenium flavcllifolium, Asplenium flaccidum, Asplenium fontanum, Asplenium foresiense, Asplenium formosum, Asplenium fragile, Asplenium friesiorum, Asplenium gemmiferum, Asplenium griffithianum, Asplenium gulingense, Asplenium hallbergii, Asplenium harpeodes, Asplenium hastatum, Asplenium haughtonii, Asplenium haurakiense, Asplenium hemionitis. Asplenium hemitomum, Asplenium heterochroum, Asplenium heteroresiliens, Asplenium hispanicum, Asplenium hobdyi, Asplenium hookerianum, Asplenium hostmannii, Asplenium hybridum, Asplenium incisum, Asplenium interjectum, Asplenium jahandiezii, Asplenium juglandifolium, Asplenium kukkonenii, Asplenium laciniatum, Asplenium laetum, Asplenium lamprophyllum, Asplenium laserpitiifolium, Asplenium lividum, Asplenium lolegnamense, Asplenium loriceum), Aspleniumloxoscaphoides, transparent asp (Asplenium lucidum), crescent asp (Asplenium lunulatum), Lushan asp (Asplenium lushanense), alpine asp (Asplenium lyallii), asplenium majoricum, large asp (Asplenium majus), small-flowered asp (Asplenium mannii), marine asp (Asplenium marinum), asplenium mauritiensts, asplenium micantifrons, red-tailed asp (Asplenium milnei), single-sac asp (Asplenium monanthes), mountain asp (Asplenium montanum), multileaved asp (Asplenium myriophyllum), wrinkled-leaved bird's nest fern (Asplenium nidus), mountain wax plum asp (Asplenium nitens), hanging asp (Asplenium normale), asplenium Asplenium obliquisslmum, Asplenium obliquum, Asplenium oblongifolium, Asplenium obovatum, Asplenium obtusatum, Asplenium octoploideum, Asplenium oligophlebium, Asplenium onopteris, Asplenium palmeri, Asplenium papaverifolium, Asplenium parvifolium, Asplenium paucivenosum, Asplenium pauperequitum, Asplenium pekinense, Asplenium pellucidum, Asplenium petiolulatum, Asplenium petrarchae, Asplenium phillipsianum, narrow-leaved asplenium phyllitidis, oblique-leaved aspleniumAsplenium planicaule, Asplenium platyneuron, Asplenium polyodon, Asplenium polyphyllum, Asplenium praegracile, Asplenium praemorsum, Asplenium preussii, Asplenium prolongatum, Asplenium protensum, Asplenium pseudolaserpitiifolium, Asplenium pseudowilfordii, Asplenium pteridoides, Asplenium pteropus, Asplenium punjabense, Asplenium radicans, Asplenium resiliens, Asplenium rhizophyllum, Asplenium richardii, Asplenium Asplenium ritoense, Asplenium ruprechtii, Asplenium rutamuraria, Asplenium rutifolium, Asplenium sagittatum, Asplenium salicifolium, Asplenium sandersonii, Asplenium sarelii, Asplenium scalare, Asplenium scleropium, Asplenium scolopendrium, Asplenium seelosii, Asplenium septentrionale, Asplenium serratum, Asplenium setoi, Asplenium shimurae, Asplenium shuttleworthianum, Asplenium simplicifrons, Asplenium smedsii, Aspleniumsoleirolioides, Asplenium sphenotomum, Asplenium squamulatum, Asplenium stoloniferum, Asplenium subglandulosus, Asplenium sulcatum, Asplenium surrogatum, Asplenium tenerum, Asplenium tenuicaule, Asplenium theciferum, Asplenium thunbergii, Asplenium tricholepis, Asplenium trichomanes, Asplenium trigonopterum, Asplenium tripteropus, Asplenium variabile, Asplenium varians, Asplenium Asplenium viride, Asplenium volkensii, Asplenium vulcanicum, Asplenium wilfordii, Asplenium wrightii, Asplenium wrightioides, Asplenium x adulterinum, Asplenium x chasmophilum, Asplenium xlessinense, Asplenium x mantoniae, Asplenium x protomajoricum, Asplenium xtenuivarians, Asplenium yoshinagae, and Asplenium yunnanense.

[0050] In some embodiments, the IPD098 polypeptide is derived from species in the class Isoetopsida, order Selaginella. In some embodiments, the IPD098 polypeptide is derived from fern species in the class Isoetopsida, order Selaginella, family Selaginellaceae. In some embodiments, the IPD098 polypeptide is derived from species in the genus Selaginella. In some embodiments, the IPD098 polypeptide is derived from species of the genus *Selaginella*, which are selected from, but not limited to: *Selaginella acanthonota*, *Selaginella apoda*, *Selaginella arbuscula*, *Selaginella arenicola*, *Selaginella arizonica*, *Selaginella armata*, *Selaginella asprella*, *Selaginella biformis*, *Selaginella bigelovii*, *Selaginella braunii*, *Selaginella cinerascens*, *Selaginella cordifolia*, *Selaginella deflexa*, *Selaginella delicatula*, *Selaginella densa*, *Selaginella douglasii*, *Selaginella eatonii*, and *Selaginella*. Selaginella eclipes, Selaginella eremophila, Selaginella erythropus, Selaginella flabellata, Selaginella hansenii, Selaginella heterodonta, Selaginella kraussiana, Selaginella krugii, Selaginella laxifolia, Selaginella lepidophylla, Selaginella leucobryoides, Selaginella ludoviciana, Selaginella mutica, Selaginella oregana, Selaginella ovoideaSelaginella ovifolia, Selaginella pallescens, Selaginella peruviana, Selaginella pilifera, Selaginella plana, Selaginella plumosa, Selaginella pulcherrima, Selaginella rupestris, Selaginella rupincola, Selaginella scopulorum, Selaginella selaginoides, Selaginella sibirica, Selaginella standleyi, Selaginella stellata, Selaginella subcaulescens, Selaginella substipitata, Selaginella tenella, Selaginella tortipila, Selaginella uliginosa, Selaginella spp. Selaginella umbrosa, Selaginella uncinata, Selaginella underwoodii, Selaginella utahensis, Selaginella victoriae, Selaginella viridissima, Selaginella wallacei, Selaginella watsonii, Selaginella weatherbiana, Selaginella willdenowii, Selaginella wrightii, and Selaginella X neomexicana.

[0051] In some embodiments, the IPD098 polypeptide is derived from fern species belonging to the order Polypodiaceae, family Polypodiaceae, and genus *Pteris*. In some embodiments, the IPD098 polypeptide is derived from species within the genus *Pteris*. In some embodiments, the IPD098 polypeptide is derived from species of the genus Platycerium, which are selected from, but not limited to: Platycerium alcicorne, Platycerium andinum, Platycerium angolense, Platycerium bifurcatum, Platycerium coronarium, Platycerium elephantotis, Platycerium ellisii, Platycerium grande, Platycerium hillii, Platycerium holttumii, Platycerium madagascariense, Platycerium quadridichotomum, and Platycerium mongoosenum. Species of the genus Platycerium include: ridleyi, ES-2011, triangular staghorn fern (Platycerium stemaria), giant staghorn fern (Platycerium superbum), upright-leaved staghorn fern (Platycerium veitchii), wallichii staghorn fern (Platycerium wallichii), wandae staghorn fern (Platycerium wandae), and willinckii staghorn fern (Platycerium willinckii).

[0052] In some embodiments, the IPD098 polypeptide is derived from fungal species in the Order Eurotiales, Family Trichocomaceae, and genus Aspergillus. In some embodiments, the IPD098 polypeptide is derived from species in the genus Aspergillus. In some embodiments, the IPD098 polypeptide is derived from species of the genus *Aspergillus*, which are selected from, but not limited to: *Aspergillus acidus*, *Aspergillus aculeatinus*, *Aspergillus aculeatus*, *Aspergillus aeneus*, *Aspergillus affinis*, *Aspergillus alabamensis*, *Aspergillus alliaceus*, *Aspergillus amazonicus*, *Aspergillus ambiguus*, *Aspergillus amoenus*, *Aspergillus amstelodami*, *Aspergillus amyloliquefaciens*, *Aspergillus amylovorus*, *Aspergillus anomalus*, *Aspergillus anthodesmis*, and *Aspergillus cuspidata*. Aspergillus apicalis, Aspergillus appendiculatus, Aspergillus arachidicola, Aspergillus arenarius, Aspergillus arvii, Aspergillus asperescens, Aspergillus assulatus, Aspergillus astellatus, Aspergillus saurantiobrunneus, Aspergillus aureofulgens, Aspergillus saureolatus, Aspergillus aureoterreus, Aspergillus aureus, Aspergillus auricomus, Aspergillus australensis, Aspergillus australensisAspergillus austroafricanus, Aspergillus avenaceus, Aspergillus awamori, Aspergillus baeticus, Aspergillus bahamensis, Aspergillus biplanus, Aspergillus bisporus, Aspergillus bombycis, Aspergillus brasiliensis, Aspergillus brevipes, Aspergillus brevistipitatus, Aspergillus bridgeri, Aspergillus brunneouniseriatus, Aspergillus brunneoviolaceu, Aspergillus caelatus, Aspergillus caelatus Aspergillus caesiellus, Aspergillus caespitosus, Aspergillus calidoustus, Aspergillus campestris, Aspergillus scandidus, Aspergillus capensis, Aspergillus carbonarius, Aspergillus carneus, Aspergillus cavernicola, Aspergillus cervinus, Aspergillus chevalieri, Aspergillus chungii, Aspergillus cibarius, Aspergillus sclavatoflavus, Aspergillus dwarf Aspergillus clavatus, Aspergillus conicus, Aspergillus conjunctus, Aspergillus conversis, Aspergillus KoreanaAspergillus coremiiformis, Aspergillus costaricensis, Aspergillus costiformis, Aspergillus creber, Aspergillus cretensis, Aspergillus cristatus, Aspergillus scrrustosus, Aspergillus crystallinus, Aspergillus cvjetkovicii, Aspergillus deflectus, Aspergillus delacroixii, Aspergillus delicatus, Aspergillus densus, Aspergillus dentatulus, Aspergillus depauperatus, Aspergillus Aspergillus dessyi, Aspergillus digitatus, Aspergillus dimorphicus, Aspergillus diplocystis, Aspergillus discophorus, Aspergillus disjunctus, Aspergillus diversus, Aspergillus dorothicus, Aspergillus dubius, Aspergillus duricaulis, Aspergillus dybowskii, Aspergillus eburneocremeus, Aspergillus eburneus, Aspergillus echinosporus, Aspergillus echinulatus, Aspergillus ecuadorensis, Aspergillus Aspergillus effusus, Aspergillus gyptiacus, Aspergillus elatior, Aspergillus davidiiAspergillus elegans, Aspergillus ellipsoideus, Aspergillus ellipticus, Aspergillus elongatus, Aspergillus equitis, Aspergillus erythrocephalus, Aspergillus falconensis, Aspergillus fasciculatus, Aspergillus fennelliae, Aspergillus ferrugineus, Aspergillus ficuum, Aspergillus fiemonthi, Aspergillus filifera, Aspergillus fimetarius, Aspergillus fimeti, Aspergillus filifera, Aspergillus fimetarius, Aspergillus fimeti, Aspergillus fishes Aspergillus fischeri, Aspergillus fischerianus, Aspergillus flaschentraegeri, Aspergillus flavescens, Aspergillus flavidus, Aspergillus flavipes, Aspergillus flavofurcatus, Aspergillus flavoviridescens, Aspergillus flavus, Aspergillus flocculosus, Aspergillus floriformis, Aspergillus foeniculicola, Aspergillus foetidus, Aspergillus fonsecaeus, Aspergillus foutoynontii, Aspergillus foveolatus, Aspergillus freundii fresenii), Aspergillus fructus, Aspergillus fruticans, AspergillusAspergillus fruticulosus, Aspergillus fujiokensis, Aspergillus fuliginosus, Aspergillus fulvus, Aspergillus fumaricus, Aspergillus fumigatiaffinis, Aspergillus fumigatoides, Aspergillus fumigatus, Aspergillus fumisynnematus, Aspergillus fungoides, Aspergillus funniculosus, Aspergillus fuscus, Aspergillus galeritus, Aspergillus giganteus, Aspergillus gigantosulphureus, Aspergillus firta Aspergillus gigas, Aspergillus glaber, Aspergillus glaucoaffinis, Aspergillus glauconiveus, Aspergillus glaucus, Aspergillus globosus, Aspergillus godfrini, Aspergillus gorakhpurensis, Aspergillus gracilis, Aspergillus granulatus, Aspergillus granulosus, Aspergillus gratioti, Aspergillus greconis, Aspergillus griseus, Aspergillus guttifer, Aspergillus gymnosardae, Aspergillus halophilicus, Aspergillus... halophilus), Aspergillushelicothrix, Aspergillus hengieAspergillus hennebergii, Aspergillus erbariorum, Aspergillus heterocaryoticus, Aspergillus eteromorphus, Aspergillus heterothallicus, Aspergillus sheyangensis, Aspergillus hiratsukae, Aspergillus ollandicus, Aspergillus homomorphus, Aspergillus hortae, Aspergillus humicola, Aspergillus humus, Aspergillus sibericus, Aspergillus igneus, Aspergillus iizukae, Aspergillus implicatus, Aspergillus incrassatus, Aspergillus inincrassatus Aspergillus indicus, Aspergillus indohii, Aspergillus ingratus, Aspergillus insecticola, Aspergillus insuetus, Aspergillus insulicola, Aspergillus intermedius, Aspergillus inuii, Aspergillus itaconicus, Aspergillus ivoriensis, Aspergillus janus, Aspergillus japonicus, Aspergillus jeanselmei, Aspergillus jensenii, Aspergillus kambarensis, Aspergillus kanagawaensis, Aspergillus kassunensis, Aspergillus kaassunensis katsuobushi), AspergillusAspergillus keveii), Aspergillus koningii, Aspergillus laciniosus, Aspergillus lacticoffeatus, Aspergillus laneus, Aspergillus lanosus, Aspergillus laokiashanensis, Aspergillus lateralis, Aspergillus lentulus, Aspergillus lepidophyton, Aspergillus leporis, Aspergillus leucocarpus, Aspergillus lignieresii, Aspergillus longivesica, Aspergillus longobasidia, Aspergillus luchensi, Aspergillus luchuensis, Aspergillus lucida Aspergillus luteoniger, Aspergillus luteovirescens, Aspergillus lutescens, Aspergillus luteus, Aspergillus macfiei, Aspergillus macrosporus, Aspergillus malignus, Aspergillus malodoratus, Aspergillus malvaceus, Aspergillus mandshuricus, Aspergillus manginii, Aspergillus mannitosus, Aspergillus maritimus, Aspergillus mattletii, Aspergillus maximus, Aspergillus medius, Aspergillus bleacherii melitensis), Aspergillus melleus, Aspergillus mellinus, AspergillusAspergillus mencieri, Aspergillus michelii, Aspergillus microcephalus, Aspergillus microcysticus, Aspergillus microsporus, Aspergillus microthecius, Aspergillus microviridicitrinus, Aspergillus minimus, Aspergillus minisclerotigenes, Aspergillus minor, Aspergillus minutus, Aspergillus miyajii, Aspergillus miiyakoensis, Aspergillus mollis, Aspergillus montenegroi, Aspergillus montevidensis, Aspergillus mucoroides, Aspergillus mucoroides Aspergillus muelleri, Aspergillus multicolor, Aspergillus multiplicatus, Aspergillus muricatus, Aspergillus muscivora, Aspergillus mutabilis, Aspergillus mycetomi-villabruzzii, Aspergillus mycobanche, Aspergillus nakazawae, Aspergillus nantae, Aspergillus nanus, Aspergillus navahoensis, Aspergillus neobridgeri, Aspergillus neocarnoyi, Aspergillus neoellipticus, Aspergillus neosmolyte neoglaber, Aspergillus nidulans, Aspergillus nidulansAspergillus niger, Aspergillus nigrescens, Aspergillus nigricans, Aspergillus nishimurae, Aspergillus niveoglaucus, Aspergillus niveus, Aspergillus noelting, Aspergillus nominus, Aspergillus nomius, Aspergillus novofumigatus, Aspergillus novus, Aspergillus ochraceopetaliformis, Aspergillus ochraceoroseus, Aspergillus ochraceoruber, Aspergillus ochraceus, Aspergillus okazaki Aspergillus okazakii), Aspergillus solivaceofuscus, Aspergillus olivaceus, Aspergillus solivascens, Aspergillus olivicola, Aspergillus omanensis, Aspergillus sonokii, Aspergillus oosporus, Aspergillus ornatulus, Aspergillus ornatus, Aspergillus oryzae, Aspergillus ostianus, Aspergillus otanii, Aspergillus ovalispermus, Aspergillus paleaceus, Aspergillus pallidus, Aspergillus spanamensis, Aspergillus mirabilis paradoxus), Aspergillus parasiticus, Aspergillus parrulus, AspergillusAspergillus parvisclerotigenus, Aspergillus parviverruculosus, Aspergillus parvulu, Aspergillus paulistensi, Aspergillus pennicillatus, Aspergillus penicilliformis, Aspergillus pennicillioides, Aspergillus penicillioideum, Aspergillus pennicillopsis, Aspergillus periconioides, Aspergillus perniciosus, Aspergillus persii, Aspergillus petrakii, Aspergillus peyronelii, Aspergillus phaeocephalus, Aspergillus Aspergillus phialiseptatus, Aspergillus phoenicis, Aspergillus pidoplichknovii, Aspergillus piperis, Aspergillus polychromus, Aspergillus pouchetii, Aspergillus primulinus, Aspergillus profusus, Aspergillus proliferans, Aspergillus protuberus, Aspergillus pseudocaelatus, Aspergillus pseudocarbonarius, Aspergillus pseudocitricus, Aspergillus pseudoclavatus, Aspergillus pseudodeflectus, Aspergillus pseudoelatior, Aspergillus pseudoelegant pseudoelegans, AspergillusAspergillus pseudoflavus, Aspergillus pseudoglaucus, Aspergillus pseudoheteromorphus, Aspergillus pseudoniger, Aspergillus pseudonomius, Aspergillus pseudotamarii, Aspergillus pulchellus, Aspergillus pulmonum-hominis, Aspergillus pulverulentus, Aspergillus pulvinus, Aspergillus puniceus, Aspergillus purpureofuscus, Aspergillus purpureus, Aspergillus pusillus, Aspergillus puulaauensis, Aspergillus pyramidus, Aspergillus Aspergillus pyri, Aspergillus qinqixianii, Aspergillus qizutongii, Aspergillus quadricinctus, Aspergillus quadricingens, Aspergillus quadrifidus, Aspergillus quadrilineatus, Aspergillus quercinus, Aspergillus quininae, Aspergillus quitensis, Aspergillus racemosus, Aspergillus raianus, Aspergillus rambellii, Aspergillus ramosus, Aspergillus raperi, Aspergillus recurvatus, Aspergillus rehmii, Aspergillus repandus, Aspergillus pyrifolia repens, Aspergillus repensAspergillus reptans, Aspergillus restrictus, Aspergillus rhizopodus, Aspergillus robustus, Aspergillus roseoglobosus, Aspergillus roseoglobulosus, Aspergillus roseovelutinus, Aspergillus roseus, Aspergillus ruber, Aspergillus rubrobrunneus, Aspergillus rubrum, Aspergillus rufescens, Aspergillus rugulosus, Aspergillus rugulovalvus, Aspergillus rutilans, Aspergillus sacchari, Aspergillus sutilans Aspergillus saitoi, Aspergillus salviicola, Aspergillus sartoryi, Aspergillus scheelei, Aspergillus schiemanniae, Aspergillus sclerogenus, Aspergillus sclerolii carbonarius, Aspergillus sclerotioniger, Aspergillus sclerotiorum, Aspergillus sejunctus, Aspergillus septatus, Aspergillus sepultus, Aspergillus silvaticus, Aspergillus simplex, Aspergillus sojae, Aspergillus sparsus, Aspergillus spathulatus, Aspergillus filamentosa Aspergillus spectabilis, Aspergillus spelunceus, Aspergillus spuriousiiAspergillus spiculosus, Aspergillus spinosus, Aspergillus spinulosus, Aspergillus spiralis, Aspergillus stellamaris, Aspergillus stellatus, Aspergillus stellifer, Aspergillus stercoreus, Aspergillus sterigmatophorus, Aspergillus steynii, Aspergillus stramenius, Aspergillus striatulus, Aspergillus striatus, Aspergillus stromatoides, Aspergillus strychni, Aspergillus subfuscus, Aspergillus subgriseus, Aspergillus awl-shaped mold. Aspergillus sublatus, Aspergillus sublevisporus, Aspergillus subolivaceus, Aspergillus subsessilis, Aspergillus subunguis, Aspergillus subversicolor, Aspergillus sulphureus, Aspergillus sunderbanii, Aspergillus sydowii, Aspergillus sylvaticus, Aspergillus syncephalis, Aspergillus tabacinus, Aspergillus taichungensis, Aspergillus takakii, Aspergillus staklimakanensis, Aspergillus tamari, Aspergillus tapirirae, Aspergillus tardus, AspergillusAspergillus tatenoi, Aspergillus tennesseensis, Aspergillus terrestris, Aspergillus terreus, Aspergillus terricola, Aspergillus testaceocolorans, Aspergillus stetrazonus, Aspergillus thermomutatus, Aspergillus thomi, Aspergillus tiraboschii, Aspergillus togoensis, Aspergillus tokelau, Aspergillus tonophilus, Aspergillus toxicarius, Aspergillus stritici, Aspergillus tsurutae, Aspergillus tuberculatus, Aspergillus tubingensis, Aspergillus tunetanus, Aspergillus Aspergillus udagawae, Aspergillus umbrinus, Aspergillus umbrosus, Aspergillus undulatus, Aspergillus unguis, Aspergillus unilateralis, Aspergillus usamii, Aspergillus ustilago, Aspergillus ustus, Aspergillus uvarum, Aspergillus vadensis, Aspergillus vancampenhoutii, Aspergillus varanasensis, Aspergillus variabilis, Aspergillus varians, Aspergillus variecolor, Aspergillus variegatus, Aspergillus variabilis velutinus), Aspergillus toxicusAspergillus venezuelensis, Aspergillus versicolor, Aspergillus vinosobubalinus, Aspergillus violaceobrunneus, Aspergillus violaceofuscus, Aspergillus violaceus, Aspergillus virens, Aspergillus viridigriseus, Aspergillus viridinutans, Aspergillus vitellinus, Aspergillus vitis, Aspergillus vitricola, Aspergillus wangduanlii, Aspergillus warcupii, Aspergillus wehmeri, Aspergillus wanu. Aspergillus welwitschiae, Aspergillus wentii, Aspergillus westendorpii, Aspergillus westerdijkiae, Aspergillus xerophilus, Aspergillus yezoensis, Aspergillus zhaoqingensis, and Aspergillus zonatus.

