Insecticidal proteins and methods for their use

Abstract

This disclosure relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These pesticidal proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal formulations and in the production of transgenic pest-resistant plants. Methods to create or alter pesticidal proteins are provided for altered or enhanced pesticidal activity.

Description

Attorney Docket: 212196-WO-SEC-1 INSECTICIDAL PROTEINS AND METHODS FOR THEIR USE REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The official copy of the sequence listing is submitted electronically via DOCX as an XML formatted sequence listing with a file named “212196-US-PRV-1_SequenceListing” created on May 29, 2024, and having a size of 117,000 bytes and is filed in computer readable form concurrently with the specification. The sequence listing contained in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety. FIELD

[0002] This disclosure relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These pesticidal proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal formulations and in the production of transgenic pest- resistant plants. Methods to create or alter pesticidal proteins are provided for altered or enhanced pesticidal activity. BACKGROUND

[0003] Biological control of insect pests of agricultural significance using a microbial agent, such as fungi, bacteria or another species of insect affords an environmentally friendly and commercially attractive alternative to synthetic chemical pesticides. Generally speaking, the use of biopesticides presents a lower risk of pollution and environmental hazards and biopesticides provide greater target specificity than is characteristic of traditional broad-spectrum chemical insecticides. In addition, biopesticides often cost less to produce and thus improve economic yield for a wide variety of crops.

[0004] Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillus thuringiensis (Bt) and Bacillus popilliae are among the most successful biocontrol agents discovered to date. Insect pathogenicity has also been attributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B. cereus. Microbial insecticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives to chemical pestAttorney Docket: 212196-WO-SEC-1 control.

[0005] Crop plants have been developed with enhanced insect resistance by genetically engineering crop plants to produce pesticidal proteins from Bacillus. For example, corn and cotton plants have been genetically engineered to produce pesticidal proteins isolated from strains of Bacillus thuringiensis. These genetically engineered crops are now widely used in agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect-control methods. While they have proven to be very successful commercially, these genetically engineered, insect-resistant crop plants may provide resistance to only a narrow range of the economically important insect pests. In some cases, insects can develop resistance to different insecticidal compounds, which raises the need to identify alternative biological control agents for pest control.

[0006] Accordingly, there remains a need for new pesticidal proteins, for example from new sources, with different ranges of insecticidal activity against insect pests, e.g., insecticidal proteins which are active against a variety of insects in the order Lepidoptera and the order Coleoptera, including but not limited to insect pests that have developed resistance to existing insecticides. SUMMARY

[0007] In one aspect, compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions may include nucleic acid molecules encoding sequences for pesticidal and insecticidal polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. Compositions also may include the pesticidal polypeptide sequences and antibodies to those polypeptides. The nucleic acid sequences may be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant. Compositions may also comprise transformed bacteria, plants, plant cells, tissues and seeds.

[0008] In one aspect, isolated or recombinant nucleic acid molecules are provided encoding polypeptides comprising an amino acid sequence having at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to aAttorney Docket: 212196-WO-SEC-1 sequence selected from the group consisting of SEQ ID Nos: 1-104, and fragments thereof, including amino acid substitutions, deletions, insertions, and fusions.

[0009] In another embodiment, provided are DNA constructs comprising a polynucleotide which encodes an insect inhibitory protein comprising a polypeptide comprising an amino acid sequence having at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID Nos: 1-104.

[0010] In one embodiment, provided are host cells comprising a polynucleotide which encodes an insect inhibitory protein comprising a polypeptide comprising an amino acid sequence having at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID Nos: 1-104.

[0011] In one embodiment, provided are transgenic plants comprising polynucleotide which encodes an insect inhibitory protein comprising a polypeptide comprising an amino acid sequence having at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID Nos: 1-104.

[0012] In one embodiment, provided are expression cassettes comprising a polynucleotide which encodes an insect inhibitory protein comprising a polypeptide comprising an amino acid sequence having at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID Nos: SEQ ID Nos: 1-104.