[0053] As used herein, “sufficient homology” refers to an amino acid sequence that, using standard parameters and one of the alignment procedures described herein, has at least about 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 with a reference sequence. In some embodiments, the sequence homology refers to the full-length sequence of the IPD098 peptide.

[0054] In some embodiments, the IPD098 peptide is associated with SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 107. NO: 117 has at least about 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 compared to other sequences. When used in conjunction with sequence identity percentages, the term “about” means / - 0.5%. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of proteins, taking into account amino acid similarity, etc.

[0055] IPD108 protein and its variants and fragments

[0056] This disclosure covers the IPD108 polypeptide. As used interchangeably herein, “IPD108 polypeptide” and “IPD108 protein” refer to a polypeptide having insecticidal activity (including, but not limited to, insecticidal activity against one or more insect pests of the Lepidoptera and / or Coleoptera) and being sufficiently homologous to the IPD108Aa polypeptide of SEQ ID NO: 131. Various IPD108 polypeptides are considered. Sources of IPD108 polypeptides or related proteins include ferns or other primitive plant species selected from, but not limited to, species of the genera *Selaginella*, *Athyrium*, or *Onoclea*.

[0057] In some embodiments, the IPD108 polypeptide is derived from species in the class Isomorpha, order Selaginellae. In some embodiments, the IPD108 polypeptide is derived from fern species in the class Isomorpha, order Selaginellae, and family Selaginellaceae. In some embodiments, the IPD108 polypeptide is derived from species in the genus Selaginella. In some embodiments, the IPD108 polypeptide is derived from species of the genus *Selaginella*, which are selected from, but not limited to: *Selaginella acanthonota*, *Selaginella stenoptera*, *Selaginella arborescens*, *Selaginella dunes*, *Selaginella serrata*, *Selaginella spicata*, *Selaginella serrata*, *Selaginella dimorpha*, *Selaginella bipinnata*, *Selaginella bryoides*, *Selaginella serrata ... pulcherrima, rock selaginella, hairy selaginella, cliff cherry selaginella, European selaginella, Siberian selaginella, Selaginella stanense, flowering selaginella, Selaginella subcaulescens, Selaginella substipitata, moldy selaginella, Selaginella tortipila, sparrow tongue selaginella, shade selaginella, Selaginella selaginella, scalloped tail selaginella, Utah selaginella, Victoria selaginella, Selaginella viridissima, Selaginella watsonii, Selaginella weatherbiana, vine selaginella, Selaginella wrightii, such as Selaginella X neomexicana.

[0058] In some embodiments, the IPD108 polypeptide is derived from species in the class Polypodiaceae, order Polypodiaceae. In some embodiments, the IPD108 polypeptide is derived from fern species in the class Polypodiaceae, order Polypodiaceae, and family Aquilegidae. In some embodiments, the IPD108 polypeptide is derived from species in the genus Aquilegidae. In some embodiments, the IPD108 polypeptide is derived from species of the genus *Athyrium*, which are selected from, but are not limited to: *Athyrium arisanense*, *Athyrium atkinsonii*, *Athyrium biserrulatum*, *Athyrium brevifrons*, *Athyrium chingianum*, *Athyrium clarkei*, *Athyrium clivicola*, *Athyrium cryptoogrammoides*, *Athyrium cumingianum*, *Athyrium cuspidatum*, *Athyrium deltoidofrons*, *Athyrium distentifolium*, *Athyrium dolosa*, *Athyrium epirachis*, and *Athyrium*. Athyrium fangii, Athyrium filix-femina, Athyrium frangulum, Athyrium giraldii, Athyrium iseanum, Athyrium kirisimaense, Athyrium kuratae, Athyrium masamunei, Athyrium melanolepis, Athyrium monomachi, Athyrium multidentatum, Athyrium nakanoi, Athyrium neglectum, Athyrium nigripes, Athyrium nikkoense, Athyrium niponicum, Athyrium niponicum nyalamense), Athyriumoblitescens, Athyrium otophorum, Athyrium palustre, Athyrium pinetorum, Athyrium pubicostatum, Athyrium reflexipinnum, Athyrium rhachidosorum, Athyrium rupestre, Athyrium scandicinum, Athyrium setullgerum, Athyrium sheareri, Athyrium silvicola, Athyrium sinense, Athyrium skinneri, species of the genus Athyrium, species of the genus Athyrium YCL-2009, Athyrium spinulosum, Athyrium strigillosum, Athyrium Athyrium subrigescens, Athyrium subtriangulare, Athyrium supraspinescens, Athyrium tashiroi, Athyrium tozanense, Athyrium vidalii, Athyrium viridescentipes, Athyrium wardii, Athyrium x akiense, Athyrium x hisatsuanum, Athyrium xtokashikii, Athyrium yokoscense, and Athyrium yui.

[0059] In some embodiments, the IPD108 peptide is derived from fern species belonging to the class Polypodiaceae, order Polypodiaceae, and family Onoclea. In some embodiments, the IPD108 peptide is derived from species belonging to the genus Onoclea. In some embodiments, the IPD108 peptide is derived from Onoclea sensibilis, Onoclea orientalis, Onoclea interrupta, Onoclea hintonii, or Onoclea sensibilis.

[0060] As used herein, “sufficient homology” refers to an amino acid sequence that, using standard parameters and one of the alignment procedures described herein, has at least about 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 with a reference sequence. In some embodiments, the sequence homology refers to the full-length sequence of the IPD108 peptide.

[0061] In some embodiments, the IPD108 peptide is associated with SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, or SEQ ID NO: 135. NO: 135 has at least about 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 compared to other sequences. When used in conjunction with sequence identity percentages, the term “about” means / - 0.5%. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of proteins, taking into account amino acid similarity, etc.

[0062] IPD109 protein and its variants and fragments

[0063] This disclosure covers the IPD109 polypeptide. As used interchangeably herein, “IPD109 polypeptide” and “IPD109 protein” refer to a polypeptide having insecticidal activity (including, but not limited to, insecticidal activity against one or more insect pests of the Lepidoptera and / or Coleoptera) and being sufficiently homologous to the IPD109Aa polypeptide of SEQ ID NO: 138. Various IPD109 polypeptides are considered. Sources of IPD109 polypeptides or related proteins include, but are not limited to, ferns or other primitive plant species selected from, but not limited to, Selaginella species.

[0064] In some embodiments, the IPD109 polypeptide is derived from species in the class Isomorpha, order Selaginellae. In some embodiments, the IPD109 polypeptide is derived from fern species in the class Isomorpha, order Selaginellae, family Selaginellaceae. In some embodiments, the IPD109 polypeptide is derived from species in the genus Selaginella. In some embodiments, the IPD109 polypeptide is derived from species of the genus *Selaginella*, which are selected from, but not limited to: *Selaginella acanthonota*, *Selaginella acanthonota*, *Selaginella stenoptera*, *Selaginella stenoptera*, *Selaginella dunes*, *Selaginella serrata*, *Selaginella serrata*, *Selaginella dimorpha*, *Selaginella bipinnata*, *Selaginella bryoides*, *Selaginella serrata ... pulcherrima, rock selaginella, hairy selaginella, cliff cherry selaginella, European selaginella, Siberian selaginella, Selaginella stanense, flowering selaginella, Selaginella subcaulescens, Selaginella substipitata, moldy selaginella, Selaginella tortipila, bird tongue selaginella, shade selaginella, Selaginella selaginella, scalloped tail selaginella, Utah selaginella, Victoria selaginella, Selaginella viridissima, Selaginella watsonii, Selaginella weatherbiana, vine selaginella, Selaginella wrightii, and Selaginella X neomexicana.

[0065] As used herein, “sufficient homology” refers to an amino acid sequence that, using standard parameters and one of the alignment procedures described herein, has at least about 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 with a reference sequence. In some embodiments, the sequence homology refers to the full-length sequence of the IPD109 peptide.

[0066] In some embodiments, the IPD109 polypeptide has at least about 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 with SEQ ID NO: 138. When used in conjunction with the percentage of sequence identity, the term “about” means / -0.5%. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding homology of proteins, taking into account amino acid similarity, etc.

[0067] In some embodiments, the ClustalW algorithm is used in Vector The program suite (Invitrogen Corporation, Carlsbad, California) The module calculates the sequence identity under all default parameters. In some embodiments, the ClustalW algorithm is used in Vector. Program Suite (Ingenium Inc., Carlsbad, California) The module calculates sequence identity across the full-length polypeptide under all default parameters.

[0068] As used herein, the terms “protein,” “peptide molecule,” or “polypeptide” include any molecule comprising five or more amino acids. It is well known in the art that protein, peptide, or polypeptide molecules can be modified, including post-translational modifications such as, but not limited to, disulfide bond formation, glycosylation, phosphorylation, or oligomerization. Therefore, as used herein, the terms “protein,” “peptide molecule,” or “polypeptide” include any protein modified by any biological or non-biological process. The terms “amino acid” and “multiple amino acids” refer to all naturally occurring L-amino acids.

[0069] As used herein, “recombinant protein” refers to a protein that is no longer in its natural environment (e.g., in vitro or in recombinant bacterial or plant host cells). Peptides that are substantially free of cellular material include formulations of proteins having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-harm-killing bioproteins (also referred to herein as “contamination proteins”).

[0070] As used herein, “variant” refers to a protein or polypeptide having 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 greater identical to the parent amino acid sequence.

[0071] In some embodiments, the ClustalW algorithm is used in Vector Program Suite (Ingenium Inc., Carlsbad, California) The module calculates sequence identity across the full-length polypeptide under all default parameters.

[0072] Methods for such operations are generally known in the art. For example, amino acid sequence variants of proteins can be prepared by mutations in DNA. This can also be accomplished through one of several forms of mutagenesis and / or in directed evolution.

[0073] In some respects, changes encoded in the amino acid sequence will have substantially no effect on the function of the protein. Such variants will possess the desired necrotic activity. However, it should be understood that these techniques can be used on the compositions of this disclosure to improve the ability of proteins to confer necrotic activity. For example, conserved amino acid substitutions can be made at one or more, predicted, non-essential amino acid residues. “Non-essential” amino acid residues are residues that can be altered from the wild-type sequence of a protein without changing its biological activity. Alignment of the amino acid sequences of protein homologs (e.g., Figures 2-6) allows the identification of highly conserved residues among natural homologs in this family, as well as residues or regions tolerant of amino acid diversity. “Conserved amino acid substitution” is a substitution in which said 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 (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic acid, glutamine); amino acids with non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); and amino acids with small aliphatic, nonpolar, or slightly polar residues (e.g., alanine, serine, threonine, proline). Amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with large aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cysteine); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine); amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); amino acids with large aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0074] Amino acid substitutions can occur in non-conserved regions that retain function. Typically, such substitutions are not targeted at conserved amino acid residues or amino acid residues within a conserved motif, where such residues are essential for protein activity. Examples of conserved residues that may be essential for protein activity include, for example, residues that are identical across all proteins included in a comparison with toxins that are sequence-similar or related to the embodiments (e.g., identical residues in a homologous protein comparison). Examples of conserved residues that allow for conserved amino acid substitutions and still retain activity include, for example, residues with only conserved substitutions across all proteins included in a comparison with toxins that are sequence-similar or related to the embodiments (e.g., only conserved substitutions across all proteins in a homologous protein comparison). However, those skilled in the art will understand that functional variants may have minor conserved or non-conserved changes within the conserved residues. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), which is incorporated herein by reference.

[0075] When making such changes, the hydrophilicity index of the amino acids can be considered. The importance of the hydrophilic amino acid index in conferring interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, (1982) J MolBiol. [Journal of Molecular Biology], 157(1): 105-32). It is accepted that the relative hydrophilicity of the amino acids contributes to the secondary structure of the resulting protein, which in turn defines the protein's interactions with other molecules (e.g., enzymes, matrix, receptors, DNA, antibodies, antigens, etc.).

[0076] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydrophilicity indices or scores, and still produce proteins with similar biological activities, i.e., still yielding proteins with biologically equivalent functions. Each amino acid is assigned a hydrophilicity index (Kyte and Doolittle, ibid.) based on its hydrophobicity and charge characteristics. They are: 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); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). When making this change, amino acid substitutions with a hydrophilicity index of 2 or less are preferred, particularly preferred to be 1 or less, and even more particularly preferred to be 0.5 or less.

[0077] It is also understood in the art that similar amino acid substitutions can be effectively performed based on hydrophilicity. U.S. Patent No. 4,554,101 illustrates that the maximum local average hydrophilicity of a protein (e.g., governed by the hydrophilicity of its adjacent amino acids) is related to the biological properties of the protein.

[0078] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​have been assigned to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0, +0.1); glutamic acid (+3.0, +0.1); serine (+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).

[0079] Alternatively, the protein sequence of various proteins at the amino or carboxyl terminus can be altered without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modern molecular methods, such as PCR, which includes PCR amplification that alters or elongates the protein-coding sequence by incorporating the amino acid-coding sequence into the oligonucleotide used in the PCR amplification. Alternatively, the added protein sequence can include the complete protein-coding sequence, such as those commonly used in the art to generate protein fusions. Such fusion proteins are often used to (1) increase the expression of a target protein; (2) introduce binding domains, enzyme activity, or epitopes to facilitate protein purification, protein detection, or other experimental uses known in the art; and (3) target the secretion or translation of a protein to subcellular organelles, such as the periplasmic space of Gram-negative bacteria, mitochondria or chloroplasts of plants, or the endoplasmic reticulum of eukaryotic cells, the latter often leading to protein glycosylation.

[0080] The variant nucleotide and amino acid sequences disclosed herein also encompass sequences derived from procedures that induce mutagenesis and recombination, such as DNA shuffling. Through such procedures, new polypeptides with desired properties can be created using one or more different homologous polypeptide coding regions. In this manner, recombinant polynucleotide libraries are generated from a population of related sequence polynucleotides containing sequence regions that have basic sequence identity and are capable of homologous recombination in vitro or in vivo. For example, using this method, sequence motifs encoding a target domain can be shuffled between pest-killing genes and other known pest-killing genes to obtain new genes encoding proteins with improved target properties, such as increased insecticidal activity. Such DNA shuffling strategies are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 91: 10747-10751; Stemmer, (1994) Nature [Nature] 370: 389-391; Ctameri et al., (1997) Nature Biotech. [Nature Biotechnology] 15: 436-438; Moore et al., (1997) J Mol Biol [Journal of Molecular Biology] 272: 336-347; Zhang et al., (1997) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 94: 4504-4509; Crameri et al., (1998) Nature [Nature] 391: 288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458.

[0081] Domain exchange or shuffling is another mechanism for producing altered peptides. Exchanging domains between peptide homologs can result in hybrid or chimeric toxins with improved insecticidal activity or target profiles. The methods used to generate recombinant proteins and test their necrotic activity are well known in the art (see, for example, Naimov et al., (2001) Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 67: 5328-5330; de Maagd et al., (1996) Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 62: 1537-1543; Ge et al., (1991) J. Biol. Chem. [Journal of Biochemistry] 266: 17954-17958; Schnepf et al., (1990) J. Biol. Chem. [Journal of Biochemistry] 265: 20923-20930; Rang et al., (1999) Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 65: 2918-2925).

[0082] Phylogenetic analysis, sequence motif discovery, and structural analysis of an insecticidal protein family. Sequence and structural analysis methods can be used, comprising four parts: phylogenetic tree construction, protein sequence motif discovery, secondary structure prediction, and protein sequence-secondary structure alignment. Detailed descriptions of each part are provided below.

[0083] Phylogenetic tree construction

[0084] Phylogenetic analysis can be performed using the software MEGA5. Protein sequences can be analyzed using ClustalW version 2 (Larkin MA et al. (2007) Bioinformatics 23(21): 2947-2948) for multiple sequence alignment. Evolutionary history is then inferred using maximum likelihood estimation based on a JTT matrix-based model. A tree with the highest log-likelihood values ​​is obtained, exported in Newick format, and further processed to extract sequence IDs in the same order as those appearing in the tree. Several clades representing subfamilies can be manually identified for each insecticidal protein family.

[0085] Protein sequence motif discovery

[0086] Protein sequences were reordered based on a previously constructed phylogenetic tree and input into the motif analysis tool MEME (Multiple EM for motif extraction) (Bailey TL 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. MEME settings were as follows: minimum number of sites 2, minimum motif width 5, and maximum number of motifs 30. Sequence motifs unique to each subfamily were identified visually. The distribution of motifs throughout the gene family can be viewed on an HTML webpage. Motifs were numbered relative to their E-value ranking.

[0087] Secondary structure prediction

[0088] PSIPRED, the top-ranked secondary structure prediction method (Jones DT. (1999) J. Mol. Biol. [Journal of Molecular Biology] 292: 195-202), can be used for protein secondary structure prediction. This tool uses two feedforward neural networks based on the output of PSI-BLAST to provide accurate structure predictions. The PSI-BLAST database was created by removing low-complexity, transmembrane, and coiled-coil regions from Uniref100. PSIPRED results include the predicted secondary structures (α-helix: H, β-chain: E, and helix: C) and the corresponding confidence score for each amino acid in a given protein sequence.

[0089] Comparison of protein sequence and secondary structure

[0090] Scripts can be developed to generate vacancy secondary structure alignments for all proteins based on multiple protein sequence alignments from step 1. All aligned protein sequences and structures are then linked into a single FASTA file and imported into MEGA for visualization and identification of conserved structures.

[0091] In some embodiments, the IPD059, IPD098, IPD108, or IPD109 peptides have modified physical properties. As used herein, the term "physical property" refers to any parameter suitable for describing the physicochemical characteristics of a protein. As used herein, "target physical property" and "target property" are used interchangeably to refer to the physical properties of the protein being studied and / or modified. Examples of physical properties include, but are not limited to: 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 hydrophobic density, total count of ionized groups on the surface, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second isotropic coefficient. Examples of physical properties also include increased expression, increased solubility, reduced phytotoxicity, and digestibility of the proteolytic fragments in the insect gut of the IPD059, IPD098, IPD108, or IPD109 peptides. Models simulating gastric digestion are known to those skilled in the art (Fuchs, RL and JD. Astwood. Food Technology 50: 83-88, 1996; Astwood, JD et al., Nature Biotechnology 14: 1269-1273, 1996; Fu TJ et al., J. Agric Food Chem. 50: 7154-7160, 2002).

[0092] In some embodiments, variants include polypeptides that differ in amino acid sequence due to mutagenesis. The variant proteins covered by this disclosure possess biological activity, meaning they retain the biological activity required of the natural protein (i.e., pest-killing activity). In some embodiments, the variants 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 natural protein. In some embodiments, the variants may have improved activity compared to the natural protein.

[0093] Bacterial genes typically have multiple methionine start codons near the start of the read frame. Translation initiation at one or more of these start codons usually results in the production of a functional protein. These start codons may include the ATG codon. However, bacteria (e.g., Bacillus species) also recognize the GTG codon as a start codon, and proteins translated at the GTG codon contain methionine at the first amino acid. In rare cases, translation in bacterial systems may begin at the TTG codon, although in this event TTG encodes methionine. Furthermore, it is not typically determined a priori which of these codons are naturally used in bacteria. Therefore, it should be understood that using one of the alternative methionine codons may also result in the production of pest-killing proteins. These pest-killing proteins are covered in this disclosure and can be used in the methods of this disclosure. It should be understood that when expressed in plants, it is necessary to change the alternative start codon to ATG for proper translation.

[0094] Those skilled in the art will understand that polynucleotide coding sequences can be modified to add a codon after the methionine start codon to create a restriction enzyme site for recombinant cloning purposes and / or for expression purposes. In some embodiments, the polypeptides disclosed herein further include an alanine residue after the translation initiation factor methionine.

[0095] In some embodiments, the translation initiation factor methionine of the disclosed peptides is cleaved post-translation. Those skilled in the art will understand that in many cellular expression systems, the N-terminal translation initiation factor methionine can be removed by methionine aminopeptidase.