[0013] In one embodiment, provided are methods for controlling a Coleopteran insect, said method comprising the steps of: (a) providing an insect inhibitory amount of an insect inhibitory protein, wherein said insect inhibitory protein comprises a polypeptide comprising an amino acid sequence having at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID Nos: 1-104; and (b) contacting said Lepidopteran insect with said inhibitory amount of said insect inhibitory protein, thereby controlling said Lepidopteran insect. In one exemplary embodiment, the Lepidopteran pest is selected from European corn borerAttorney Docket: 212196-WO-SEC-1 (Ostrinia nubilalis; ECB), corn earworm (Helicoverpa zea; CEW), black cutworm (Agrotis ipsilon; BCW), fall armyworm (Spodoptera frugiperda; FAW), Soybean looper (Pseudoplusia includens; SBL) and Velvetbean caterpillar (Anticarsia gemmatalis; VBC)). In another exemplary embodiment, a Coleopteran pest is selected from Western corn rootworm (Diabrotica virgifera; WCRW). In yet another exemplary embodiment, a Hemipteran pest is selected from southern green stink bug (Nezara viridula; SGSB). DETAILED DESCRIPTION

[0014] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

[0015] The present disclosure is drawn to compositions and methods for controlling pests. The methods involve transforming organisms with nucleic acid sequences encoding insecticidal polypeptides. In particular, the nucleic acid sequences of the embodiments are useful for preparing plants and microorganisms that possess pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. The compositions include pesticidal nucleic acids and proteins of fern species. The nucleic acid sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes, and for the generation of altered insecticidal polypeptides by utilizing aspects of certain methods known in the art, such as site directed mutagenesis, domain swapping, or DNA shuffling. The insecticidal polypeptides find use in controlling or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with pesticidal activity. Insect pests of interest include, but are not limited to, Lepidoptera species including but not limited to: Corn Earworm, (CEW) (Helicoverpa zea), European Corn Borer (ECB) (Ostrinia nubialis), diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper, e.g., Pseudoplusia includens Walker; and velvet bean caterpillar e.g., Anticarsia gemmatalis Hübner and Coleoptera species including but notAttorney Docket: 212196-WO-SEC-1 limited to Western corn rootworm (Diabrotica virgifera) - WCRW, Southern corn rootworm (Diabrotica undecimpunctata howardi) – SCRW, and Northern corn rootworm (Diabrotica barberi) - NCRW.

[0016] By “pesticidal toxin” or “pesticidal protein” is used herein to refer to a toxin that has toxic activity against one or more pests. For example, pests may include members of the Lepidoptera, Diptera, Hemiptera and Coleoptera orders or the Nematoda phylum or a protein that has homology to such a protein. Pesticidal proteins have been isolated from organisms including, for example, Bacillus sp., Bacillus thurengiensis (“Bt”), Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae.

[0017] In some embodiments a insecticidal polypeptide includes an amino acid sequence deduced from the full-length nucleic acid sequence disclosed herein and amino acid sequences that are shorter than the full-length sequences, either due to the use of an alternate downstream start site or due to processing that produces a shorter protein having pesticidal activity. Processing may occur in the organism in which the protein is expressed in or in the pest after ingestion of the protein.

[0018] In some embodiments, an engineered polypeptide is disclosed herein. In one embodiment, the engineered polypeptides disclosed herein have an altered spectrum of activity. In another embodiment, the engineered polypeptide has an altered amount of pesticidal activity. In some embodiments, the engineered polypeptide has an altered mode of action or site of action. In some embodiments, the engineered polypeptide has an altered solubility.

[0019] Thus, provided herein are isolated or recombinant nucleic acid sequences encoding engineered polypeptides conferring pesticidal activity. Also provided are the amino acid sequences of engineered polypeptides. The polypeptides resulting from translation of these engineered genes allows cells to control or kill pests that ingest it.