[0096] In some embodiments, a chimeric polypeptide is provided that includes regions of at least two different IPD059, IPD098, IPD108, or IPD109 polypeptides of this disclosure. In some embodiments, a chimeric polypeptide is provided that includes regions of at least two different IPD098 polypeptides of this disclosure. In some embodiments, a chimeric polypeptide is provided that includes regions of at least two different IPD059, IPD098, IPD108, or IPD109 polypeptides of this disclosure. In some embodiments, a chimeric polypeptide is provided that includes regions of at least two different IPD059, IPD098, IPD108, or IPD109 polypeptides of this disclosure.

[0097] In other embodiments, the polypeptides of this disclosure can be expressed as precursor proteins having intercalation sequences that catalyze multi-step post-translational protein splicing. Protein splicing involves the excision of the intercalation sequence from the polypeptide, accompanied by the attachment of flanking sequences to generate a new polypeptide (Chong et al., (1996) J. Biol. Chem., 271: 22159-22168). This intercalation sequence, or protein splicing element, referred to as an inteptide, catalyzes its own excision through the following three coordinated reactions at the N-terminal and C-terminal splice sites: acyl rearrangement of the N-terminal cysteine ​​or serine; transesterification reaction forming a branched ester or thioester intermediate between the two ends; and peptide bond cleavage that releases the inteptide via cyclization with the C-terminal asparagine of the inteptide (Evans et al., (2000) J. Biol. Chem., 275: 9091-9094). Elucidating the mechanisms of protein splicing has led to numerous integrity-based applications (Comb et al., U.S. Patent No. 5,496,714; Comb et al., U.S. Patent No. 5,834,247; Camarero and Muir, (1999) J. Amer. Chem. Soc. [Journal of the American Chemical Society] 121: 5597-5598; Chong et al., (1997) Gene [Gene] 192: 271-281; ​​Chong et al., (1998) Nucleic Acids Res. [Nucleic Acid Research] 26: 5109-5115; Chong et al., (1998) J. Biol. Chem. 273: 10567-10577; 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. [Protein Science] 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 [Proceedings of the National Academy of Sciences] 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. [Journal of the European Society for Molecular Biology] 17: 918-926; Southworth et al., (1999) Biotechniques [Biotechnology] 27: 110-120; Wood et al., (1999) Nat Biotechnol. [Nature Biotechnology] 17: 889-892; Wu et al., (1998a) Proc. Natl. Acad. Scci. USA [Proceedings of the National Academy of Sciences of the United States of America] 95: 9226-9231; Wu et al., (1998b) Biochim Biophys Acta Biochimica et Biophysica Sinica 1387: 422-432; Xu et al., (1999) Proc. Natl. Acad. Scci. USA 96: 388-393; Yamazaki et al., (1998) J. Am. Chem. Soc. 120: 5591-5592. For the application of integrins in plant transgenics, see Yang et al. (Transgene Research 15: 583-593 (2006)) and Evans et al. (Annu. Rev. Plant Biol. 56: 375-392 (2005)).

[0098] In another embodiment, the polypeptide of this disclosure may be encoded by two separate genes, wherein the inteins of the precursor protein are derived from two genes called split inteins, and the two portions of the precursor are linked by peptide bonds. This peptide bond formation is achieved through intein-mediated trans-splicing. For this purpose, first and second expression cassettes comprising two separate genes further encode inteins capable of mediating protein trans-splicing. Through trans-splicing, the protein and polypeptide encoded by the first and second fragments are linked by peptide bonds. The trans-splicing inteins may be selected from the nucleolar and organelle genomes of various organisms, including eukaryotes, archaea, and bacteria. Available inteins are listed at neb.com / neb / inteins.html, which can be accessed on the World Wide Web using the "www" prefix. The nucleotide sequence encoding the intein may be split into 5′ and 3′ portions, which encode the 5′ and 3′ portions of the intein, respectively. Sequence portions not desired for intein splicing (e.g., homing endonuclease domains) may be deleted. The inteptide-coding sequence is split so that the 5′ and 3′ portions can be trans-spliced. To select a suitable splitting site for the inteptide-coding sequence, considerations disclosed by Southworth et al., (1998) EMBO J. [Journal of the European Society for Molecular Biology] 17: 918-926 can be followed. In constructing the first and second expression cassettes, the 5′ inteptide-coding sequence is attached to the 3′ end of a first fragment encoding the N-terminal portion of the disclosed IPD059, IPD098, IPD108, or IPD109 polypeptide, and the 3′ inteptide-coding sequence is attached to the 5′ end of a second fragment encoding the C-terminal portion of the IPD059, IPD098, IPD108, or IPD109 polypeptide.

[0099] Generally, any fragmented inteptide can be used to design trans-splicing couples, including any naturally occurring or artificially derived fragmented inteptides. Several naturally occurring fragmented inteptides are known, such as the fragmented inteptide of the DnaE gene from the Synechocystis species PCC6803 (see Wu et al., (1998) Proc Natl Acad Sci USA. [Proceedings of the National Academy of Sciences] 95(16): 9226-31 and Evans et al., (2000) J Biol Chem. [Journal of Biochemistry] 275(13): 9091-4) and the fragmented inteptide of the DnaE gene from Nostoc punctiforme (see Iwai et al., (2006) FEBS Lett. [Communications of the European Federation of Biochemical Societies] 580(7): 1853-8). Non-fragmented inteins are artificially split in the laboratory to produce new fragmented inteins, for example: artificially split Ssp DnaB inteins (see, Wu et al., (1998) Biochim Biophys Acta. [Acta Biochim Biophysica Sinica] 1387: 422-32) and split Sce VMA inteins (see, Brenzel et al., (2006) Biochemistry. [Biochemistry] 45(6): 1571-8) and artificially split fungal microinteins (see, Elleuche et al., (2007) Biochem Biophys ResCommun. [Biochem Biophysica Research Communications] 355(3): 830-4). There are also intein databases that catalog known inteins (see, for example, an online database available at bioinformatics.weizmann.ac.il / ~pietro / inteins / Inteinstable.html, which can be accessed on the World Wide Web using the "www" prefix).

[0100] Naturally occurring non-fragmented inteins may possess endonuclease or other enzymatic activities, which can typically be removed when designing artificially fragmented inteins. Such miniature or minimized fragmented inteins are well known in the art and are typically less than 200 amino acid residues long (see, Wu et al., (1998) Biochim Biophys Acta. [Chinese Journal of Biochemistry and Biophysics] 1387: 422-32). Suitable fragmented inteins can have peptide elements added to their structure to enable further purification, provided that these elements do not inhibit the splicing of the fragmented inteins or are added in a manner that allows them to be removed prior to splicing. Protein splicing has been reported using proteins including: bacterial integrin-like (BIL) domains (see, Amitai et al., (2003) Mol Microbiol. [Molecular Microbiology] 47: 61-73) and hedgehog self-processing domains (when referred to as the Hog / integrin superfamily or HINT family, the latter being combined with integrins; see, Dassa et al., (2004) J Biol Chem. [Journal of Biochemistry] 279: 32001-7) and domains that can also be used to prepare artificially split integrins. In particular, non-splicing members of these families can be modified by molecular biological methods to introduce or restore splicing activity in these related species. Recent studies have shown that splicing can be observed when the N-terminal cleaved integument component is allowed to react with a C-terminal cleaved integument component that is not found in nature as its "partner"; for example, splicing has been observed using a partner with as little as 30% to 50% homology to the "natural" splicing partner (see, Dassa et al., (2007) Biochemistry, 46(1): 322-30). Other such mixtures of different cleaved integuments have been shown to be mixtures that do not react with another (see, Brenzel et al., (2006) Biochemistry, 45(6): 1571-8). However, determining whether a particular pair of peptides can bind to each other to provide a functional integument using conventional methods and without the need for creative skill is within the capabilities of those skilled in the art.

[0101] In some embodiments, the polypeptides disclosed herein are circularly arranged variants. Advances in recombinant DNA methods have made it possible to study the effects of sequence transposition on protein folding, structure, and function. The method used to create new sequences is similar to that used to associate naturally occurring protein pairs by linear shuffling of their amino acid sequences (Cunningham et al., (1979) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 76: 3218-3222; Teather and Erfle, (1990) J. Bacteriol. [Journal of Bacteriology] 172: 3837-3841; Schimming et al., (1992) Eur. J. Biochem. [European Journal of Biochemistry] 204: 13-19; Yamiuchi and Minamikawa, (1991) FEBS Lett. [Federation of European Biochemical Societies Newsletter] 260: 127-130; MacGregor et al., (1996) FEBS Lett. [Federation of European Biochemical Societies Newsletter] 378: 263-266). Goldenberg and Creighton described the first in vitro application of this type of protein rearrangement (J. Mol. Biol. [Journal of Molecular Biology] 165: 407-413, 1983). In the variant that creates a circular arrangement, a new N-terminus is selected at an internal site (breakpoint) of the original sequence. The new sequence has the same amino acid order as the original sequence from the breakpoint until it reaches the original C-terminus or an amino acid in or near it. At this point, the new sequence is attached directly or through another part of the sequence (linker) to the original N-terminus or an amino acid in or near it, and the new sequence continues with the same sequence as the original sequence until it reaches the point at or near the N-terminal amino acid of the breakpoint site of the original sequence, where this residue forms the new C-terminus of the chain. The length of the amino acid sequence of the linker can be selected empirically, guided by structural information, or by using a combination of both methods. When no structural information is available, small series of connectors can be prepared for testing using the ability to design and take necessary verification without compromising the conformational flexibility of the biotoxic peptide (conformal flexibility; Karplus and Schulz, (1985) Naturwissenschaften [Natural Sciences] 72: 212-213), with the length of the design spanning from 0 to The range varies and the designed sequence is selected to correspond to surface exposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol. [Molecular Immunology] 20: 483-489; Kyte and Doolittle, (1982) J. Mol. Biol. [Journal of Molecular Biology] 157: 105-132; solvent-exposed surface area, Lee and Richards, (1971) J. Mol. Biol. [Journal of Molecular Biology] 55: 379-400). Assuming an average translation of 2.0 to 100 mg / L per residue. This means that the length to be tested is between 0 and 30 residues, with 0 to 15 residues being the preferred range. An example of such an empirical series would be the construction of linkers using box sequences repeated n times, such as Gly-Gly-Gly-Ser, where n is 1, 2, 3, or 4. Those skilled in the art will recognize that there are many such sequences of different lengths or compositions that can be used as linkers, with the main consideration being that they are neither too long nor too short (see Sandhu, (1992) Critical Rev. Biotech. [Biotechnology Review] 12: 437-462); if they are too long, entropy effects may destabilize the three-dimensional folding and may also make the folding kinetically impractical, and if they are too short, they may destabilize the molecule due to torsion or spatial strain. Those skilled in protein structural information analysis will recognize that the distance between chain ends, defined as the distance between c-α carbons, can be used to define the length of the sequence to be used, or at least limit the number of possibilities that must be tested in the empirical selection of linkers. Therefore, they will also recognize that in cases where the position of the polypeptide chain terminus is sometimes ambiguous in the structural model derived from X-ray diffraction or NMR spectroscopy data, and when this is indeed the case, it is necessary to consider this situation in order to correctly estimate the required linker length. From those residues whose positions are clearly defined, two residues sequentially close to the chain terminus are selected, and the approximate length of the linker between them is calculated using the distance between their c-α carbons. Using the calculated length as a guide, residues within a certain range (using 2 to 1000 carbons per residue) are then selected. The linkers are calculated. These linkers can consist of the original sequence, shortened or lengthened if necessary, and when lengthened, additional flexible and hydrophilic residues can be selected as described above; or optionally, the original sequence can be replaced by a series of linkers, an example being the Gly-Gly-Gly-Ser box method mentioned above; or optionally, a combination of the original sequence and the new sequence with an appropriate total length can be used. Sequences of biocidal polypeptides capable of folding into a biologically active state can be prepared by appropriately selecting the start (amino terminus) and end (carboxyl terminus) positions from the original polypeptide chain, while using linker sequences as described above. The amino and carboxyl terms are selected from common extensions of sequences called breakpoint regions using the guidelines described below. Thus, novel amino acid sequences are generated by selecting amino and carboxyl terms within the same breakpoint region. In many cases, the selection of the new terminus will result in the original position of the carboxyl terminus being adjacent to the position of the amino terminus. However, those skilled in the art will recognize that the selection of the terminus anywhere within the region may be effective, and these will effectively result in the deletion or addition of the amino or carboxyl portion of the new sequence. The folding of the major amino acid sequence of a protein into a three-dimensional structure necessary for expressing its biological function is a core principle of molecular biology. Methods for obtaining and interpreting three-dimensional structural information using X-ray diffraction of a single protein crystal or nuclear magnetic resonance spectroscopy of a protein solution are known to those skilled in the art. Examples of structural information associated with the identification of breakpoint regions include the location and type of protein secondary structures (α and 3–10 helices, parallel and antiparallel β-sheets, chain reversals and turns, and loops; Kabsch and Sander, (1983) Biopolymers 22: 2577–2637); the solvent exposure of amino acid residues, the degree and type of interaction between residues (Chothia, (1984) Ann. Rev. Biochem. 53: 537–572); and the static and dynamic distribution of conformations along the polypeptide chain (Alber and Mathews, (1987) Methods Enzymol. 154: 511–533). In some cases, other information regarding the solvent exposure of residues is known; one example is the post-translational attachment site of carbohydrates, which is necessarily located on the surface of the protein. When experimental structural information is unavailable or impossible to obtain, methods can also be used to analyze the primary amino acid sequence to predict protein tertiary and secondary structures, solvent accessibility, and the occurrence of turns and loops.When direct structural methods are not feasible, biochemical methods are sometimes applicable to empirically determine surface exposure; for example, surface exposure can be inferred by identifying chain break sites after limited proteolysis (Gentile and Salvatore, (1993) Eur. J. Biochem. [European Journal of Biochemistry] 218: 603-621). Using experimentally derived structural information or predictive methods (e.g., Srinivisan and Rose, (1995) Proteins: Struct., Funct. & Genetics [Proteins: Structure, Function & Genetics] 22: 81-99), parental amino acid sequences are examined to classify regions based on whether they are essential for maintaining secondary and tertiary structures. Sequence occurrences in regions known to involve periodic secondary structures (α and 3–10 helices, parallel and antiparallel β-sheets) are regions that should be avoided. Similarly, regions observed or predicted to have amino acid sequences with low solvent exposure are more likely to be part of the so-called hydrophobic core of the protein, and amino and carboxyl ends should also be avoided. In contrast, regions known or predicted to be surface-internal turns or loops, and especially those regions known to be undesirable for biological activity, are preferred sites for locating polypeptide chain ends. The continuous extension of the amino acid sequence preferred based on the above criteria is called a breakpoint region. Polynucleotides encoding circularly arranged IPD059, IPD098, IPD108, or IPD109 polypeptides with novel N-terminals / C-termini, comprising a linker region separating the original C-terminus from the N-terminus, can be prepared by essentially following the method described in Mullins et al., (1994) J. Am. Chem. Soc. [Journal of the American Chemical Society] 116: 5529-5533. Multiple steps of polymerase chain reaction (PCR) amplification are used to rearrange the DNA sequence encoding the primary amino acid sequence of the protein. Polynucleotides encoding circularly arranged IPD059, IPD098, IPD108, or IPD109 polypeptides with novel N-terminals / C-termini, comprising a linker region separating the original C-terminus from the N-terminus, can be prepared based on the tandem repeat method described by Horlick et al., (1992) Protein Eng. 5: 427-431. Polymerase chain reaction (PCR) amplification of the novel N-terminal / C-terminal gene is then performed using the tandem repeat template DNA.

[0102] In another embodiment, a fusion protein is provided, wherein the amino acid sequence of the fusion protein includes an amino acid sequence containing a polypeptide of the present disclosure. Methods for designing and constructing fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art. The polynucleotide encoding the polypeptide of an embodiment may be fused to a signal sequence that directs the polypeptide of the embodiment to a specific compartment of a prokaryotic or eukaryotic cell and / or directs the secretion of the polypeptide of the embodiment from a prokaryotic or eukaryotic cell. For example, in *E. coli*, it may be desirable to direct protein expression into the periplasmic space. Examples of signal sequences or proteins (or fragments thereof) that can be fused with the polypeptide of the present disclosure to direct the expression of the polypeptide into the bacterial periplasmic space include, but are not limited to: the pelB signal sequence, the maltose-binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the heat-labile enterotoxin B subunit of *E. coli* periplasmic, and the signal sequence of alkaline phosphatase. Several vectors for constructing fusion proteins that direct protein localization are commercially available, such as the pMAL series vectors (particularly the pMAL-p series) available from New England Biolabs. In specific embodiments, the IPD059, IPD098, IPD108, or IPD109 peptides may be fused to the pelB pectic acid lyase signal sequence to increase the efficiency of expression and purification of such peptides in Gram-negative bacteria (see U.S. Patents 5,576,195 and 5,846,818). Plant plasmid transport peptide / peptide fusions are well known in the art. Apoplast transport peptides, such as rice or barley α-amylase secretion signals, are also well known in the art. Plasmid transport peptides are typically N-terminally fused to a target peptide (e.g., a fusion coupler). In one embodiment, the fusion protein consists essentially of a plasmid transport peptide and a target peptide of this disclosure. In another embodiment, the fusion protein comprises a plasmid transport peptide and a target peptide. In such embodiments, the plasmid transport peptide is preferably located at the N-terminus of the fusion protein. However, additional amino acid residues may be located at the N-terminus of the plasmid transport peptide, provided that the fusion protein at least partially targets the plasmid. In specific embodiments, the plasmid transport peptide is located at the N-terminal half, N-terminal third, or N-terminal quarter of the fusion protein. When a plasmid is inserted, most or all of the plasmid transport peptide is typically cleaved from the fusion protein. Due to specific intercellular conditions or the particular combination of transport peptide / fusion partner used, the cleavage site may vary slightly between plant species at different developmental stages. In one embodiment, the plasmid transport peptide cleavage is uniform, such that the cleavage site is identical throughout the fusion protein population. In another embodiment, the plasmid transport peptide is not uniform, such that the cleavage site differs by 1-10 amino acids throughout the fusion protein population. The plasmid transport peptide can be recombinantly fused to a second protein in one of several ways.For example, restriction endonuclease recognition sites can be introduced into the nucleotide sequence of the transport peptide corresponding to its C-terminus, and identical or compatible sites can be engineered to the nucleotide sequence of the protein to be targeted at its N-terminus. Care must be taken to design these sites to ensure that the coding sequences of the transport peptide and the second protein remain “in-the-box” to allow for the synthesis of the desired fusion protein. In some cases, when introducing new restriction sites, it is preferable to remove the initiation factor methionine of the second protein. Introducing restriction endonuclease recognition sites on two parent molecules, and their subsequent ligation via recombinant DNA technology, can result in the addition of one or more additional amino acids between the transport peptide and the second protein. This generally does not affect targeting activity, as long as the transport peptide cleavage site remains accessible, and the addition of these additional amino acids at its N-terminus does not alter the function of the second protein. Alternatively, those skilled in the art can use gene synthesis (Stemmer et al., (1995) Gene 164:49-53) or similar methods to generate precise cleavage sites between the transport peptide and the second protein (with or without its initiator methionine). Additionally, transport peptide fusions can intentionally include amino acids downstream of the cleavage site. The amino acids at the N-terminus of a mature protein can affect the ability of a transport peptide to target the protein to the plasmid and / or the efficiency of protein cleavage after input. This may depend on the protein to be targeted. See, for example, Comai et al., (1988) J. Biol. Chem. 263(29): 15104-9.

[0103] In some embodiments, the disclosed polypeptide is fused with a heterologous signal peptide or a heterologous transport peptide.

[0104] In some embodiments, a fusion protein comprising a polypeptide of the present disclosure is provided, the fusion protein being represented by a formula selected from the group consisting of:

[0105] R 1 LR 2 R 2 -LR 1 R 1 -R 2 Or R 2 -R 1

[0106] Where R 1 It is a disclosed polypeptide, and R 2 It is the target protein. In some embodiments, R 1 and R 2 This is the disclosed polypeptide. The R... 1 The peptide is fused directly or via the linker (L) segment to R 2Peptide. The term "direct" is defined as the fusion of peptides without peptide linkers. Therefore, "L" indicates fusion with R. 1 and R 2 In-frame fusion of chemically bound or polypeptide regions, most commonly, L is R 1 and R 2 R via amide bond 1 The carboxyl terminus of L is attached to the amino terminus of L, and the carboxyl terminus of L is attached to R. 2 A linear peptide at the amino terminus. "In-frame fusion" refers to R... 1 and R 2 There is no translation termination or interruption between reading frames. The linker group (L) is typically a polypeptide with a length between 1 and 500 amino acids. The linker connecting the two molecules is preferably designed to (1) allow the two molecules to fold and function independently of each other, (2) not have a tendency to develop ordered secondary structures that could interfere with the functional domains of the two proteins, (3) have minimal hydrophobic or charged features that can interact with the functional protein domains, and (4) provide R 1 and R 2 Spatial separation makes R 1 and R 2 It can interact simultaneously with the corresponding receptor on a single cell. Typically, the surface amino acids in the flexible protein region include Gly, Asn, and Ser. Almost any arrangement of amino acid sequences containing Gly, Asn, and Ser is expected to meet the criteria for the linker sequence described above. Other neutral amino acids such as Thr and Ala can also be used as linker sequences. Additional amino acids can also be included in the linker because unique restriction sites are added to the linker sequence to facilitate the construction of the fusion body.