[0020] Also provided herein are isolated or recombinant nucleic acid sequences encoding IPD115, IPD119, IPD125, or IPD130 polypeptides with insecticidal activity. Also provided are the amino acid sequences of IPD115, IPD119, IPD125, or IPD130 polypeptides. Toxin Proteins and Variants and Fragments Thereof

[0021] IPD115, IPD119, IPD125, or IPD130 polypeptides are encompassed by the disclosure. “IPD115, IPD119, IPD125, or IPD130 polypeptide,” and “IPD115, IPD119, IPD125, or IPD130 protein” as used herein interchangeably refers to a polypeptide(s) having insecticidal activityAttorney Docket: 212196-WO-SEC-1 including but not limited to insecticidal activity against one or more insect pests of the Lepidoptera and / or Coleoptera orders. A variety of IPD115, IPD119, IPD125, or IPD130 polypeptides are contemplated. Sources of IPD115, IPD119, IPD125, or IPD130 polypeptides or related proteins include fern or other primitive plant species.

[0022] In some embodiments, the IPD115, IPD119, IPD125, or IPD130 polypeptide is derived from a species in the Class Isoetopsida Order Selaginales. In some embodiments, the IPD120 polypeptide is derived from a fern species in the Class Isoetopsida, Order Selaginales, Family Selaginellaceae. In some embodiments, the IPD120 polypeptide is derived from a species in the Genus Selaginella. In some embodiments, the IPD120 polypeptide is derived from a Selaginella species 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, 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 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 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.

[0023] Variant polypeptides are encompassed by the disclosure. “ Variant polypeptides,” and “engineered toxin protein” as used herein interchangeably refers to a polypeptide(s) having insecticidal activity including but not limited to insecticidal activity against one or more insect pests of the Lepidoptera and / or Coleoptera orders, and has been engineered from IPD115, IPD119, IPD125, or IPD130.Attorney Docket: 212196-WO-SEC-1

[0024] “Sufficiently identical” is used herein to refer to an amino acid sequence that has at least about 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. In one embodiment the variant polypeptide has at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to any one of SEQ ID NOs: 1-104. The term “about” when used herein in context with percent sequence identity means + / - 1.0%.

[0025] A “recombinant protein” is used herein to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

[0026] “Substantially free of cellular material” as used herein refers to a polypeptide including preparations of protein having less than about 30%, 20%, 10% or 5% (by dry weight) of non- pesticidal protein (also referred to herein as a “contaminating protein”).

[0027] “Fragments” or “biologically active portions” include polypeptide fragments comprising amino acid sequences sufficiently identical to any one of IPD115, IPD119, IPD125, or IPD130 polypeptide and that exhibit insecticidal activity. “Fragments” or “biologically active portions” of IPD115, IPD119, IPD125, or IPD130 polypeptides includes fragments comprising amino acid sequences sufficiently identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-104, wherein the IPD115, IPD119, IPD125, or IPD130 polypeptide has insecticidal activity. Such biologically active portions can be prepared by recombinant techniques and evaluated for insecticidal activity. In some embodiments, the IPD115, IPD119, IPD125, or IPD130 polypeptide fragment is an N-terminal and / or a C-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more amino acids from the N-terminus and / or C-terminus relative to any one of SEQ ID NOs: 1-104, e.g., by proteolysis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion of a start codon, and / or insertion of a stop codon. In some embodiments, the IPD115, IPD119, IPD125, or IPD130 polypeptide fragment is an N-terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 amino acids from the N-terminus of any one of SEQ ID NOs: 1-104. In some embodiments,Attorney Docket: 212196-WO-SEC-1 the IPD115, IPD119, IPD125, or IPD130 polypeptide fragment is an N-terminal and / or a C- terminal truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or more amino acids from the N-terminus and / or C-terminus relative to any one of SEQ ID NOs: 1-104.

[0028] “Variants” as used herein refers to proteins or polypeptides 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 parental amino acid sequence. In one embodiment, "variant" is intended to mean a protein or polypeptide derived from a native protein or polypeptide by deletion or addition of one or more amino acids at one or more internal sites in the native protein or polypeptide and / or substitution of one or more amino acids at one or more sites in a native protein or polypeptide. Variants encompassed by the present disclosure exhibit a biological activity of the native protein or polypeptide sequence.