[0107] In some embodiments, the linker comprises a sequence selected from the group consisting of (Gly3Ser)n, (Gly4Ser)n, (Gly5Ser)n, (GlynSer)n, or (AlaGlySer)n, where n is an integer. An example of a highly flexible linker is a (GlySer)-rich spacer region present within the pIII protein of filamentous bacteriophages (e.g., phage M13 or fd) (Schaller et al., 1975). This region provides a long, flexible spacer region between two domains of the pIII surface protein. Linkers are also included that include endopeptidase recognition sequences. Such cleavage sites can be valuable for separating the individual components of the fusion to determine whether they are properly folded and active in vitro. Examples of various endopeptidases include, but are not limited to: plasmin, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostridial protease, rennet, collagenase, Russell's viper venom protease, postproline cleavage enzyme, V8 protease, thrombin, and factor Xa. In some embodiments, the linker comprises the amino acid EEKKN (SEQ ID NO: 156) from a multigene expression vector (MGEV), which is cleaved by a vacuolar protease as disclosed in U.S. Patent Application Publication No. US 2007 / 0277263. In other embodiments, peptide linker segments from hinge regions of heavy chain immunoglobulins IgG, IgA, IgM, IgD, or IgE provide angular relationships between the attached polypeptides. Hinge regions where cysteine ​​residues are replaced by serine residues are particularly useful. The linkers of this disclosure comprise sequences derived from the hinge region of mouse IgG γ2b, wherein cysteine ​​residues have been replaced by serine residues. Fusion proteins are not limited by the form, size, or number of linker sequences used, and the only requirement for the linker is that it does not adversely interfere with the folding and function of the fused individual molecules.

[0108] Nucleic acid molecules and their variants and fragments

[0109] Provided are isolated or recombinant nucleic acid molecules containing nucleic acid sequences encoding polypeptides or their biologically active portions, and nucleic acid molecules sufficient to be used as hybridization probes to identify nucleic acid molecules encoding proteins having regions of sequence homology. As used herein, the term "nucleic acid molecule" refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plasmid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA), as well as DNA or RNA analogs produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0110] As used herein, “isolated” nucleic acid molecule (or DNA) refers to a nucleic acid sequence (or DNA) that is no longer in its natural environment (e.g., in vitro). As used herein, “recombinant” nucleic acid molecule (or DNA) refers to a nucleic acid sequence (or DNA) in a recombinant bacterial or plant host cell. In some embodiments, “isolated” or “recombinant” nucleic acids do not contain sequences naturally located flanking the nucleic acid in the genomic DNA of an organism derived from the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) (preferably sequences encoding proteins). For the purposes of this disclosure, “isolated” or “recombinant” excludes isolated chromosomes when used to refer to nucleic acid molecules. For example, in various embodiments, a recombinant nucleic acid molecule encoding a polypeptide of this disclosure may comprise a nucleic acid sequence of less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb, said nucleic acid sequence being naturally located flanking the nucleic acid molecule in the genomic DNA of a cell derived from the nucleic acid.

[0111] In some embodiments, the isolated nucleic acid molecule encoding the polypeptide of this disclosure has one or more variations in its nucleic acid sequence compared to a natural or genomic nucleic acid sequence. In some embodiments, the variations in the natural or genomic nucleic acid sequence include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to amino acid substitutions, insertions, deletions, and / or additions compared to a natural or genomic sequence; removal of one or more introns; deletion of one or more upstream or downstream regulatory regions; and deletion of 5′ and / or 3′ untranslated regions associated with a genomic nucleic acid sequence. In some embodiments, the nucleic acid molecule encoding the polypeptide of this disclosure is a non-genomic sequence.

[0112] Various polynucleotides encoding the polypeptides or related proteins disclosed herein are considered. When operably linked to suitable promoters, transcription terminators, and / or polyadenylated sequences, these polynucleotides can be used to produce the polypeptides disclosed herein in host cells. These polynucleotides can also be used as probes for isolating homologous or substantially homologous polynucleotides encoding the polypeptides or related proteins disclosed herein.

[0113] Polynucleotide encoding IPD059 polypeptide

[0114] One source of the polynucleotide encoding the IPD059 polypeptide or related protein is a fern or other primitive plant species selected from, but not limited to, species of the genera *Polygonum*, *Adiantum*, *Hylocereus*, *Aristolochia*, or *Adiantum*, containing the polynucleotide encoded by SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 6 ...67, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or the IPD059 polypeptide of SEQ ID NO: 75 NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDIPD059 polynucleotides of NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID The polynucleotide NO: 36 or SEQ ID NO: 37 is used to express the IPD059 polypeptide in recombinant bacterial host cells (including but not limited to Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas, and Rhizobium bacterial host cells). The polynucleotide can also be used as a probe for isolating homologous or substantially homologous polynucleotides encoding the IPD059 polypeptide or related proteins. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from ferns or other primitive plant species, selected from, but not limited to, species of the genera *Polypodium*, *Adiantum*, *Adiantum*, *Adiantum*, or *Pteridium*.

[0115] The polynucleotide encoding the IPD059 polypeptide can also be synthesized de novo from the IPD059 polypeptide sequence. The sequence of the polynucleotide gene can be deduced from the IPD059 polypeptide sequence using the genetic code. Computer programs such as "BackTranslate" (GCG) can be used to deduce this. TMThe package (Acclerys, Inc., San Diego, California) can be used to convert peptide sequences into corresponding nucleotide sequences encoding said peptides. Examples of IPD059 polypeptide sequences that can be used to obtain the corresponding nucleotide coding sequence include, but are not limited to, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 6 ...9, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: IPD059 polypeptides of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 78. Furthermore, the synthetic IPD059 polynucleotide sequence of this disclosure can be designed for expression in plants.

[0116] In some embodiments, the nucleic acid molecule encoding the IPD059 polypeptide is a molecule having the following properties: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 5. Polynucleotides, variants, fragments, and complementary sequences of the sequences shown in SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37. As used herein, a “complementary sequence” is a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence, such that it can hybridize with said given nucleic acid sequence to form a stable double strand. As used herein, a “polynucleotide sequence variant” is a nucleic acid sequence that encodes the same polypeptide except for the degeneracy of the genetic code.

[0117] In some embodiments, the nucleic acid molecule encoding the IPD059 polypeptide is a non-genomic nucleic acid sequence. As used herein, a “non-genomic nucleic acid sequence” or “non-genomic nucleic acid molecule” or “non-genomic polynucleotide” refers to a nucleic acid molecule having one or more variations in its nucleic acid sequence compared to a natural or genomic nucleic acid sequence. In some embodiments, the alterations to the natural or genomic nucleic acid molecule include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence by introducing at least one amino acid substitution, insertion, deletion, and / or addition compared to the natural 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 associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of 5′ and / or 3′ untranslated regions associated with the genomic nucleic acid sequence; insertion of heterologous 5′ and / or 3′ untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is cDNA.

[0118] In some embodiments, the nucleic acid molecule encoding the IPD059 polypeptide is a non-genomic polynucleotide having the following nucleotide sequence, wherein the nucleotide sequence is identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 3 ... NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID The nucleic acid sequence of NO:37 has 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 greater identity, wherein the IPD059 polypeptide has insecticidal activity.

[0119] In some embodiments, the nucleic acid molecule encodes SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ The IPD059 polypeptide has the amino acid sequence of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 78.Compared to SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID The natural amino acid at the corresponding position of SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 78, said amino acid sequence having 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 or more amino acid substitutions, deletions and / or insertions.

[0120] Nucleic acid molecules encoding transcription and / or translation products are also provided, which are subsequently spliced ​​to ultimately produce a functional IPD059 polypeptide. Splicing can be performed in vitro or in vivo and can involve cis or trans splicing. The substrate for splicing can be a polynucleotide (e.g., RNA transcript) or a polypeptide. An example of cis splicing of a polynucleotide is removing introns inserted into the coding sequence and splicing two flanking exon regions to produce the IPD059 polypeptide coding sequence. An example of trans splicing is encrypting the polynucleotide by separating the coding sequence into two or more fragments, which can be transcribed individually and then spliced ​​to form a full-length pest-killing coding sequence. Cis or trans splicing of the polypeptide can be facilitated using splicing enhancer sequences that can be introduced into the construct (US Patent Nos. 6,365,377 and 6,531,316). Therefore, in some embodiments, the polynucleotide does not directly encode the full-length IPD059 polypeptide, but rather encodes one or more fragments of the IPD059 polypeptide. These polynucleotides can be used to express functional IPD059 peptides via mechanisms involving splicing, where splicing can occur at the level of polynucleotides (e.g., introns / exons) and / or peptides (e.g., intepids / exons). This can be used, for example, to control the expression of pest-killing activity, because if all the necessary fragments are expressed in an environment that allows the splicing process to produce a functional product, only the functional pest-killing peptide is expressed. In another example, introducing one or more insert sequences into a polynucleotide can facilitate recombination with low-homology polynucleotides; the use of introns or intepids targeting the insert sequences facilitates the removal of the intercalation sequences, thereby restoring the function of the encoded variant.

[0121] Nucleic acid molecules that are fragments of these nucleic acid sequences encoding the IPD059 polypeptide are also included in the examples. As used herein, “fragment” refers to a portion of the nucleic acid sequence encoding the IPD059 polypeptide. The fragment of the nucleic acid sequence may encode the biologically active portion of the IPD059 polypeptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below. The nucleic acid molecule that is a fragment of the nucleic acid sequence encoding the IPD059 polypeptide contains at least about 150, 180, 210, 240, 270, 300, 330, or 360 consecutive nucleotides, or up to the number of nucleotides present in the full-length nucleic acid sequence encoding the IPD059 polypeptide disclosed herein, depending on the intended use. As used herein, “consecutive nucleotides” refers to nucleotide residues that are adjacent to each other. The nucleic acid sequence fragments of the examples will encode protein fragments that retain the biological activity of the IPD059 polypeptide and thus retain its insecticidal activity. As used herein, "retained insecticidal activity" refers to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95%, or higher insecticidal activity of the full-length IPD059Aa polypeptide (SEQ ID NO: 39). In some embodiments, the insecticidal activity is targeted at Lepidoptera species. In one embodiment, the insecticidal activity is targeted at Coleoptera species.

[0122] In some embodiments, the IPD059 polypeptide consists of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID Nucleic acid sequences that are sufficiently homologous to the nucleic acid sequences NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37 encode a nucleic acid sequence. As used herein, “sufficient homology” means an amino acid or nucleic acid sequence that, using standard parameters and one of the alignment procedures described herein, has 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 with a reference sequence. Those skilled in the art will recognize that these values ​​can be appropriately adjusted by taking into account degeneracy, amino acid similarity, reading frame localization, etc., to determine the corresponding homology of proteins encoded by the two nucleic acid sequences. In some embodiments, the sequence homology refers to the full-length sequence of the polynucleotide encoding the IPD059 polypeptide or to the full-length sequence of the IPD059 polypeptide.

[0123] In some embodiments, the nucleic acid encodes the following IPD059 polypeptide: the IPD059 polypeptide is associated with SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 6 ...9, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: 69, SEQ ID NO: Compared with SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 78, it has 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 identity.

[0124] In some embodiments, the ClustalW algorithm is used in Vector Program Suite (Ingenium Inc., Carlsbad, California) The sequence identity is calculated in the module with all default parameters. In some embodiments, the ClustalW algorithm is used to calculate sequence identity across the full-length peptide in the ALIGNX module of the Vector NTI program suite (Ingenieur, Carlsbad, CA) with all default parameters.

[0125] To determine the percentage of identity between two or more amino acid sequences or two or more nucleic acid sequences, sequences are aligned for optimal comparison purposes. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of identity = number of identical positions / total number of positions (e.g., overlapping positions × 100)).

[0126] In one embodiment, the two sequences have the same length. In another embodiment, the comparison is performed across the entire reference sequence (e.g., across the entire SEQ ID NO: 39). Techniques similar to those described below can be used to determine the percentage identity between the two sequences, with or without gaps. In calculating percentage identity, exact matches are typically counted.

[0127] Another non-limiting example of a mathematical algorithm for comparing sequences is the one described in the following literature: Needleman and Wunsch, (1970) J. Mol. Biol. [Journal of Molecular Biology] 48(3): 443-453, using GAP version 10 software to determine sequence identity or similarity with the following default parameters: for identity % and similarity % of nucleic acid sequences, a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmpii scoring matrix; for identity % or similarity % of amino acid sequences, a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring procedure. Equivalent procedures may also be used. As used herein, “equivalent procedure” means any sequence comparison procedure that produces an alignment for any two sequences in question that, when compared with the corresponding alignment produced by GAP version 10, has the same nucleotide residue pairings and the same percentage of sequence identity.

[0128] In some embodiments, the IPD059 polynucleotide encodes an IPD059 polypeptide comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity with the amino acid sequence throughout the entire length of SEQ ID NO: 39.

[0129] In some embodiments, a polynucleotide encoding a chimeric polypeptide is provided, the chimeric polypeptide comprising regions of at least two different IPD059 polypeptides disclosed herein.

[0130] In some embodiments, a polynucleotide encoding a chimeric polypeptide is provided, the chimeric polypeptide comprising regions selected from at least two different IPD059 polypeptides: SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 6 ... NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 78.

[0131] In some embodiments, the IPD059 polynucleotide encodes SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID The IPD059 polypeptide has the amino acid sequence of NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ ID NO: 78.

[0132] The polynucleotide encoding the IPD098 polypeptide

[0133] One source of the polynucleotide encoding the IPD098 polypeptide or related protein is a fern or other primitive plant species selected from, but not limited to, species of the genera *Acer tectorum*, *Pteris*, *Aspergillus*, or *Selaginella*, containing SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117 respectively. The IPD098 polynucleotide can be expressed in recombinant bacterial hosts (including, but not limited to, Agrobacterium, Bacillus, Escherichia coli, Salmonella, Pseudomonas, and Rhizobium host cells) using SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 94. The IPD098 polypeptide can also be expressed in recombinant bacterial host cells (including, but not limited to, Agrobacterium, Bacillus, Escherichia coli, Salmonella, Pseudomonas, and Rhizobium host cells). The polynucleotides can also be used as probes for isolating homologous or substantially homologous polynucleotides encoding the IPD098 polypeptide or related proteins. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from ferns or other primitive plant species, selected from, but not limited to, species of the genera *Polygonum*, *Adiantum*, *Adiantum*, *Adiantum*, or *Pteris*.

[0134] The polynucleotide encoding the IPD098 polypeptide can also be synthesized de novo from the IPD098 polypeptide sequence. The sequence of the polynucleotide gene can be deduced from the IPD098 polypeptide sequence using the genetic code. Computer programs such as "BackTranslate" (GCG) can be used to deduce this. TM The present invention (Aclere, Inc., San Diego, California) can be used to convert peptide sequences into corresponding nucleotide sequences encoding said peptides. Examples of IPD098 polypeptide sequences that can be used to obtain the corresponding nucleotide-encoding sequences include, but are not limited to, the IPD098 polypeptides of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117. Furthermore, the synthetic IPD098 polynucleotide sequences of this disclosure can be engineered for expression in plants.

[0135] In some embodiments, the nucleic acid molecule encoding the IPD098 polypeptide is a polynucleotide, variants thereof, fragments thereof, and complementary sequences having the sequences shown in SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 94. As used herein, a “complementary sequence” refers to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize with said given nucleic acid sequence to form a stable double strand. As used herein, a “polynucleotide sequence variant” refers to a nucleic acid sequence that encodes the same polypeptide except for the degeneracy of the genetic code.

[0136] In some embodiments, the nucleic acid molecule encoding the IPD098 polypeptide is a non-genomic nucleic acid sequence. As used herein, “non-genomic nucleic acid sequence” or “non-genomic nucleic acid molecule” or “non-genomic polynucleotide” refers to a nucleic acid molecule having one or more variations in its nucleic acid sequence compared to a natural or genomic nucleic acid sequence. In some embodiments, the alterations to the natural or genomic nucleic acid molecule include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence by introducing at least one amino acid substitution, insertion, deletion, and / or addition compared to the natural 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 associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of 5′ and / or 3′ untranslated regions associated with the genomic nucleic acid sequence; insertion of heterologous 5′ and / or 3′ untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is cDNA.

[0137] In some embodiments, the nucleic acid molecule encoding the IPD098 polypeptide is a non-genomic polynucleotide having the following nucleotide sequence, wherein the nucleotide sequence is identical to SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 94. The nucleic acid sequence of IDNO:94 has 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 greater identity, wherein the IPD098 polypeptide has insecticidal activity.

[0138] In some embodiments, the nucleic acid molecule encodes an IPD098 polypeptide comprising the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117, compared to the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 117. The amino acid sequence has natural amino acids at the corresponding positions of SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117, said amino acid sequence having 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 or more amino acid substitutions, deletions and / or insertions.

[0139] Nucleic acid molecules encoding transcription and / or translation products are also provided, which are subsequently spliced ​​to ultimately produce a functional IPD098 polypeptide. Splicing can be performed in vitro or in vivo and can involve cis or trans splicing. The substrate for splicing can be a polynucleotide (e.g., RNA transcript) or a polypeptide. An example of cis splicing of a polynucleotide is removing introns inserted into the coding sequence and splicing two flanking exon regions to produce the IPD098 polypeptide coding sequence. An example of trans splicing is encrypting the polynucleotide by separating the coding sequence into two or more fragments, which can be transcribed individually and then spliced ​​to form a full-length pest-killing coding sequence. Cis or trans splicing of the polypeptide can be facilitated using splicing enhancer sequences that can be introduced into the construct (US Patent Nos. 6,365,377 and 6,531,316). Therefore, in some embodiments, the polynucleotide does not directly encode the full-length IPD098 polypeptide, but rather encodes one or more fragments of the IPD098 polypeptide. These polynucleotides can be used to express functional IPD098 peptides via mechanisms involving splicing, where splicing can occur at the level of polynucleotides (e.g., introns / exons) and / or peptides (e.g., intepids / exons). This can be used, for example, to control the expression of pest-killing activity, because if all the necessary fragments are expressed in an environment that allows the splicing process to produce a functional product, only the functional pest-killing peptide is expressed. In another example, introducing one or more insert sequences into a polynucleotide can facilitate recombination with low-homology polynucleotides; the use of introns or intepids targeting the insert sequences facilitates the removal of the intercalation sequences, thereby restoring the function of the encoded variant.

[0140] Nucleic acid molecules that are fragments of these nucleic acid sequences encoding the IPD098 polypeptide are also included in the examples. As used herein, “fragment” refers to a portion of the nucleic acid sequence encoding the IPD098 polypeptide. The fragment of the nucleic acid sequence may encode the biologically active portion of the IPD098 polypeptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below. The nucleic acid molecule that is a fragment of the nucleic acid sequence encoding the IPD098 polypeptide contains at least about 150, 180, 210, 240, 270, 300, 330, or 360 consecutive nucleotides, or up to the number of nucleotides present in the full-length nucleic acid sequence encoding the IPD098 polypeptide disclosed herein, depending on the intended use. As used herein, “consecutive nucleotides” refers to nucleotide residues that are adjacent to each other. The nucleic acid sequence fragments of the examples will encode protein fragments that retain the biological activity of the IPD098 polypeptide and thus retain its insecticidal activity. As used herein, "retained insecticidal activity" refers to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95%, or higher insecticidal activity of the full-length IPD098Aa polypeptide (SEQ ID NO: 102). In some embodiments, the insecticidal activity is targeted at Lepidoptera species. In one embodiment, the insecticidal activity is targeted at Coleoptera species.

[0141] In some embodiments, the IPD098 polypeptide is encoded by a nucleic acid sequence that is sufficiently homologous to the nucleic acid sequences of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, or SEQ ID NO: 94. As used herein, “sufficient homology” means an amino acid or nucleic acid sequence that, using standard parameters and one of the alignment procedures described herein, has 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 with a reference sequence. Those skilled in the art will recognize that these values ​​can be appropriately adjusted by taking into account degeneracy, amino acid similarity, reading frame localization, etc., to determine the corresponding homology of proteins encoded by the two nucleic acid sequences. In some embodiments, the sequence homology refers to the full-length sequence of the polynucleotide encoding the IPD098 polypeptide or to the full-length sequence of the IPD098 polypeptide.

[0142] In some embodiments, the nucleic acid encodes the following IPD098 polypeptide: the IPD098 polypeptide is associated with SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 108. NO:117 has 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 identity compared to it.

[0143] In some embodiments, the ClustalW algorithm is used in Vector Program Suite (Ingenium Inc., Carlsbad, California) The sequence identity is calculated in the module with all default parameters. In some embodiments, the ClustalW algorithm is used to calculate sequence identity across the full-length peptide in the ALIGNX module of the Vector NTI program suite (Ingenieur, Carlsbad, CA) with all default parameters.

[0144] To determine the percentage of identity between two or more amino acid sequences or two or more nucleic acid sequences, sequences are aligned for optimal comparison purposes. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of identity = number of identical positions / total number of positions (e.g., overlapping positions × 100)).

[0145] In one embodiment, the two sequences have the same length. In another embodiment, the comparison is performed across the entire reference sequence (e.g., across the entire SEQ ID NO: 102). Techniques similar to those described below can be used to determine the percentage identity between the two sequences, with or without gaps. In calculating percentage identity, exact matches are typically counted.