[0029] In some embodiments an IPD115, IPD119, IPD125, or IPD130 polypeptide comprises an amino acid sequence having at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the full length or a fragment of the amino acid sequence of any one of SEQ ID NOs: 1-104, wherein the IPD115, IPD119, IPD125, or IPD130 polypeptide has insecticidal activity.

[0030] In some embodiments an IPD115, IPD119, IPD125, or IPD130 polypeptide comprises an amino acid sequence of any one or more of SEQ ID NOs: 1-104 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, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or more amino acid substitutions compared to the amino acid at the corresponding position of any one or more of the respective SEQ ID NOs: 1-104.

[0031] Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a IPD115, IPD119, IPD125, or IPD130 polypeptide can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis,Attorney Docket: 212196-WO-SEC-1 such as for example site-specific double strand break technology, and / or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will possess a desired pesticidal activity. However, it is understood that the ability of a IPD115, IPD119, IPD125, or IPD130 polypeptide to confer pesticidal activity or other polypeptide physical property may be improved or altered by the use of such techniques upon the compositions of this disclosure.

[0032] Conservative amino acid substitutions may be made at one or more predicted nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of a IPD115, IPD119, IPD125, or IPD130 polypeptide without altering the biological activity. A “conservative amino acid substitution” is one in which the 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); acidic side chains (e.g., aspartic acid, glutamic acid); polar, negatively charged residues and their amides (e.g., aspartic acid, asparagine, glutamic, acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); small aliphatic, nonpolar or slightly polar residues (e.g., Alanine, serine, threonine, proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); large aliphatic, nonpolar residues (e.g., methionine, leucine, isoleucine, valine, cystine); beta-branched side chains (e.g., threonine, valine, isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine); large aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).

[0033] Amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related toxins to the sequences of the embodiments (e.g., residues that are identical in an alignment of homologous proteins). Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or relatedAttorney Docket: 212196-WO-SEC-1 toxins to the sequences of the embodiments (e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins). However, one of skill in the art would understand that functional variants may have minor conserved or nonconserved alterations in the conserved residues.

[0034] Alternatively, alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification. Alternatively, the protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions. Such fusion proteins are often used to (1) increase expression of a protein of interest (2) introduce a binding domain, enzymatic activity or epitope to facilitate either protein purification, protein detection or other experimental uses known in the art (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, mitochondria or chloroplasts of plants or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.

[0035] Variant nucleotide and amino acid sequences of the disclosure also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different shuffled IPD115, IPD119, IPD125, or IPD130 polypeptide coding regions can be used to create a new shuffled IPD115, IPD119, IPD125, or IPD130 polypeptide respectively, possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer, (1994) Nature 370:389- 391; and US Patent Numbers 5,605,793 and 5,837,458.

[0036] Domain swapping as shuffling is another mechanism for generating altered toxinAttorney Docket: 212196-WO-SEC-1 polypeptides. Domains may be swapped between one or more homologs of IPD115, IPD119, IPD125, or IPD130 polypeptides resulting in hybrid or chimeric toxins with altered insecticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for pesticidal activity are known in the art (see, for example, Naimov, et al., (2001) Appl. Environ. Microbiol.67:5328-5330; de Maagd, et al., (1996) Appl. Environ. Microbiol.62:1537-1543; Ge, et al., (1991) J. Biol. Chem.266:17954-17958; Schnepf, et al., (1990) J. Biol. Chem.265:20923- 21010; Rang, et al., 91999) Appl. Environ. Microbiol.65:2918-2925).