[0146] Another non-limiting example of a mathematical algorithm for comparing sequences is the one described in the following literature: Needleman and Wunsch, (1970) J. Mol. Biol. [Journal of Molecular Biology] 48(3): 443-453, using GAP version 10 software to determine sequence identity or similarity with the following default parameters: for identity % and similarity % of nucleic acid sequences, a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmpii scoring matrix; for identity % or similarity % of amino acid sequences, a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring procedure. Equivalent procedures may also be used. As used herein, “equivalent procedure” means any sequence comparison procedure that produces an alignment for any two sequences in question that, when compared with the corresponding alignment produced by GAP version 10, has the same nucleotide residue pairings and the same percentage of sequence identity.

[0147] In some embodiments, the IPD098 polynucleotide encodes an IPD098 polypeptide comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity with the entire length of the amino acid sequence throughout SEQ ID NO: 102.

[0148] In some embodiments, a polynucleotide encoding a chimeric polypeptide is provided, the chimeric polypeptide comprising regions of at least two different IPD098 polypeptides disclosed herein.

[0149] In some embodiments, a polynucleotide encoding a chimeric polypeptide is provided, the chimeric polypeptide comprising a region selected from at least two different IPD098 polypeptides: SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117.

[0150] In some embodiments, the IPD098 polynucleotide encodes an IPD098 polypeptide comprising the amino acid sequences of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117.

[0151] Polynucleotide encoding IPD108 polypeptide

[0152] One source of the polynucleotide encoding the IPD108 polypeptide or related protein is a fern or other primitive plant species selected from, but not limited to, species of the genera *Selaginella*, *Acer tectorum*, or *Dryopteris*, containing IPD108 polynucleotides encoding SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129 of the IPD108 polypeptide encoded by SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, or SEQ ID NO: 135, respectively. The polynucleotides SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129 can be used to express the IPD108 polypeptide in recombinant bacterial hosts (including but not limited to *Agrobacterium*, *Bacillus*, *Escherichia coli*, *Salmonella*, *Pseudomonas*, and *Rhizobium* bacterial host cells). The polynucleotides can also be used as probes for isolating homologous or substantially homologous polynucleotides encoding the IPD108 polypeptide or related proteins. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from ferns or other primitive plant species, selected from, but not limited to, species of the genera *Selaginella*, *Acer*, or *Dryopteris*.

[0153] The polynucleotide encoding the IPD108 polypeptide can also be synthesized de novo from the IPD108 polypeptide sequence. The sequence of the polynucleotide gene can be deduced from the IPD108 polypeptide sequence using the genetic code. Computer programs such as "BackTranslate" (GCG) can be used to deduce this. TM The IPD108 polypeptide sequence disclosed herein (from Aclair, Inc., San Diego, California) can be used to convert a peptide sequence into a corresponding nucleotide sequence encoding the peptide. Examples of IPD108 polypeptide sequences that can be used to obtain the corresponding nucleotide-encoding sequence include, but are not limited to, the IPD108 polypeptides of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135. Furthermore, the synthetic IPD108 polynucleotide sequence of this disclosure can be engineered for expression in plants.

[0154] In some embodiments, the nucleic acid molecule encoding the IPD108 polypeptide is a polynucleotide, variants thereof, fragments, and complementary sequences having the sequences shown in SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129. As used herein, a “complementary sequence” refers to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize with said given nucleic acid sequence to form a stable duplex. As used herein, a “polynucleotide sequence variant” refers to a nucleic acid sequence that encodes the same polypeptide except for the degeneracy of the genetic code.

[0155] In some embodiments, the nucleic acid molecule encoding the IPD108 polypeptide is a non-genomic nucleic acid sequence. As used herein, “non-genomic nucleic acid sequence” or “non-genomic nucleic acid molecule” or “non-genomic polynucleotide” refers to a nucleic acid molecule having one or more variations in its nucleic acid sequence compared to a natural or genomic nucleic acid sequence. In some embodiments, the alterations to the natural or genomic nucleic acid molecule include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence by introducing at least one amino acid substitution, insertion, deletion, and / or addition compared to the natural 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 associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of 5′ and / or 3′ untranslated regions associated with the genomic nucleic acid sequence; insertion of heterologous 5′ and / or 3′ untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is cDNA.

[0156] In some embodiments, the nucleic acid molecule encoding the IPD108 polypeptide is a non-genomic polynucleotide having the following nucleotide sequence, wherein the nucleotide sequence is consistent with SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 128. The nucleic acid sequence of NO:129 has 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 greater identity, wherein the IPD108 polypeptide has insecticidal activity.

[0157] In some embodiments, the nucleic acid molecule encodes an IPD108 polypeptide comprising the amino acid sequences of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135, compared to the amino acid sequences of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135. The natural amino acid at the corresponding position of NO: 135, wherein the amino acid sequence has 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 or more amino acid substitutions, deletions and / or insertions.

[0158] Nucleic acid molecules encoding transcription and / or translation products are also provided, which are subsequently spliced ​​to ultimately produce a functional IPD108 polypeptide. Splicing can be performed in vitro or in vivo and can involve cis or trans splicing. The substrate for splicing can be a polynucleotide (e.g., RNA transcript) or a polypeptide. An example of cis splicing of a polynucleotide is the removal of introns inserted into the coding sequence and splicing two flanking exon regions to produce the IPD108 polypeptide coding sequence. An example of trans splicing is the encryption of the polynucleotide by separating the coding sequence into two or more fragments, which can be transcribed individually and then spliced ​​to form a full-length pest-killing coding sequence. Cis or trans splicing of the polypeptide can be facilitated using splicing enhancer sequences that can be introduced into the construct (US Patent Nos. 6,365,377 and 6,531,316). Therefore, in some embodiments, the polynucleotide does not directly encode the full-length IPD108 polypeptide, but rather encodes one or more fragments of the IPD108 polypeptide. These polynucleotides can be used to express functional IPD108 peptides via mechanisms involving splicing, where splicing can occur at the level of polynucleotides (e.g., introns / exons) and / or peptides (e.g., intepids / exons). This can be used, for example, to control the expression of pest-killing activity, because if all the necessary fragments are expressed in an environment that allows the splicing process to produce a functional product, only the functional pest-killing peptide is expressed. In another example, introducing one or more insert sequences into a polynucleotide can facilitate recombination with low-homology polynucleotides; the use of introns or intepids targeting the insert sequences facilitates the removal of the intercalation sequences, thereby restoring the function of the encoded variant.

[0159] Nucleic acid molecules that are fragments of these nucleic acid sequences encoding the IPD108 polypeptide are also covered in the examples. As used herein, “fragment” refers to a portion of the nucleic acid sequence encoding the IPD108 polypeptide. The fragment of the nucleic acid sequence may encode the biologically active portion of the IPD108 polypeptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below. The nucleic acid molecule that is a fragment of the nucleic acid sequence encoding the IPD108 polypeptide contains at least about 150, 180, 210, 240, 270, 300, 330, or 360 consecutive nucleotides, or up to the number of nucleotides present in the full-length nucleic acid sequence encoding the IPD108 polypeptide disclosed herein, depending on the intended use. As used herein, “consecutive nucleotides” refers to nucleotide residues that are adjacent to each other. The nucleic acid sequence fragments of the examples will encode protein fragments that retain the biological activity of the IPD108 polypeptide and thus retain its insecticidal activity. As used herein, "retained insecticidal activity" refers to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95%, or higher insecticidal activity of the full-length IPD1 08Aa polypeptide (SEQ ID NO: 131). In some embodiments, the insecticidal activity is targeted at Lepidoptera species. In one embodiment, the insecticidal activity is targeted at Coleoptera species.

[0160] In some embodiments, the IPD108 polypeptide is encoded by a nucleic acid sequence that is sufficiently homologous to the nucleic acid sequences of SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, or SEQ ID NO: 129. As used herein, “sufficient homology” means an amino acid or nucleic acid sequence that, using standard parameters and one of the alignment procedures described herein, has 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 to a reference sequence. Those skilled in the art will recognize that these values ​​can be appropriately adjusted by taking into account degeneracy, amino acid similarity, reading frame localization, etc., to determine the corresponding homology of proteins encoded by the two nucleic acid sequences. In some embodiments, the sequence homology refers to the full-length sequence of the polynucleotide encoding the IPD108 polypeptide or to the full-length sequence of the IPD108 polypeptide.

[0161] In some embodiments, the nucleic acid encodes the following IPD108 polypeptide: the IPD108 polypeptide has 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 identity with SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, or SEQ ID NO: 135.

[0162] In some embodiments, the ClustaIW algorithm is used in Vector Program Suite (Ingenium Inc., Carlsbad, California) The sequence identity is calculated in the module with all default parameters. In some embodiments, the ClustalW algorithm is used to calculate sequence identity across the full-length peptide in the ALIGNX module of the Vector NTI program suite (Ingenieur, Carlsbad, CA) with all default parameters.

[0163] To determine the percentage of identity between two or more amino acid sequences or two or more nucleic acid sequences, sequences are aligned for optimal comparison purposes. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of identity = number of identical positions / total number of positions (e.g., overlapping positions × 100)).

[0164] In one embodiment, the two sequences have the same length. In another embodiment, the comparison is performed across the entire reference sequence (e.g., across the entire SEQ ID NO: 131). Techniques similar to those described below can be used to determine the percentage identity between the two sequences, with or without gaps. In calculating percentage identity, exact matches are typically counted.

[0165] Another non-limiting example of a mathematical algorithm for comparing sequences is the one described in the following literature: Needleman and Wunsch, (1970) J. Mol. Biol. [Journal of Molecular Biology] 48(3): 443-453, using GAP version 10 software to determine sequence identity or similarity with the following default parameters: for identity % and similarity % of nucleic acid sequences, a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmpii scoring matrix; for identity % or similarity % of amino acid sequences, a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring procedure. Equivalent procedures may also be used. As used herein, “equivalent procedure” means any sequence comparison procedure that produces an alignment for any two sequences in question that, when compared with the corresponding alignment produced by GAP version 10, has the same nucleotide residue pairings and the same percentage of sequence identity.

[0166] In some embodiments, the IPD108 polynucleotide encodes an IPD108 polypeptide comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity with the entire length of the amino acid sequence throughout SEQ ID NO: 131.

[0167] In some embodiments, a polynucleotide encoding a chimeric polypeptide is provided, the chimeric polypeptide comprising regions of at least two different IPD108 polypeptides disclosed herein.

[0168] In some embodiments, a polynucleotide encoding a chimeric polypeptide is provided, the chimeric polypeptide comprising a region selected from at least two different IPD108 polypeptides: SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO: 135.

[0169] In some embodiments, the IPD108 polynucleotide encodes an IPD108 polypeptide comprising the amino acid sequence of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, or SEQ ID NO: 135.

[0170] Polynucleotide encoding IPD109 polypeptide

[0171] One source of the polynucleotide encoding the IPD109 polypeptide or related protein is a fern or other primitive plant species selected from, but not limited to, *Selaginella* species, containing the IPD109 polynucleotide of SEQ ID NO: 137 encoding the IPD109 polypeptide of SEQ ID NO: 138. The polynucleotide of SEQ ID NO: 137 can be used to express the IPD109 polypeptide in recombinant bacterial hosts (including but not limited to *Agrobacterium*, *Bacillus*, *Escherichia*, *Salmonella*, *Pseudomonas*, and *Rhizobium* bacterial host cells). The polynucleotide can also be used as a probe for isolating homologous or substantially homologous polynucleotides encoding the IPD109 polypeptide or related protein. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from ferns or other primitive plant species selected from, but not limited to, *Selaginella* species.

[0172] The polynucleotide encoding the IPD109 polypeptide can also be synthesized de novo from the IPD109 polypeptide sequence. The sequence of the polynucleotide gene can be deduced from the IPD109 polypeptide sequence using the genetic code. Computer programs such as "BackTranslate" (GCG) can be used to deduce this. TM The IPD109 polypeptide sequence (from Aclair, Inc., San Diego, California) can be used to convert a peptide sequence into a corresponding nucleotide sequence encoding the peptide. Examples of IPD109 polypeptide sequences that can be used to obtain the corresponding nucleotide-encoding sequence include, but are not limited to, the IPD109 polypeptide of SEQ ID NO: 137. Furthermore, the synthetic IPD109 polynucleotide sequence of this disclosure can be engineered for expression in plants.

[0173] In some embodiments, the nucleic acid molecule encoding the IPD109 polypeptide is a polynucleotide having the sequence shown in SEQ ID NO: 137, and variants, fragments, and complementary sequences thereof. As used herein, a “complementary sequence” refers to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize with said given nucleic acid sequence to form a stable double strand. As used herein, a “polynucleotide sequence variant” refers to a nucleic acid sequence that encodes the same polypeptide except for the degeneracy of the genetic code.

[0174] In some embodiments, the nucleic acid molecule encoding the IPD109 polypeptide is a non-genomic nucleic acid sequence. As used herein, “non-genomic nucleic acid sequence” or “non-genomic nucleic acid molecule” or “non-genomic polynucleotide” refers to a nucleic acid molecule having one or more variations in its nucleic acid sequence compared to a natural or genomic nucleic acid sequence. In some embodiments, the alterations to the natural or genomic nucleic acid molecule include, but are not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; optimization of the nucleic acid sequence for expression in plants; changes in the nucleic acid sequence by introducing at least one amino acid substitution, insertion, deletion, and / or addition compared to the natural 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 associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of 5′ and / or 3′ untranslated regions associated with the genomic nucleic acid sequence; insertion of heterologous 5′ and / or 3′ untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence. In some embodiments, the non-genomic nucleic acid molecule is cDNA.

[0175] In some embodiments, the nucleic acid molecule encoding the IPD109 polypeptide is a non-genomic polynucleotide having the following nucleotide sequence, which is consistent with SEQ ID NO: ... The nucleic acid sequence of NO: 137 has 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 greater identity, wherein the IPD109 polypeptide has insecticidal activity.

[0176] In some embodiments, the nucleic acid molecule encodes an IPD109 polypeptide comprising the amino acid sequence of SEQ ID NO: 138, wherein, compared to the native amino acid at the corresponding position in SEQ ID NO: 138, the amino acid sequence has 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 or more amino acid substitutions, deletions and / or insertions.

[0177] Nucleic acid molecules encoding transcription and / or translation products are also provided, which are subsequently spliced ​​to ultimately produce a functional IPD109 polypeptide. Splicing can be performed in vitro or in vivo and can involve cis or trans splicing. The substrate used for splicing can be a polynucleotide (e.g., RNA transcript) or a polypeptide. An example of cis splicing of a polynucleotide is removing introns inserted into the coding sequence and splicing two flanking exon regions to produce the IPD109 polypeptide coding sequence. An example of trans splicing is encrypting the polynucleotide by separating the coding sequence into two or more fragments, which can be transcribed individually and then spliced ​​to form a full-length pest-killing coding sequence. Cis or trans splicing of the polypeptide can be facilitated using splicing enhancer sequences that can be introduced into the construct (US Patent Nos. 6,365,377 and 6,531,316). Therefore, in some embodiments, the polynucleotide does not directly encode the full-length IPD109 polypeptide, but rather encodes one or more fragments of the IPD109 polypeptide. These polynucleotides can be used to express functional IPD109 peptides via mechanisms involving splicing, where splicing can occur at the level of polynucleotides (e.g., introns / exons) and / or peptides (e.g., intepids / exons). This can be used, for example, to control the expression of pest-killing activity, because if all the necessary fragments are expressed in an environment that allows the splicing process to produce a functional product, only the functional pest-killing peptide is expressed. In another example, introducing one or more insert sequences into a polynucleotide can facilitate recombination with low-homology polynucleotides; the use of introns or intepids targeting the insert sequences facilitates the removal of the intercalation sequences, thereby restoring the function of the encoded variant.

[0178] Nucleic acid molecules that are fragments of these nucleic acid sequences encoding the IPD109 polypeptide are also included in the examples. As used herein, “fragment” refers to a portion of the nucleic acid sequence encoding the IPD109 polypeptide. The fragment of the nucleic acid sequence may encode the biologically active portion of the IPD109 polypeptide, or it may be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below. The nucleic acid molecule that is a fragment of the nucleic acid sequence encoding the IPD109 polypeptide contains at least about 150, 180, 210, 240, 270, 300, 330, or 360 consecutive nucleotides, or up to the number of nucleotides present in the full-length nucleic acid sequence encoding the IPD109 polypeptide disclosed herein, depending on the intended use. As used herein, “consecutive nucleotides” refers to nucleotide residues that are adjacent to each other. The nucleic acid sequence fragments of the examples will encode protein fragments that retain the biological activity of the IPD109 polypeptide and thus retain its insecticidal activity. As used herein, "retained insecticidal activity" refers to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95%, or higher insecticidal activity of the full-length IPD109Aa polypeptide (SEQ ID NO: 138). In some embodiments, the insecticidal activity is targeted at Lepidoptera species. In one embodiment, the insecticidal activity is targeted at Coleoptera species.

[0179] In some embodiments, the IPD109 polypeptide is encoded by a nucleic acid sequence that is sufficiently homologous to the nucleic acid sequence of SEQ ID NO: 137. As used herein, “sufficient homology” means an amino acid or nucleic acid sequence that, using standard parameters and one of the alignment procedures described herein, has 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 with a reference sequence. Those skilled in the art will recognize that these values ​​can be appropriately adjusted by taking into account degeneracy, amino acid similarity, reading frame localization, etc., to determine the corresponding homology of proteins encoded by the two nucleic acid sequences. In some embodiments, the sequence homology refers to the full-length sequence of the polynucleotide encoding the IPD109 polypeptide or to the full-length sequence of the IPD109 polypeptide.

[0180] In some embodiments, the nucleic acid encodes the following IPD109 polypeptide: the IPD109 polypeptide has 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 identity with SEQ ID NO: 138.

[0181] In some embodiments, the ClustalW algorithm is used in Vector Program Suite (Ingenium Inc., Carlsbad, California) The sequence identity is calculated in the module with all default parameters. In some embodiments, the ClustalW algorithm is used to calculate sequence identity across the full-length peptide in the ALIGNX module of the Vector NTI program suite (Ingenieur, Carlsbad, CA) with all default parameters.

[0182] To determine the percentage of identity between two or more amino acid sequences or two or more nucleic acid sequences, sequences are aligned for optimal comparison purposes. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of identity = number of identical positions / total number of positions (e.g., overlapping positions × 100)).

[0183] In another embodiment, the comparison is performed across the entire reference sequence (e.g., across the entire SEQ ID NO: 138). Techniques similar to those described below can be used to determine the percentage identity between two sequences, with or without gaps. In calculating percentage identity, exact matches are typically counted.

[0184] Another non-limiting example of a mathematical algorithm for comparing sequences is the one described in the following literature: Needleman and Wunsch, (1970) J. Mol. Biol. [Journal of Molecular Biology] 48(3): 443-453, using GAP version 10 software to determine sequence identity or similarity with the following default parameters: for identity % and similarity % of nucleic acid sequences, a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmpii scoring matrix; for identity % or similarity % of amino acid sequences, a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring procedure. Equivalent procedures may also be used. As used herein, “equivalent procedure” means any sequence comparison procedure that produces an alignment for any two sequences in question that, when compared with the corresponding alignment produced by GAP version 10, has the same nucleotide residue pairings and the same percentage of sequence identity.

[0185] In some embodiments, the IPD109 polynucleotide encodes an IPD109 polypeptide comprising an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity with the entire length of the amino acid sequence throughout SEQ ID NO: 138.

[0186] In some embodiments, a polynucleotide encoding a chimeric polypeptide is provided, the chimeric polypeptide comprising regions of at least two different IPD109 polypeptides disclosed herein.

[0187] In some embodiments, the IPD109 polynucleotide encodes an IPD109 polypeptide comprising the amino acid sequence of SEQ ID NO: 138.

[0188] The embodiments also cover nucleic acid molecules encoding variants of the polypeptides of this disclosure. "Variants" of the polypeptides of this disclosure encoding nucleic acid sequences include those sequences encoding the polypeptides of this disclosure but exhibiting conserved differences due to the degeneracy of the genetic code, as well as those 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, which, for example, are generated by directed mutagenesis but still encode the disclosed polypeptides discussed below.

[0189] Those skilled in the art will further understand that changes can be introduced through mutations in the nucleotide sequence, resulting in variations in the amino acid sequence of the encoded polypeptide of this disclosure without altering the biological activity of the protein. Therefore, variant nucleic acid molecules can be generated by introducing one or more nucleotide substitutions, additions, and / or deletions into the corresponding nucleic acid sequences disclosed herein, thereby introducing one or more amino acid substitutions, additions, or deletions into the encoded protein. Mutations can be introduced using standard techniques, such as directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also covered by this disclosure.

[0190] Alternatively, variant nucleic acid sequences can be prepared by randomly introducing mutations along all or part of the coding sequence (e.g., through saturation mutagenesis), and the resulting mutants can be screened to confer pest-killing activity to identify the ability of mutants that retain activity. Following mutagenesis, the encoded protein can be recombinantly expressed, and the activity of the protein can be determined using standard assay techniques.

[0191] The polynucleotides and fragments thereof disclosed herein may be optionally used as substrates for various recombination and recursive recombination reactions, in addition to standard cloning methods described, for example, by Ausubel, Berger, and Sambrook, i.e., to produce additional biocidal polypeptide homologs and fragments thereof with desired properties. Various such reactions are known, including those developed by the inventors and their colleagues. Methods for producing variants of any of the nucleic acids listed herein (methods involving recursively recombinating such polynucleotides with a second (or more) polynucleotide to form a variant polynucleotide library) are also embodiments of this disclosure, as are the resulting libraries, the cells containing said libraries, and any recombinant polynucleotides produced by such methods. Additionally, such methods may optionally include selecting variant polynucleotides from such libraries based on biocidal activity, as in which such recursive recombination is performed in vitro or in vivo.

[0192] Various diversity generation schemes (including nucleic acid recursive recombination schemes) are available and well described in the art. These procedures can be used alone and / or in combination to generate one or more variants of nucleic acids or nucleic acid assemblies, as well as variants of the encoded proteins. Individually or holistically, these procedures provide robust and widely applicable methods for generating diverse nucleic acids and nucleic acid assemblies (including, for example, nucleic acid libraries), which can be used, for example, for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells, and / or organisms with novel and / or improved characteristics.

[0193] Although distinctions and classifications have been made in the subsequent discussion for clarity, it should be understood that the techniques described are generally not mutually exclusive. In fact, various methods can be used individually or in combination, in parallel or in series, to obtain different sequence variants.

[0194] The result of any diversity generation procedure described herein may be the production of one or more nucleic acids, which can select or screen nucleic acids having or conferring desired properties or nucleic acids encoding proteins having or conferring desired properties. Following diversification by one or more methods described herein or otherwise available to a person skilled in the art, any of the produced nucleic acids can be selected for desired activity or property (e.g., pest-killing activity) or such activity at a desired pH. This may include the identification by any assay in the art that can, for example, be detected in an automated or automatable manner, see, for example, the discussion of insecticidal activity screening below. Various related (or even unrelated) properties may be evaluated in series or in parallel by the practitioner at their discretion.

[0195] Descriptions of various diversity-generating procedures for producing modified nucleic acid sequences (e.g., those encoding polypeptides or fragments thereof with cytotoxic biological activity) can be found in the following publications and references cited therein: Soong et al., (2000) Nat Genet 25(4): 436-439; Stemmer et al., (1999) Tumor Targeting 4: 1-4; 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 [Contemporary Views in Biochemistry] 3: 284-290; Christians et al., (1999) Nat Biotechnol [Nature Biotechnology] 17: 259-264; Crameri et al., (1998) Nature [Nature] 391: 288-291; Crameri et al., (1997) Nat Biotechnol [Nature Biotechnology] 15: 436-438; Zhang et al., (1997) PNASUSA [Proceedings of the National Academy of Sciences of the United States of America] 94: 4504-4509; Patten et al., (1997) Curr Opin Biotechnol [Contemporary Views in Biotechnology] 8: 724-733; Crameri et al., (1996) Nat Med [Nature Medicine] 2: 100-103; Crameri et al., (1996) Nat Biotechnol [Nature Biotechnology] 14: 315-319; Gates et al., (1996) J Mol Biol [Journal of Molecular Biology] 255: 373-386; Stemmer, (1996) "Sexual PCR and Assembly PCR" in: The Encyclopedia of Molecular Biology.[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.

[0196] Mutation methods that generate diversity include, for example, directed mutagenesis (Ling et al., (1997) Anal Biochem 254(2): 157-178; Dale et al., (1996) Methods Mol Biol 57: 369-374; Smith, (1985) Ann 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 (edited by Eckstein and Lilley, Springer). Verlag (Springer Publishing Company, Berlin); Mutagenesis using templates containing uracil (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 [Nucleic Acid Research] 10: 6487-6500); DNA mutagenesis modified with thiophosphate (Taylor et al., (1985) Nucleic Acids Res [Nucleic Acid Research] 13: 8749-8764; Taylor et al., (1985) Nucleic Acids Res [Nucleic Acid Research] 13: 8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucleic Acids Res [Nucleic Acid Research] 14: 9679-9698;Sayers et al., (1988) Nucl Acids Res [Nucleic Acid Research] 16: 791-802 and Sayers et al., (1988) Nucl Acids Res [Nucleic Acid Research] 16: 803-814); mutagenesis using nicked double-stranded DNA (Kramer et al., (1984) Nucl Acids Res [Nucleic Acid Research] 12: 9441-9456; Kramer and Fritz, (1987) Methods Enzymol [Enzymatic Methods] 154: 350-367; Kramer et al., (1988) Nucl Acids Res [Nucleic Acid Research] 16: 7207 and Fritz et al., (1988) Nucl Acids Res [Nucleic Acid Research] 16: 6987-6999). Other suitable methods include point mismatch repair (Kramer et al., (1984) Cell 38: 879-887), mutagenesis using host strains with repair defects (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 [Philosophical Transactions of the Royal Society of London Series A] 317: 415-423), mutagenesis through whole-genome synthesis (Nambiar et al., (1984) Science 223: 1299-1301; Sakamar and Khorana, (1988) Nucle Acids Research 14: 6361-6372; Wells et al., (1985) Gene 34: 315-323 and; (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). Further details of many of the above methods can be found in Methods Enzymol, Vol. 154, which also describes useful controls for troubleshooting using various mutagenesis methods.

[0197] Further details regarding the various methods of generating diversity can be found in the following U.S. patents, PCT publications and applications, and EPO publications: U.S. Patent No. 5,723,323, U.S. Patent No. 5,763,192, U.S. Patent No. 5,814,476, U.S. Patent No. 5,817,483, U.S. Patent No. 5,824,514, U.S. Patent No. 5,976,862, U.S. Patent No. 5,605,793, U.S. Patent No. 5,811,238, U.S. Patent No. 5,830,721, U.S. Patent No. 5,834,252, U.S. Patent No. 5,837,458, WO 1995 / 22625, WO 1996 / 33207, WO 1997 / 20078, WO 1997 / 35966, WO 1999 / 41402, WO 1999 / 41383, WO 1999 / 41369、WO 1999 / 41368、EP 752008、EP 0932670、WO 1999 / 23107、WO 1999 / 21979、WO 1998 / 31837、WO 1998 / 27230、WO 1998 / 27230、WO 2000 / 00632、WO 2000 / 09679、WO 1998 / 42832、WO 1999 / 29902、WO 1998 / 41653、WO 1998 / 41622、WO1998 / 42727、WO 2000 / 18906、WO 2000 / 04190、WO 2000 / 42561、WO 2000 / 42559, WO 2000 / 42560, WO 2001 / 23401, and PCT / US01 / 06775.

[0198] The nucleotide sequences of the examples can also be used to isolate corresponding sequences from ferns or other primitive plants. In this way, such sequences can be identified using methods such as PCR, hybridization, etc. (based on their sequence homology with the sequences shown herein). The examples cover sequences selected based on sequence identity with all or fragments of the sequences shown herein. Such sequences include sequences that are orthologs of publicly disclosed sequences. The term "ortholog" refers to a gene that originates from a common ancestral gene and is found in different species due to speciation. Genes found in different species are considered orthologs when their nucleotide sequences and / or the protein sequences they encode share a basic identity as defined elsewhere herein. The function of orthologs is generally highly conserved among species.

[0199] In PCR methods, oligonucleotide primers can be designed for PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any target organism. Methods for designing PCR primers and for PCR cloning are generally known in the art and are disclosed in Sambrook et al., (1989) *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter referred to as “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, and partially mismatched primers.

[0200] To identify potential insecticidal peptides from ferns or other primitive plants, Western blotting and / or ELISA methods can be used to screen cell lysates of the ferns or other primitive plants with antibodies generated against the peptides disclosed herein. This type of assay can be performed in a high-throughput manner. Positive samples can be further analyzed using various techniques, such as antibody-based protein purification and identification. Methods for generating antibodies are well known in the art and are discussed below.

[0201] Alternatively, mass spectrometry-based protein identification methods can be used with protocols in the literature for identifying homologs of the peptides disclosed herein (Scott Patterson, (1998), 10.22, 1-24, Current Protocol in Molecular Biology, John Wiley & Son Inc). Specifically, MS data from a given cell lysate or a sample enriched to a desired molecular weight (excised from an SDS-PAGE gel of the relevant molecular weight band) are combined with sequence information of the peptides and their homologs disclosed herein using LC-MS / MS-based protein identification methods. Any match in the peptide sequence indicates the possibility of a homologous protein in the sample. Additional techniques (protein purification and molecular biology) can be used to isolate the protein and identify the sequences of the homologs.

[0202] In hybridization methods, all or part of the harmful organism-killing nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for constructing such cDNA and genomic libraries are generally known in the art and disclosed in Sambrook and Russell, (2001), ibid. Hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides and can be labeled with a detectable group (such as 32P or any other detectable label, such as other radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors). Probes used for hybridization can be prepared by labeling synthetic oligonucleotides based on nucleic acid sequences encoding polypeptides disclosed herein. Degenerate primers, designed based on conserved nucleotides or amino acid residues in the nucleic acid sequence or the encoded amino acid sequence, can also be used. The probe typically comprises a region of a nucleic acid sequence that, under stringent conditions, hybridizes to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, or 200 consecutive nucleotides of a nucleic acid sequence or fragment or variant thereof encoding the disclosed IPD059, IPD098, IPD108, or IPD109 polypeptide. Methods for preparing the probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, (2001), ibid., which is incorporated herein by reference.

[0203] For example, the complete nucleic acid sequence encoding the polypeptide disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing with the corresponding nucleic acid sequence encoding the polypeptide sequence disclosed herein and the messenger RNA. To achieve specific hybridization under different conditions, such probes include sequences that are unique and preferably at least about 10 nucleotides in length, or at least about 20 nucleotides in length. Such probes can be used to amplify the corresponding pest-killing sequence 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 plated DNA libraries (patches or colonies; see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, NY).

[0204] Hybridization of such sequences can be performed under stringent conditions. As used herein, “stringent conditions” or “stringent hybridization conditions” refer to conditions under which the probe hybridizes to its target sequence to a detectably higher degree than it hybridizes to other sequences (e.g., at least 2 times higher than background). Stringent conditions are sequence-dependent and will vary under different conditions. By controlling the stringency of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can be adjusted to allow for some mismatches in the sequences in order to detect a lower degree of similarity (heterologous detection). Typically, the probe length is less than about 1000 nucleotides, preferably less than 500 nucleotides.

[0205] Composition

[0206] It also includes compositions comprising at least one IPD059, IPD098, IPD108, or IPD109 polypeptide of the present disclosure, or a chimeric polypeptide comprising IPD059, IPD098, IPD108, or IPD109. In some embodiments, the composition comprises an agricultural carrier containing at least one IPD059, IPD098, IPD108, or IPD109 polypeptide of the present disclosure.

[0207] Antibody

[0208] This also covers antibodies against the IPD059, IPD098, IPD108, or IPD109 polypeptides or variants or fragments thereof found in the embodiments. The antibodies of this disclosure include polyclonal and monoclonal antibodies and fragments thereof that retain their ability to bind to the IPD059, IPD098, IPD108, or IPD109 polypeptides found in the insect gut. It is believed that antibodies, monoclonal antibodies, or fragments thereof can bind to molecules provided that the antibody, monoclonal antibody, or fragment thereof can specifically react with the molecule to bind the molecule to the antibody, monoclonal antibody, or fragment thereof. The terms “antibody” (Ab) or “monoclonal antibody” (Mab) are intended to include complete molecules and fragments thereof or binding regions or domains (e.g., like Fab and F(ab).sub.2 fragments) capable of binding haptens. Such fragments are typically produced by proteolytic cleavage (e.g., papain or pepsin). Alternatively, hapten-binding fragments can be produced by applying recombinant DNA technology or by synthetic chemistry. Methods for preparing the antibodies of this disclosure are generally known in the art. For example, see Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory, NY (1988), and the references cited therein. Standard references outlining the general principles of immunology include: Klein, J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley & Son, New York (1982); Dennett et al., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (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 U.S. Patent Nos. 4,196,265; 4,609,893; 4,713,325; 4,714,681; 4,716,111; 4,716,117 and 4,720,459. Antibodies against IPD059, IPD098, IPD108 or IPD109 peptides or their antigen-binding moieties can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique described in Kohler and Milstein, (1975) Nature 256:495. Other techniques for producing monoclonal antibodies can also be used, such as viral or oncogenic transformation of B lymphocytes. The animal system used for preparing hybridomas is the mouse system. Immunoimmunization protocols and techniques for isolating spleen cells for fusion are known in the art. Fusion couples (e.g., mouse myeloma cells) and fusion methods are also known. The antibodies and monoclonal antibodies disclosed herein can be prepared using IPD059, IPD098, IPD108 or IPD109 peptides as antigens.

[0209] A kit is provided for detecting the presence of peptides IPD059, IPD098, IPD108, or IPD109 in a sample, or for detecting the presence of nucleotide sequences encoding peptides IPD059, IPD098, IPD108, or IPD109. In one embodiment, the kit provides antibody-based reagents for detecting the presence of peptides IPD059, IPD098, IPD108, or IPD109 in a tissue sample. In another embodiment, the kit provides labeled nucleic acid probes for detecting the presence of one or more polynucleotides encoding peptides IPD059, IPD098, IPD108, or IPD109. The kit is provided with appropriate reagents and controls for performing the detection method, and instructions for use of the kit.

[0210] Receptor identification and isolation

[0211] Receptors for the IPD059, IPD098, IPD108, or IPD109 peptides or variants or fragments thereof, as described in the embodiments, are also covered. Methods for identifying the receptors are well known in the art (see, Hofmann et al., (1988) Eur. J. Biochem. [European Journal of Biochemistry] 173: 85-91; Gill et al., (1995) J. Biol. Chem. [Journal of Biochemistry] 27277-27282). Receptors recognizing the IPD059, IPD098, IPD108, or IPD109 peptides can be identified and isolated using brush border membrane vesicles from susceptible insects. In addition to the radiolabeling methods listed in the cited literature, the IPD059, IPD098, IPD108, or IPD109 peptides can be labeled with fluorescent dyes and other common markers such as streptavidin. Brush border membrane vesicles (BBMVs) of susceptible insects (such as soybean borer and stink bug) can be prepared according to the protocols listed in the references, separated on SDS-PAGE gels, and imprinted on suitable membranes. Labeled IPD059, IPD098, IPD108, or IPD109 peptides can be incubated with the imprinted BBMV membrane, and the labeled IPD059, IPD098, IPD108, or IPD109 peptides can be identified using labeled reporter genes. Identification of one or more protein bands interacting with IPD059, IPD098, IPD108, or IPD109 peptides can be performed using N-terminal amino acid-based gas chromatography sequencing or mass spectrometry-based protein identification methods (Patterson, (1998) 10.22, 1-24, Current Protocol in Molecular Biology, John Willie & Son). Once the protein is identified, the corresponding gene can be cloned from the genomic DNA or cDNA library of susceptible insects, and the binding affinity can be directly measured using IPD059, IPD098, IPD108, or IPD109 peptides. The receptor function of the insecticidal activity of IPD059, IPD098, IPD108, or IPD109 peptides can be verified by RNAi-type gene knockout (Rajagopal et al., (2002) J. Biol. Chem., 277: 46849-46851).

[0212] Nucleotide constructs, expression cassettes, and vectors

[0213] The use of the term "nucleotide construct" herein is not intended to limit the embodiments to nucleotide constructs containing DNA. Those skilled in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides, as well as combinations of ribonucleotides and deoxyribonucleotides, can also be used in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments further encompass all complementary forms of such constructs, molecules, and sequences. Furthermore, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences that can be used in the plant transformation methods of the embodiments, including but not limited to those composed of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. 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-loop structures, etc.

[0214] Other embodiments involve transformed organisms, such as those selected from plant and insect cells, bacteria, yeast, baculoviruses, protozoa, nematodes, and algae. The transformed organism comprises: the DNA molecule of the embodiments, an expression cassette containing the DNA molecule, or a vector containing the expression cassette, which can be stably incorporated into the genome of the transformed organism.

[0215] The DNA construct provides sequences of the embodiments for expression in a target organism. The construct will include regulatory sequences at the 5′ and 3′ angles operably linked to the sequences of the embodiments. As used herein, the term “operably linked” refers to a functional link between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. Typically, operably linked means that the linked nucleic acid sequences are contiguous and, if necessary, link two protein-coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be co-transformed into an organism. Alternatively, one or more additional genes may be provided on multiple DNA constructs.

[0216] The provided DNA construct has multiple restriction sites for inserting the IPD059, IPD098, IPD108, or IPD109 polypeptide gene sequence of this disclosure into a transcriptionally regulated region. The DNA construct may additionally contain a selective marker gene.

[0217] Following a transcriptional direction from 5′ to 3′, the DNA construct will typically include: a transcription and translation initiation region (i.e., a promoter), the DNA sequence of the embodiment, and a transcription and translation termination region (i.e., a termination region) that functions in the organism used as a host. The transcription initiation region (i.e., the promoter) may be natural, similar, exogenous, or heterologous, depending on the host organism and / or sequence of the embodiment. Furthermore, the promoter may be a natural sequence, or alternatively, a synthetic sequence. As used herein, the term "exogenous" means that the promoter is not found in the natural organism in which it is introduced. In the case where the promoter is "exogenous" or "heterogeneous" with respect to the sequence of the embodiment, it means that the promoter is not natural or naturally occurring with respect to the operably linked sequence of the embodiment. As used herein, chimeric genes contain a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. When the promoter is a natural sequence, the expression of the operably linked sequence changes from wild-type expression, resulting in a phenotypic alteration.

[0218] In some embodiments, the DNA construct comprises a polynucleotide encoding a polypeptide of the embodiment, such as IPD059, IPD098, IPD108, or IPD109.

[0219] In some embodiments, the DNA construct comprises: a polynucleotide encoding SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 7 ... ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQID NO:78 contains an IPD059 polypeptide with 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 greater sequence identity; and a heterologous regulatory element.

[0220] In some embodiments, the DNA construct comprises: a polynucleotide encoding SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, 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, or SEQ ID NO: 104. NO:124 contains an IPD098 polypeptide with 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 greater sequence identity; and a heterologous regulatory element.

[0221] In some embodiments, the DNA construct comprises: a polynucleotide encoding SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 107. NO:117 contains an IPD098 polypeptide with 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 greater sequence identity; and a heterologous regulatory element.

[0222] In some embodiments, the DNA construct comprises: a polynucleotide encoding SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, or SEQ ID NO: 136. NO:136 has an IPD108 polypeptide with 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 greater sequence identity; and a heterologous regulatory element.

[0223] In some embodiments, the DNA construct comprises: a polynucleotide encoding SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, or SEQ ID NO: 135. NO:135 contains an IPD108 polypeptide with 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 greater sequence identity; and a heterologous regulatory element.

[0224] In some embodiments, the DNA construct comprises: a polynucleotide encoding an IPD109 polypeptide having 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 greater sequence identity with SEQ ID NO: 138; and a heterologous regulatory element.

[0225] In some embodiments, the DNA construct comprises a polynucleotide encoding a chimeric polypeptide of the embodiment, such as IPD059, IPD098, IPD108, or IPD109.

[0226] In some embodiments, the DNA construct comprises a polynucleotide encoding a fusion protein containing a polypeptide of the embodiment, such as IPD059, IPD098, IPD108, or IPD109.

[0227] In some embodiments, the DNA construct comprises: a polynucleotide encoding SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, or SEQ ID NO: 124. IDNO:124 is a polypeptide having 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 greater sequence identity; and a heterologous regulatory element.

[0228] In some embodiments, the DNA construct comprises: a polynucleotide encoding a polypeptide having 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 greater sequence identity with SEQ ID NO: 136; and a heterologous regulatory element.

[0229] In some embodiments, the DNA construct comprises: a polynucleotide encoding a polypeptide having 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 greater sequence identity with SEQ ID NO: 136; and a heterologous regulatory element.

[0230] In some embodiments, the DNA construct may further include a transcriptional enhancer sequence. As used herein, the term "enhancer" refers to a DNA sequence that can stimulate promoter activity and may be an innate or heterologous element of a promoter inserted to enhance promoter level or tissue specificity. Various enhancers are known in the art, including, for example, introns that have gene expression-enhancing properties in plants (US Patent Application Publication No. 2009 / 0144863), ubiquitin introns (i.e., corn ubiquitin intron 1 (see, for example, NCBI sequence S94464)), ω enhancers or ω major enhancers (Gallie et al., (1989) Molecular Biology of RNA, ed.: Cech (Liss, NY) 237-256 and Gallie et al., (1987) Gene 60: 217-25), CaMV 35S enhancers (see, for example, Benfey et al., (1990) EMBO J. 9: 1685-96) and enhancers also available in US Patent No. 7,803,992, each of which is incorporated herein by reference. The above list of transcriptional enhancers is not intended to be restrictive. Any appropriate transcription enhancer may be used in the examples.

[0231] The termination region may be natural for the transcription start region, natural for the target DNA sequence to which it is operatively linked, natural for the plant host, or derived from another source (i.e., exogenous or heterologous to the promoter, target sequence, plant host, or any combination thereof).

[0232] Convenient termination regions can be obtained from Ti plasmids of Agrobacterium tumefaciens, such as the termination regions of octopaline synthase and carmine synthase. See also Guerineau et al., (1991) Mol Gen. Genet. [Molecular Genetics and General Genetics] 262: 141-144; Proudfoot, (1991) Cell [Cell] 64: 671-674; Sanfacon et al., (1991) Genes Dev. [Genes and Development] 5: 141-149; Mogen et al., (1990) Plant Cell [Plant Cell] 2: 1261-1272; Munroe et al., (1990) Gene [Genes] 91: 151-158; Ballas et al., (1989) Nucleic Acids Res. [Nucleic Acid Research] 17: 7891-7903; and Joshi et al., (1987) Nucleic Acid Res. [Nucleic Acid Research] 15: 9627-9639.