[0037] Phylogenetic, sequence motif, and structural analyses of insecticidal protein families. A sequence and structure analysis method can be employed, which is composed of four components: phylogenetic tree construction, protein sequence motifs finding, secondary structure prediction, and alignment of protein sequences and secondary structures. Details about each component are illustrated below. 1) Phylogenetic tree construction

[0038] The phylogenetic analysis can be performed using the software MEGA5. Protein sequences can be subjected to ClustalW version 2 analysis (Larkin M.A et al (2007) Bioinformatics 23(21): 2947-2948) for multiple sequence alignment. The evolutionary history is then inferred by the Maximum Likelihood method based on the JTT matrix-based model. The tree with the highest log likelihood is obtained, exported in Newick format, and further processed to extract the sequence IDs in the same order as they appeared in the tree. A few clades representing sub-families can be manually identified for each insecticidal protein family. 2) Protein sequence motifs finding

[0039] Protein sequences are re-ordered according to the phylogenetic tree built previously, and fed to the MOTIF analysis tool MEME (Multiple EM for MOTIF Elicitation) (Bailey T.L., and Elkan C., Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, pp. 28-36, AAAI Press, Menlo Park, California, 1994.) for identification of key sequence motifs. MEME is setup as follows: Minimum number of sites 2, Minimum motif width 5, and Maximum number of motifs 30. Sequence motifs unique to each sub-family were identified by visual observation. The distribution of MOTIFs across the entire gene family couldAttorney Docket: 212196-WO-SEC-1 be visualized in HTML webpage. The MOTIFs are numbered relative to the ranking of the E- value for each MOTIF. 3) Secondary structure prediction

[0040] PSIPRED, top ranked secondary structure prediction method (Jones DT. (1999) J. Mol. Biol. 292: 195-202), can be used for protein secondary structure prediction. The tool provides accurate structure prediction using two feed-forward neural networks based on the PSI-BLAST output. The PSI-BLAST database is created by removing low-complexity, transmembrane, and coiled-coil regions in Uniref100. The PSIPRED results contain the predicted secondary structures (Alpha helix: H, Beta strand: E, and Coil: C) and the corresponding confidence scores for each amino acid in a given protein sequence. 4) Alignment of protein sequences and secondary structures

[0041] A script can be developed to generate gapped secondary structure alignment according to the multiple protein sequence alignment from step 1 for all proteins. All aligned protein sequences and structures are concatenated into a single FASTA file, and then imported into MEGA for visualization and identification of conserved structures.

[0042] In some embodiments the variant IPD115, IPD119, IPD125, or IPD130 polypeptide has a modified physical property. As used herein, the term “physical property” refers to any parameter suitable for describing the physical-chemical characteristics of a protein. As used herein, “physical property of interest” and “property of interest” are used interchangeably to refer to physical properties of proteins that are being investigated 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 hydrophobic residue distribution on the protein surface, surface charge density, surface hydrophobicity density, total count of surface ionizable groups, surface tension, protein size and its distribution in solution, melting temperature, heat capacity, and second virial coefficient. Examples of physical properties also include, variant IPD115, IPD119, IPD125, or IPD130 polypeptide having increased expression, increased solubility, decreased phytotoxicity, and digestibility of proteolytic fragments in an insect gut. Models for digestion by simulated gastric fluids are known to one skilled in the art (Fuchs, R.L. and J.D. Astwood. Food Technology 50: 83-Attorney Docket: 212196-WO-SEC-1 88, 1996; Astwood, J.D., et al Nature Biotechnology 14: 1269-1273, 1996; Fu TJ et al J. Agric Food Chem.50: 7154-7160, 2002).

[0043] In some embodiments variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the disclosure are biologically active, that is they continue to possess a desired biological activity (i.e. pesticidal activity) of the native protein. In some embodiment the variant will have at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 80% or more of the insecticidal activity of the native protein. In some embodiments, the variants may have improved activity over the native protein.

[0044] Bacterial genes quite often possess multiple methionine initiation codons in proximity to the start of the open reading frame. Often, translation initiation at one or more of these start codons will lead to generation of a functional protein. These start codons can include ATG codons. However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. On rare occasions, translation in bacterial systems can initiate at a TTG codon, though in this event the TTG encodes a methionine. Furthermore, it is not often determined a priori which of these codons are used naturally in the bacterium. Thus, it is understood that use of one of the alternate methionine codons may also lead to generation of pesticidal proteins. These pesticidal proteins are encompassed in the present disclosure and may be used in the methods of the present disclosure. It will be understood that, when expressed in plants, it will be necessary to alter the alternate start codon to ATG for proper translation.