[0233] Nucleic acids can be optimized to increase expression in the host organism when appropriate. Therefore, in the case of a plant as the host organism, synthetic nucleic acids can be synthesized using plant-preferred codons to improve expression. For a discussion of host-preferred use, see, for example, Campbell and Gowri, (1990) Plant Physiol. [Plant Physiology] 92: 1-11. For example, while the nucleic acid sequences of the examples can be expressed in both monocotyledonous and dicotyledonous plant species, the sequences can be modified to account for specific preferences and GC content preferences of monocotyledonous or dicotyledonous plants, as these preferences have shown differences (Murray et al. (1989) Nucleic Acids Res. [Nucleic Acid Research] 17: 477-498). Thus, corn preference for specific amino acids can be derived from known gene sequences of corn. Twenty-eight genes from corn plants are listed in Table 4 of Murray et al. (ibid.). 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., MolBio Rep. 37: 677-684, 2010, which are incorporated herein by reference. The usage table for maize (Zea maize) can also be found at kazusa.or.jp / / cgi-bin / show.cgi?species=4577, which can be accessed using the www prefix. The usage table for soybean can be found at kazusa.or.jp / / cgi-bin / show.cgi?species=3847&aa=1&style=N, which can be accessed using the www prefix.

[0234] In some embodiments, the recombinant nucleic acid molecule encoding the IPD059, IPD098, IPD108, or IPD109 polypeptide has a corn-optimized codon.

[0235] Other sequence modifications are known to enhance gene expression in a cell host. These include the removal of sequences encoding pseudopolyadenylation signals, exon-intron splicing site signals, transposon-like repeat sequences, and other well-characterized sequences that may be detrimental to gene expression. The GC content of a sequence can be adjusted to the average level for a given cell host, as calculated by referencing known genes expressed in said host cells. As used herein, the term “host cell” refers to a cell that contains a vector and supports the replication and / or expression of the expression vector. Host cells can be prokaryotic cells such as *Escherichia coli*, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a corn host cell. When possible, sequences are modified to avoid predictable hairpin secondary mRNA structures.

[0236] The expression cassette may additionally contain a 5′ leader sequence. Such leader sequences can enhance translation. Translation leader sequences are known in the art and include: microRNA leader sequences, such as the EMCV leader sequence (5′ untranslated region of encephalomyocarditis) (Elroy-Stein et al., (1989) Proc. Natl. Acad. Sci. USA, 86: 6126-6130); potato virus Y leader sequences, such as the TEV leader sequence (tobacco etch virus) (Gallie et al., (1995) Gene 165(2): 233-238), the MDMV leader sequence (corn dwarf mosaic virus), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al., (1991) Nature 353: 90-94); and untranslated leader sequences from the coat protein mRNA of alfalfa mosaic virus (AMV RNA). 4)(Jobling et al., (1987) Nature 325: 622-625); Tobacco mosaic virus leader sequence (TMV))(Gallie et al., (1989) Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader sequence (MCMV))(Lommel et al., (1991) Virology 81: 382-385). See also, Della-Cioppa et al., (1987) Plant Physiol. 84: 965-968. Such constructs may also contain a “signal sequence” or “leader sequence” to facilitate the co-translation or post-translational transport of the peptide to certain intracellular structures, such as chloroplasts (or other plastids), endoplasmic reticulum, or Golgi apparatus.

[0237] As used herein, a “signal sequence” refers to a sequence known or suspected of causing co-translational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, accompanied by some form of glycosylation. Prototoxins are typically synthesized from bacterial insecticidal toxins, which are then activated by proteolysis in the gut of the target pest (Chang, (1987) Methods Enzymol. [Enzymological Methods] 153: 507-516). In some embodiments, the signal sequence is located within the natural sequence or may be derived from the sequence of the embodiment. As used herein, the term “leader sequence” refers to any sequence that, upon translation, produces an amino acid sequence sufficient to initiate co-translational transport of the peptide chain with subcellular organelles. Thus, this includes leader sequences that target transport and / or glycosylation by entering the endoplasmic reticulum, vacuoles, plastids (including chloroplasts, mitochondria), etc. Nuclear-encoded proteins targeting the thylakoid cavity of chloroplasts have a characteristic binary transport peptide consisting of a matrix-targeting signal peptide and a cavity-targeting signal peptide. Matrix-targeting information is located at the amino-proximal portion of the transport peptide. The cavity-targeting signal peptide is located at the carboxyl-proximal portion of the transport peptide and contains all the information for targeting the cavity. Recent studies in higher plant chloroplast proteomics have made progress in identifying many nuclear-encoded cavity proteins (Kieselbach et al., FEBSLETT [European Federation of Biochemical Societies Communications] 480: 271-276, 2000; Peltier et al., Plant Cell [Plant Cell] 12: 319-341, 2000; Bricker et al., Biochim. Biophys Acta [Acta Biochimica et Biophysica Sinica] 1503: 350-356, 2001), and according to this disclosure, the cavity-targeting signal peptide of the nuclear-encoded cavity proteins may be used. Kieselbach et al., Photosynthesis Research [Photosynthesis Research] 78: 249-264, 2003 reported approximately 80 proteins from the genus *Arabidopsis*, as well as homologous proteins from spinach and peas. Specifically, Table 2 of this disclosure (which is incorporated herein by reference) discloses 85 proteins from the chloroplast lumen identified by their accession numbers (see also U.S. Patent Application Publication 2009 / 09044298). Additionally, a recently published draft version of the rice genome (Goff et al., Science 296: 92-100, 2002) is a suitable source for lumen-targeting signal peptides that can be used according to this disclosure.

[0238] Suitable chloroplast transport peptides (CTPs) known to those skilled in the art also include chimeric CTs, including but not limited to: N-terminal, central, or C-terminal domains of CTPs derived from: rice 1-deoxy-D-xylulose-5-phosphate synthase, rice superoxide dismutase, rice soluble starch synthase, rice NADP-dependent malate enzyme, rice phosphate 2-dehydro-3-deoxyheptanoate aldolase 2, rice L-ascorbate peroxidase 5, rice phosphoglucan hydrochloride 2-kinase, maize ssRUBISCO, maize β-glucosidase, maize malate dehydrogenase, and maize thioredoxin M-type (U.S. Patent Application Publication 2012 / 0304336).

[0239] The expression of IPD059, IPD098, IPD108, or IPD109 polypeptide genes targeting chloroplasts can be optimized to address the differences in usage between the plant nucleus and this organelle. In this way, target nucleic acids can be synthesized using chloroplast-preferred sequences.

[0240] In preparing expression cassettes, various DNA fragments can be manipulated to provide DNA sequences that are in the appropriate orientation and at the appropriate time within the appropriate reading frame. This can be achieved by using adapters or linkers to ligate the DNA fragments, or by employing other techniques to provide convenient restriction sites, remove redundant DNA, and eliminate restriction sites. For this purpose, in vitro mutagenesis, primer repair, restriction enzyme digestion, annealing, and substitution (e.g., conversion and transversion) can be employed.

[0241] Many promoters are available for implementing the embodiments described. Promoters can be selected based on the desired results. Nucleic acids can be combined with constitutive, tissue-biased, inducible, or other promoters for expression in a host organism. Suitable constitutive promoters for use in plant host cells include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999 / 43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al., (1985) Nature 313: 810-812); rice actin (McElroy et al., (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al., (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al., (1992) Plant Mol. Biol. [Plant Molecular Biology] 18: 675-689); pEMU (Last et al., (1991) Theor. Appl. Genet. [Theoretical and Applied Genetics] 81: 581-588); MAS (Velten et al., (1984) EMBO J. [Journal of the European Society for Molecular Biology] 3: 2723-2730); ALS promoter (US Patent No. 5,659,026), etc. Other constitutive promoters include, for example, those discussed in the following US Patent Nos.: 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611.

[0242] Depending on the desired results, gene expression from an inducible promoter may be beneficial. Of particular interest in regulating nucleotide sequences used in this embodiment for expression in plants is the wound-inducible promoter. These wound-inducible promoters can respond to damage caused by insect feeding and include the potato protease inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath. [Annals of Plant Pathology] 28: 425-449; Duan et al., (1996) Nature Biotechnology [Nature Biotechnology] 14: 494-498); wun1 and wun2, US Patent No. 5,428,148; win1 and win2 (Stanford et al., (1989) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 215: 200-208); systemin (McGurl et al., (1992) Science [Science] 225: 1570-1573); WIP1 (Rohmeier et al., (1993) Plant Mol.Biol. [Plant Molecular Biology] 22: 783-792; Eckelkamp et al., (1993) FEBS Letters [European Federation of Biochemical Societies Communications] 323: 73-76); MPI gene (Corderok et al., (1994) Plant J. [Plant Journal] 6(2): 141-150), etc. The above references are incorporated into this article by way of citation.

[0243] Furthermore, pathogen-inducible promoters can be used in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those derived from pathogenesis-associated proteins (PR proteins) that are induced upon pathogen infection; for example, PR proteins, SAR proteins, β-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al., (1983) Neth. J. Plant Pathol. [Dutch Journal of Plant Pathology] 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, which is incorporated herein by reference.

[0244] Of particular interest are promoters expressed locally at or near the site of pathogen infection. See, for example, Marineau et al., (1987) Plant Mol. Biol. [Plant Molecular Biology] 9: 335-342; Matton et al., (1989) Molecular Plant-Microbe Interactions [Molecular Plant-Microbe Interactions] 2: 325-331; Somsisch et al., (1986) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 83: 2427-2430; Somsisch et al., (1988) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 2: 93-98; and Yang, (1996) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 93: 14972-14977. See also Chen et al., (1996) Plant J. 10: 955-966; Zhang et al., (1994) Proc. Natl. Acad. Sci. USA 91: 2507-2511; Warner et al., (1993) Plant J. 3: 191-201; Siebertz et al., (1989) Plant Cell 1: 961-968; U.S. Patent No. 5,750,386 (nematode-induced type) and the references cited therein. Of particular interest is the inducible promoter of the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordera et al., (1992) Physiol. Mol. Plant Path. [Physiology and Molecular Plant Pathology] 41: 189-200).

[0245] Chemically regulated promoters can be used to regulate gene expression in plants by applying exogenous chemical regulators. Depending on the target, the promoter can be a chemically inducible promoter, in which a chemical is applied to induce gene expression, or a chemically repressive promoter, in which a chemical is applied to suppress gene expression. Chemically inducible promoters are known in the art and include, but are not limited to, the corn In2-2 promoter activated by a benzenesulfonamide herbicide safener, the corn GST promoter activated by a hydrophobic electrophilic compound used as a pre-emergence herbicide, and the tobacco PR-1a promoter activated by salicylic acid. Other targeted chemical regulatory promoters include steroid response promoters (see, for example, glucocorticoid-inducible promoters in Schena et al., (1991) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 88: 10421-10425 and McNellis et al., (1998) Plant J [Journal of Plant Science] 14(2): 247-257) and tetracycline-inducible and tetracycline-inhibiting promoters (see, for example, Gatz et al., (1991) Mol. Gen. Genet. [Molecular Genetics and Genomics] 227: 229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156), which are incorporated herein by reference.

[0246] Tissue-biased promoters can be used to target enhanced expression of IPD059, IPD098, IPD108, or IPD109 peptides in specific plant tissues. Tissue-biased promoters include those described in the following literature: Yamamoto et al., (1997) Plant J. [Plant Journal] 12(2): 255-265; Kawamata et al., (1997) Plant Cell Physiol. [Plant Cell Physiology] 38(7): 792-803; Hansen et al., (1997) Mol. Gen Genet. [Molecular and General Genetics] 254(3): 337-343; Russell et al., (1997) Transgenic Res. [Transgenic Research] 6(2): 157-168; Rinehart et al., (1996) Plant Physiol. [Plant Physiology] 112(3): 133l-1341; Van Camp et al., (1996) Plant Physiol. [Plant Physiology] 112(2): 525-535; Canevascini et al., (1996) Plant Physiol. [Plant Physiology] 112(2): 513-524; Yamamoto et al., (1994) Plant Cell Physiol. [Plant Cell Physiology] 35(5): 773-778; Lam, (1994) Results Probl. Cell Differ. [Results and Problems of Cell Differentiation] 20: 181-196; Orozco et al., (1993) Plant Mol Biol. [Plant Molecular Biology] 23(6): 1129-1138; Matsuoka et al., (1993) Proc Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90(20): 9586-9590; and Guevara-Garcia et al., (1993) Plant J. [Plant Journal] 4(3): 495-505. If necessary, such promoters can be modified for weak expression.

[0247] Leaf-biased 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.

[0248] Root-biased or root-specific promoters are known and can be selected from many available promoters from the literature or re-isolated from different compatible species. See, for example, Hire et al., (1992) PlantMol.Biol. 20(2): 207-218 (Root-specific glutamine synthase gene in soybean); Keller and Baumgartner, (1991) Plant Cell 3(10): 1051-1061 (Root-specific control element in GRP 1.8 gene of common bean); Sanger et al., (1990) Plant Mol.Biol. 14(3): 433-443 (Root-specific promoter of mannan synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al., (1991) Plant Cell 3(1): 11-22 (Full-length cDNA clone encoding cytosolic glutamine synthase (GS) 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 nitrogen-fixing non-leguminous plant Parasponia andersonii and the related non-nitrogen-fixing non-leguminous plant Trematomentosa. The promoters of these genes are linked to β-glucuronidase reporter genes and have been introduced into both the non-leguminous crop Nicotiana tabacum and the leguminous crop Lotus corniculatus, and root-specific promoter activity has been preserved in both cases. Leach and Aoyagi, (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1): 69-76). They concluded that enhancers and tissue-preferred DNA determinants are dissociated in the promoter. Teeri et al. (1989) used gene fusion with lacZ to show that Agrobacterium T-DNA genes encoding octopus alkaloid synthase are active, especially in the epidermis of the root tip, and that the TR2′ gene is root-specific in intact plants and stimulated by trauma in leaf tissues, a particularly desirable combination of features for use with insecticidal or larval-killing genes (see EMBO J. [Journal of the European Society for Molecular Biology] 8(2): 343-350).The TR1′ gene fused with nptII (neomycin phosphotransferase II) exhibits similar characteristics. Other root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol. Biol. [Plant Molecular Biology] 29(4): 759-772); and the rolB promoter (Capana et al., (1994) Plant Mol. Biol. [Plant Molecular Biology] 25(4): 681-691). See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Root-preferred regulatory sequences in Arabidopsis thaliana are disclosed in US20130117883.

[0249] “Seed-biased” promoters include seed-specific promoters (those that are active during seed development, such as promoters of seed storage proteins) and seed germination promoters (those that are active during seed germination). See Thompson et al., (1989) BioEssays 10:108, which is incorporated herein by reference. Such seed-biased promoters include, but are not limited to, Cim1 (cytokinin-induced information); cZ19B1 (19kDa zein); and milps (inositol-1-phosphate synthase) (see U.S. Patent No. 6,225,529, which is incorporated herein by reference). γ-zein and Glb-1 are endosperm-specific promoters. For dicotyledons, seed-specific promoters include, but are not limited to: Kunitz trypsin inhibitor 3 (KTi3) (Jofuku and Goldberg, (1989) PlantCell 1: 1079-1093), genistein, rapeseed protein, β-conglycinin, genistein 1, genistein lectins, cruciferous proteins, etc. For monocotyledons, seed-specific promoters include, but are not limited to: 15kDa zein, 22kDa zein, 27kDa zein, g-zein, waxes, condensation 1, condensation 2, globulin 1, etc. 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 pBAN seed coat promoter from the genus Arabidopsis; and early seed promoters from the genus Arabidopsis, p26, p63, and p63tr (US Patent Nos. 7,294,760 and 7,847,153). Promoters that exhibit “preferential” expression in a particular tissue are expressed at a higher level in that tissue than in at least one other plant tissue. Some tissue-preferential promoters are expressed almost exclusively in a specific tissue.

[0250] Weak promoters can be used when low levels of expression are required. Generally, as used herein, the term "weak promoter" refers to a promoter that drives expression of the coding sequence at low levels. Low levels of expression are defined as levels between approximately 1 / 1000 of transcripts and approximately 1 / 100,000 of transcripts and approximately 1 / 500,000 of transcripts. Alternatively, it should be recognized that the term "weak promoter" also encompasses promoters that drive expression only in a few cells but not in others, resulting in low overall expression. When a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to reduce expression levels.

[0251] Such weakly constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO1999 / 43838 and U.S. Patent No. 6,072,050), the core 35S CaMV promoter, etc. Other constitutive promoters include, for example, those disclosed in the following patent documents: U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611, which are incorporated herein by reference.

[0252] The list of promoters above is not intended to be limiting. Any suitable promoter can be used in the embodiments.

[0253] Typically, expression cassettes contain selective marker genes for selecting transformed cells. Selective marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicide compounds such as glufosinate, bromosulfuron, imidazolinone, and 2,4-dichlorophenoxyacetic acid (2,4-D). Other examples of suitable selective marker genes include, but are not limited to, genes encoding tolerance to: chloramphenicol (Herrera Estrella et al., (1983) EMBO J. [Journal of the European Society for Molecular Biology] 2: 987-992); methotrexate (Herrera Estrella et al., (1983) Nature 303: 209-213 and Meijer et al., (1991) Plant Mol. Biol. [Plant Molecular Biology] 16: 807-820); streptomycin (Jones et al., (1987) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 210: 86-91); spectinomycin (Bretagne-Sagnard et al., (1996) Transgenic Res. [Transgenic Research] 5: 131-137); bleomycin (Hille et al., (1990) Plant Mol. Biol. [Plant Molecular Biology] 7: 171-176); sulfonamides (Guerineau et al., (1990) Plant Mol. Biol. [Plant Molecular Biology] 15: 127-136); bromonazine (Stalker et al., (1988) Science 242: 419-423); glyphosate (Shaw et al., (1986) Science 233: 478-481 and US patent application serial numbers 10 / 004,357 and 10 / 427,692); glufosinate (DeBlock et al., (1987) EMBO J. [Journal of the European Society for Molecular Biology] 6: 2513-2518). See also Yarranton, (1992) Curr. Opin. Biotech. [Contemporary Perspectives on Biotechnology] 3: 506-511; Christopherson et al., (1992) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 89: 6314-6318; Yao et al., (1992) Cell [Cell] 71: 63-72; Reznikoff, (1992) Mol. Microbiol.[Molecular Microbiology] 6: 2419-2422; Barkley et al., (1980) in The Operon, pp. 177-220; Hu et al., (1987) Cell 48: 555-566; Brown et al., (1987) Cell 49: 603-612; Figge et al., (1988) Cell 52: 713-722; 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) Ph.D. Thesis, University of Heidelberg, University of Heidelberg, Germany; 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. [Hot Topic Molecular Structural Biology] 10: 143-162; Degenkolb et al., (1991) Antimicrob. Agents Chemother. [Antimicrobial Agents Chemotherapy] 35: 1591-1595; Kleinschnidt et al., (1988) Biochemistry [Biochemistry] 27: 1094-1104; Bonin, (1993) Ph.D. Thesis [Doctoral Dissertation] University of Heidelberg [Germany]; Gossen et al., (1992) Proc.Natl.Acad.Sci.USA [Proceedings of the National Academy of Sciences of the United States of America] 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 publications are incorporated herein by reference.

[0254] The list of selective marker genes above is not intended to be limiting. Any selective marker gene may be used in the examples.

[0255] Plant transformation

[0256] The methods described in these embodiments involve introducing a polypeptide or polynucleotide into a plant. As used herein, "introduction" means presenting the polynucleotide or polypeptide to the plant in such a manner that the sequence enters the interior of the plant cell. The methods of these embodiments are not dependent on a specific method for introducing the polynucleotide or polypeptide into the plant, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the plant. Methods for introducing polynucleotides or polypeptides into plants are known in the art, including but not limited to stable transformation, transient transformation, and virus-mediated transformation.

[0257] As used herein, "stable transformation" means the integration of a nucleotide construct introduced into a plant into the plant's genome and its ability to be inherited by its offspring. As used herein, "transient transformation" means the introduction of a polynucleotide into the plant without integration into its genome, or the introduction of a polypeptide into the plant. As used herein, "plant" refers to the whole plant, plant organs (e.g., leaves, stems, roots), seeds, plant cells, propagules, and their embryos and offspring. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

[0258] The transformation scheme and the scheme for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocot or dicot). Suitable methods for introducing nucleotide sequences into plant cells and subsequently inserting them 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 Patent Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al., (1984) EMBO J 3: 2717-2722), and ballistic particle acceleration (see, for example, US Patent Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes et al., (1995) Plant Cell, Tissue, and Organ Culture: Fundamental Methods, edited by Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al., (1988) Biotechnology 6: 923-926; and Lecl transformation (WO 00 / 28058). For potato transformation, see Tu et al., (1998) Plant Molecular Biology 37: 829-838 and Chong et al., (2000) Transgenic Research 9: 71-78. Other transformation methods can be found in the following literature: Weissinger et al., (1988) Ann. Rev. Genet. [Annals of Genetics] 22: 421-477; Sanford et al., (1987) Particulate Science and Technology [5: 27-37 (onion); Christou et al., (1988) Plant Physiol. [87: 671-674 (soybean); McCabe et al., (1988) Bio / Technology [6: 923-926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol.[In vitro cell biology and developmental biology] 27P: 175-182 (soybean); Singh et al., (1998) Theor. Appl. Genet. 96: 319-324 (soybean); Datta et al., (1990) Biotechnology 8: 736-740 (rice); Klein et al., (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309 (corn); Klein et al., (1988) Biotechnology 6: 559-563 (corn); US Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol. 91: 440-444 (Corn); Fromm et al., (1990) Biotechnology 8: 833-839 (Corn); Hooykaas-Van Slogteren et al., (1984) Nature (London) 311: 763-764; US Patent No. 5,736,369 (Cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al., (1985) The Experimental Manipulation of Ovule Tissues, edited by 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.[Theoretical and Applied Genetics] 84:560-566 (whisker-mediated transformation); 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 (rice); Osjoda et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of these are incorporated herein by reference.

[0259] In specific embodiments, various transient transformation methods can be used to provide the sequences of the embodiments to plants. Such transient transformation methods include, but are not limited to, the direct introduction of IPD059, IPD098, IPD108, or IPD109 polynucleotides, or variants and fragments thereof, into plants, or the introduction of IPD059, IPD098, IPD108, or IPD109 polypeptide transcripts into plants. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al., (1986) Mol Gen. Genet. [Molecular Genetics and General Genetics] 202: 179-185; Nomura et al., (1986) Plant Sci. [Plant Science] 44: 53-58; Hepler et al., (1994) Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences of the United States of America] 91: 2176-2180; and Hush et al., (1994) The Journal of Cell Science [Journal of Cell Science] 107: 775-784, all of which are incorporated herein by reference. Alternatively, IPD059, IPD098, IPD108, or IPD109 polynucleotides can be transiently transformed into plants using techniques known in the art. Such techniques include viral vector systems and precipitation of polynucleotides in a manner that prevents subsequent release of DNA. Thus, transcription can be performed from particle-bound DNA, but the frequency with which it is released to integrate into the genome is greatly reduced. Such methods include using particles coated with polyethyleneimine (PEI; Sigma, p. 3143).

[0260] Methods for targeted insertion of polynucleotides at specific locations in a plant genome are known in the art. In one embodiment, a site-specific recombination system is used to achieve the insertion of the polynucleotide at the desired genomic location. 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, the polynucleotide of the embodiments may be contained within a transfer cassette flanked by two distinct recombination sites. The transfer cassette is introduced into a plant that has stably incorporated the target site into its genome, the target site being flanked by two distinct recombination sites corresponding to the site on the transfer cassette. A suitable recombinase is provided, and the transfer cassette is integrated into the target site. Thus, the target polynucleotide is integrated at a specific chromosomal location in the plant genome.

[0261] Plant transformation vectors can consist of one or more DNA vectors required to achieve plant transformation. For example, it is common practice in the art to use plant transformation vectors composed of more than one continuous DNA fragment. These vectors are generally referred to in the art as “binary vectors.” Binary vectors, as well as vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation, where the size and complexity of the DNA fragments required for efficient transformation are considerable, and it is advantageous to isolate function onto individual DNA molecules. Binary vectors typically contain a plasmid vector containing cis-acting sequences required for T-DNA transfer (e.g., left and right boundaries), a selective marker engineered to be expressed in plant cells, and a “target gene” (a gene engineered to be expressed in plant cells where transgenic plants are desired). The sequences required for bacterial replication are also present on this plasmid vector. The cis-acting sequences are arranged in a manner that allows efficient transfer to and expression in plant cells. For example, the selective marker gene and the pest-killing gene are located between the left and right boundaries. Typically, the second plasmid vector contains a trans-acting factor that mediates T-DNA transformation from Agrobacterium to plant cells. As understood in the art, the plasmid typically contains a virulence function (Vir gene) that allows infection of plant cells by Agrobacterium, as well as DNA transfer via boundary sequence cleavage and vir-mediated DNA transfer (Hellens and Mullineaux, (2000) Trends in Plant Science 5: 446-451). Several types of Agrobacterium strains (e.g., LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. Transformation of plants by other methods such as microscopy, microinjection, electroporation, polyethylene glycol, etc., does not require a second plasmid vector.

[0262] Typically, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying appropriate selection at a maximum threshold level (depending on a selectable marker gene) to recover transformed plant cells from a population of untransformed cells. After the heterologous DNA is integrated into the plant cells, appropriate selection at a maximum threshold level is then applied in the culture medium to kill untransformed cells, and the presumed transformed cells that survived the selection treatment are isolated and proliferated by periodically transferring them to fresh culture medium. These cells transformed with the plasmid vector can be identified and proliferated through continuous passage and challenge with appropriate selection. The presence of the target heterologous gene integrated into the genome of the transgenic plant can then be confirmed using molecular and biochemical methods.

[0263] Typically, explants are transferred to freshly supplied, identical media and cultured routinely. Subsequently, after being placed on a regeneration medium supplemented with a maximum threshold level of selectant, the transformed cells differentiate into shoots. These shoots are then transferred to a selective rooting medium for the recovery of rooted shoots or plantlets. The transgenic plantlets then grow into mature plants and produce fertile seeds (e.g., Hiei et al., (1994) The Plant Journal 6: 271-282; Ishida et al., (1996) Nature Biotechnology 14: 745-750). Typically, explants are transferred to freshly supplied, identical media and cultured routinely. General descriptions of the techniques and methods used for the production of transgenic plants are found in: Ayres and Park, (1994) Critical Reviews in Plant Science 13: 219-239 and Bommineni and Jauhar, (1997) Maydica 42: 107-120. Because transformed material contains numerous cells, both transformed and untransformed cells coexist in any part of the target callus or tissue or cell population. The ability to kill untransformed cells and allow transformed cells to proliferate produces transformed plant cultures. Typically, the ability to remove untransformed cells limits the rapid recovery of transformed plant cells and the successful generation of transgenic plants.

[0264] The transformed cells can be cultured into plants using conventional methods. See, for example, McCormick et al., (1986) Plant Cell Reports 5: 81-84. These plants can then be cultured and pollinated with the same or different transformed lines, and hybrids with constitutive or inducible expression of the desired phenotypic trait can be identified. Two or more generations can be cultured to ensure that the expression of the desired phenotypic trait is stably maintained and inherited, and then the seeds are harvested to ensure that the expression of the desired phenotypic trait has been achieved.

[0265] Nucleotide constructs of the embodiments can be provided to plants by contacting them with a virus or viral nucleic acid. Typically, such methods involve incorporating the target nucleotide construct into a viral DNA or RNA molecule. It should be recognized that the recombinant protein of the embodiments described can be initially synthesized as part of a viral polyprotein, and then processed in vivo or in vitro by proteolytic hydrolysis to produce the desired IPD059, IPD098, IPD108, or IPD109 polypeptide. It is also recognized that such viral polyproteins comprising at least a portion of the amino acid sequence of the IPD059, IPD098, IPD108, or IPD109 polypeptide of the embodiments may possess the desired pest-killing activity. Such viral polyproteins and the nucleotide sequences encoding them are covered in the embodiments described. Methods for providing nucleotide constructs to plants and producing encoded proteins in plants are known in the art and involve viral DNA or RNA molecules. See, for example, U.S. Patent Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367, and 5,316,931; which are incorporated herein by reference.

[0266] Methods for transforming chloroplasts are known in the art. See, for example, Svab et al., (1990) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 87: 8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90: 913-917; Svab and Maliga, (1993) EMBO J. [Journal of the European Society for Molecular Biology] 12: 601-606. These methods rely on particle gun delivery of selectively labeled DNA and targeting the DNA to the plastid genome via homologous recombination. Alternatively, plastid transformation is achieved by transactivating silent plastid-carried transgenes through tissue-biased expression of nuclear-encoded and plastid-directed RNA synthases. This system has been reported in the following literature: McBride et al., (1994) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 91: 7301-7305.

[0267] The embodiments further relate to plant propagation materials of the transformed plants of the embodiments, including but not limited to seeds, tubers, bulbs, leaves, and cuttings of roots and buds.

[0268] The embodiments described herein can be used to transform any plant species, including but not limited to monocots and dicots. Examples of target plants include, but are not limited to, corn (Zea mays), Brassica species (e.g., Brassica napus, Brassica rapa, Brassica juncea) (especially those Brassica species that can be used as a source of seed oil), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italiea), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), and wheat (Triticum). Soybean (Glycine max), Tobacco (Nicotianatabacum), Potato (Solanum tuberosum), Peanut (Arachis hypogaea), Cotton (Gossypium barbadense, Gossypium hirsutum), Sweet potato (Ipomoea batatas), Cassava (Manihot esculenta), Coffee (Coffea species), Coconut (Cocos nucifera), Pineapple (Ananas comosus), Citrus (Citrus species), Cocoa (Theobroma cacao), Tea (Camellia sinensis), Banana (Musa species), Avocado (Persea americana), Fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (olive)Oleaeuropaea, papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (species of the genus Saccharum), oats, barley, vegetables, ornamental plants, and conifers.

[0269] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactucasativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (species of the genus Lathyrus), and members of the genus Cucumber such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and melon (C. melon). Ornamental plants include azaleas (Rhododendron species), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa species), tulips (Tulipa species), daffodils (Narcissus species), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherrima), and chrysanthemums. Conifers that can be used in practical embodiments include, for example, pine trees such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); Western hemlock (Tsugacanadensis); North American spruce (Picea glauca); redwood (Sequoia sempervirens); true fir trees such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedar trees such as western red cedar (Thuja spp.). plicata)) and Alaskan yellow cedar (Chamaecyparis nootkatensis)).The plants in the embodiments include crop plants (such as corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

[0270] Turfgrasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewing's fescue (Festuca rubra); Colonia lbentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis). Glomerata; perennial ryegrass (Lolium perenne); red foxgrass (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron) smithii); Cynodon (a species of the genus Cynodon); St. Augustine (St.Augustine grass (Stenotaphrum secundatum); Zoysia grass (a species of the genus Zoysia); Bahia grass (Paspalum notatum); Carpetgrass (Axonopus affinis); Centipede grass (Eremochloa ophiuroides); Kikuyu grass (Pennisetum clandesinum); Seashore paspalum (Paspalumvaginatum); Blue gramma (Bouteloua gracilis); Buffalo grass (Buchloe dactyloids); Sideoats gramma (Bouteloua curtipendula).

[0271] Target plants include cereal plants, oilseed plants, and legumes that provide the target seeds. Target seeds include cereal seeds such as corn, wheat, barley, rice, sorghum, rye, and millet. Oilseed plants include cotton, soybean, safflower, sunflower, brassica, maize, alfalfa, palm, coconut, flax, castor bean, and olive. Legumes include beans and peas. Beans include lentils, locust beans, fenugreek, soybean, green beans, cowpeas, mung beans, lima beans, broad beans, lentils, and chickpeas.

[0272] Plant transformation assessment

[0273] After introducing heterologous exogenous DNA into plant cells, the transformation or integration of the heterologous gene into the plant genome is confirmed by various methods (such as analyzing nucleic acids, proteins and metabolites associated with the integrated gene).

[0274] PCR analysis is a rapid method for screening transformed cells, tissues, or shoots for incorporated genes at an early stage before transplantation into soil (Sambrook and Russell, (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY). PCR is performed using oligonucleotide primers specific to the target gene or Agrobacterium vector background.

[0275] Plant transformation can be confirmed by gene DNA blot analysis (Sambrook and Russell, (2001), ibid.). Typically, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agar gel, and transferred to a nitrocellulose or nylon membrane. The membrane, or “blot,” is then probed with, for example, a radiolabeled 32P target DNA fragment to confirm the integration of the introduced gene into the plant genome according to standard techniques (Sambrook and Russell, (2001), ibid.).

[0276] In RNA blot analysis, RNA is isolated from specific tissues of the transformant, fractionated in formaldehyde agar gel, and blotted onto a nylon filter according to standard procedures conventionally used in the art (Sambrook and Russell, (2001), ibid.). The expression of RNA encoded by the pest-killing gene is then tested by methods known in the art through hybridization of the filter with a radioactive probe from the pest-killing gene (Sambrook and Russell, (2001), ibid.).

[0277] Transgenic plants can be subjected to Western blotting, biochemical assays, etc., to confirm the presence of proteins encoded by pest-killing genes using antibodies that bind to one or more epitopes present on the IPD059, IPD098, IPD108, or IPD109 polypeptides through standard procedures (Sambrook and Russell, 2001, ibid.).

[0278] Methods of introducing genome editing technology into plants

[0279] In some embodiments, genome editing technologies can be used to introduce the disclosed IPD059, IPD098, IPD108, or IPD109 polynucleotide composition into the plant genome, or genome editing technologies can be used to edit previously introduced IPD059, IPD098, IPD108, or IPD109 polynucleotides in the plant genome. For example, the disclosed polynucleotides can be introduced into the desired location in the plant genome using double-strand breakage techniques (such as TALEN, large-scale nucleases, zinc finger nucleases, CRISPR-Cas, etc.). For example, for site-specific insertion purposes, the CRISPR-Cas system can be used to introduce the disclosed polynucleotides into the desired location in the genome. The desired location in the plant genome can be any target site required for insertion, such as a genomic region suitable for breeding, or it can be a target site located within a genomic window with an existing target trait. The existing target trait may be an endogenous trait or a previously introduced trait.

[0280] In some embodiments, where the disclosed IPD059, IPD098, IPD108, or IPD109 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 can be introduced into the disclosed IPD059, IPD098, IPD108, or IPD109 polynucleotide compositions, including modifications produced using any method for introducing site-specific modifications, including but not limited to the use of gene repair oligonucleotides (e.g., U.S. Publication 2013 / 0019349) or the use of double-strand break techniques such as TALEN, large-scale nucleases, zinc finger nucleases, CRISPR-Cas, etc. Such techniques can be used to modify previously introduced polynucleotides 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 introduced polynucleotide. Additional sequences that can be added include additional expression elements (such as enhancer sequences and promoter sequences). In another embodiment, genome editing technology can be used to locate additional insecticidal active proteins within the genome of a plant near the disclosed polynucleotide compositions of IPD059, IPD098, IPD108, or IPD109 to produce molecular stacks of insecticidal active proteins.

[0281] The terms “altered target site,” “altered target sequence,” “modified target site,” and “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that contains at least one alteration when compared to an unaltered target sequence. Such “alterations” 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).

[0282] Stacking of traits in transgenic plants

[0283] Transgenic plants may contain stacks of one or more insecticidal polynucleotides disclosed herein and one or more additional polynucleotides, resulting in the production or repression of multiple polypeptide sequences. Transgenic plants containing polynucleotide sequence stacks can be obtained by one or both of conventional breeding methods or genetic engineering methods. These methods include, but are not limited to: breeding individual lines each containing the target polynucleotide, subsequently transforming transgenic plants containing genes disclosed herein with a gene, and co-transforming the genes into a single plant cell. As used herein, the term "stack" includes the presence of multiple traits in the same plant (i.e., incorporating two traits into the nuclear genome, incorporating one trait into the nuclear genome and one trait into the plastid genome, or both traits being incorporated into the plastid genome). In a non-limiting example, a "stacked trait" includes a molecular stack in which sequences are physically adjacent to each other. As used herein, a trait refers to a phenotype derived from a specific sequence or sequence group. Co-transformation of genes can be performed using a single transformation vector containing multiple genes or genes carried separately on multiple vectors. If sequences are stacked by genetically transforming plants, the target polynucleotide sequences can be combined at any time and in any order. The trait can be introduced together with the target polynucleotide provided by any combination of transformation cassettes using a co-transformation scheme. For example, if two sequences are introduced, these two sequences can be contained in separate transformation cassettes (trans) or in the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In some cases, it may be necessary to introduce a transformation cassette that will inhibit the expression of the target polynucleotide. This can be combined with any combination of other repressor or overexpression cassettes to produce the desired combination of traits in the plant. It should be further appreciated that site-specific recombination systems can be used to stack polynucleotide sequences at desired genomic locations. 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.

[0284] In some embodiments, the polynucleotide encoding the disclosed IPD059, IPD098, IPD108, or IPD109 polypeptide, alone or stacked with one or more other insect resistance traits, may be stacked with one or more other input traits (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stem strength, etc.) or output traits (e.g., increased yield, modified starch, improved oil properties, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, etc.). Therefore, the polynucleotide embodiments can be used to provide complete agronomic programs with the ability to flexibly and cost-effectively control any number of agronomic pests, resulting in improved crop quality.

[0285] Genetically modified organisms that can be stacked include, but are not limited to:

[0286] 1. Genetically modified organisms (GMOs) confer resistance or disease resistance to insects and encode:

[0287] (A) Plant resistance genes. Plant defenses are typically activated through specific interactions between the products of resistance genes (R) in plants and the products of the corresponding avirulence (Avr) genes in pathogens. Cloned resistance genes can be used to transform plant varieties to engineer plants resistant to specific pathogen strains. See, for example, Jones et al., (1994) Science 266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al., (1993) Science 262:1432 (tomato Pto gene for resistance to Pseudomonas syringaepv. tomato encodes a protein kinase); Mindrinos et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae); McDowell and Woffenden, (2003) Trends Biotechnol. [Biotechnical Trends] 21(4): 178-83 and Toyoda et al., (2002) Transgenic Res. [Transgenic Research] 11(6): 567-82. Disease-resistant plants are more resistant to pathogens than wild-type plants.

[0288] (B) Genes encoding proteins, derivatives thereof, or synthetic polypeptides modeled on Bacillus thuringiensis. See, for example, Geiser et al., (1986) Gene 48:109, which discloses the clone and nucleotide sequence of the Btδ-endotoxin gene. Furthermore, DNA molecules encoding the δ-endotoxin gene are available from the American Type Culture Collection (Rockville, Md., MD, USA), for example... Accessions 40098, 67136, 31995, and 31998. Other non-limiting examples of genetically engineered Bacillus thuringiensis transgenic strains are given in the following patents and patent applications, which are incorporated herein by reference for this purpose: U.S. Patent Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594, 6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826, and 7,105. 332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556, 7,329,736, 7,449,552, 7,468,278, 7,510,878 , 7,521,235, 7,544,862, 7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846, 7,858,849 and WO 1991 / 14778; WO 1999 / 31248; WO 2001 / 12731; WO 1999 / 24581 and WO 1997 / 40162.

[0289] Genes encoding pest-killing proteins can also be stacked, including but not limited to: insecticidal proteins from Pseudomonas species such as PSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonas protein strains CHA0 and Pf-5 (formerly Pseudomonas fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology, 10:2368-2386: Gene Bank Accession Number EU400157); from Pseudomonas taiwanensis (Liu et al., (2010) J. Agric. Food Chem., 58:12343-12349) and from Pseudomonas alkaliformis (Zhang et al., (2009) Annals Insecticidal proteins from *Microbiology* 59:45-50 and Li et al., (2007) *Plant Cell Tiss. Organ Cult.* 89:159-168; insecticidal proteins from *Proteobacterium* species and *Pathogenic Bacteria* species (Hinchliffe et al., (2010) *The Open Toxicology Journal*, 3:101-118 and Morgan et al., (2001) *Applied and Envir. Micro.* 67:2062-2069); insecticidal proteins from US patents 6,048,838 and 6,379,946; PIP-1 peptide from US 9,688,730; US 9,475,847 AfIP-1A and / or AfIP-1B peptides; US Publication No. US20160186204 PIP-47 peptide; PCT Publication No. WO2016 / 114973 IPD045, IPD064, IP...

Claims

1. An insecticidal polypeptide consisting of the amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 114 or SEQ ID NO:

115.

2. The insecticidal polypeptide of claim 1, wherein the insecticidal polypeptide is operably linked to a heterologous transit peptide or a heterologous signal polypeptide.

3. An insecticidal composition comprising the insecticidal polypeptide according to claim 1 or 2.

4. A recombinant polynucleotide encoding the insecticidal polypeptide of claim 1.

5. The recombinant polynucleotide of claim 4, wherein the polynucleotide is cDNA.

6. A DNA construct comprising the recombinant polynucleotide of claim 4 or 5 and a heterologous regulatory sequence operably linked to the recombinant polynucleotide.

7. A method for producing a transgenic plant or plant cell comprising the DNA construct according to claim 6.

8. A method of inhibiting the growth of or killing an agricultural insect pest population, the method comprising contacting the insect pest population with an insecticidally effective amount of at least one insecticidal polypeptide according to claim 1, wherein the insect pest population is corn rootworm (WCRW, Diabrotica virgifera).

9. A method of controlling damage to plants by insect pests, the method comprising providing an insecticidal polypeptide as claimed in claim 1 or 2 to an insect pest or pest population for ingestion, wherein the insecticidal polypeptide is produced by a transgenic plant and is present in at least one of the plants, and wherein the insect pest or pest population is corn rootworm (WCRW, Diabrotica virgifera).

10. A method of controlling insect infestation of a transgenic plant, the method comprising expressing in the plant a polynucleotide according to claim 4 or 5, wherein the insect is Corn Rootworm (WCRW, Diabrotica virgifera).

11. The method of claim 8, 9 or 10, wherein the insect pest or insect pest population is resistant to a Bt toxin.

12. Use of the insecticidal polypeptide of claim 1 for inhibiting the growth of or killing insects or insect populations, wherein the insects or insect populations are corn rootworms (WCRW, Diabrotica virgifera).

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