[0045] In some embodiments a variant IPD115, IPD119, IPD125, or IPD130 polypeptide comprises the amino acid sequence of any one or more of SEQ ID NOs: 1-104.

[0046] In some embodiments, chimeric polypeptides are provided comprising regions of at least two different IPD115, IPD119, IPD125, or IPD130 polypeptides of the disclosure.

[0047] In some embodiments, chimeric polypeptides are provided comprising regions of at least two different IPD115, IPD119, IPD125, or IPD130 polypeptides selected from any one or more of SEQ ID NOs: 1-104

[0048] In some embodiments, chimeric IPD115, IPD119, IPD125, or IPD130 polypeptide(s) are provided comprising an N-terminal Region of a first IPD115, IPD119, IPD125, or IPD130 polypeptide of the disclosure operably fused to a C-terminal Region of a second IPD115, IPD119,Attorney Docket: 212196-WO-SEC-1 IPD125, or IPD130 polypeptide of the disclosure.

[0049] In other embodiments the IPD115, IPD119, IPD125, or IPD130 polypeptide may be expressed as a precursor protein with an intervening sequence that catalyzes multi-step, post translational protein splicing. Protein splicing involves the excision of an intervening sequence from a polypeptide with the concomitant joining of the flanking sequences to yield a new polypeptide (Chong, et al., (1996) J. Biol. Chem., 271:22159-22168); (Evans, et al., (2000) J. Biol. Chem., 275:9091-9094). This intervening sequence or protein splicing element, referred to as inteins, which catalyze their own excision through three coordinated reactions at the N-terminal and C-terminal splice junctions: an acyl rearrangement of the N-terminal cysteine or serine; a transesterfication reaction between the two termini to form a branched ester or thioester intermediate and peptide bond cleavage coupled to cyclization of the intein C-terminal asparagine to free the intein (Evans, et al., (2000) J. Biol. Chem., 275:9091-9094. The elucidation of the mechanism of protein splicing has led to a number of intein-based applications (Comb, et al., US Patent Number 5,496,714; Comb, et al., US Patent Number 5,834,247; Camarero and Muir, (1999) J. Amer. Chem. Soc. 121:5597-5598; Chong, et al., (1997) Gene 192:271-281, Chong, et al., (1998) Nucleic Acids Res.26:5109-5115; Chong, et al., (1998) J. Biol. Chem.273:10567-10577; Cotton, et al., (1999) J. Am. Chem. Soc. 121:1100-1101; Evans, et al., (1999) J. Biol. Chem. 274:18359-18363; Evans, et al., (1999) J. Biol. Chem. 274:3923-3926; Evans, et al., (1998) Protein Sci.7:2256-2264; Evans, et al., (2000) J. Biol. Chem.275:9091-9094; Iwai and Pluckthun, (1999) FEBS Lett.459:166-172; Mathys, et al., (1999) Gene 231:1-13; Mills, et al., (1998) Proc. Natl. Acad. Sci. USA 95:3543-3548; Muir, et al., (1998) Proc. Natl. Acad. Sci. USA 95:6705-6710; Otomo, et al., (1999) Biochemistry 38:16040-16044; Otomo, et al., (1999) J. Biolmol. NMR 14:105-114; Scott, et al., (1999) Proc. Natl. Acad. Sci. USA 96:13638-13643; Severinov and Muir, (1998) J. Biol. Chem. 273:16205-16209; Shingledecker, et al., (1998) Gene 207:187-195; Southworth, et al., (1998) EMBO J.17:918-926; Southworth, et al., (1999) Biotechniques 27:110- 120; Wood, et al., (1999) Nat. Biotechnol.17:889-892; Wu, et al., (1998a) Proc. Natl. Acad. Sci. USA 95:9226-9231; Wu, et al., (1998b) Biochim Biophys Acta 1387:422-432; Xu, et al., (1999) Proc. Natl. Acad. Sci. USA 96:388-393; Yamazaki, et al., (1998) J. Am. Chem. Soc., 120:5591- 5592). For the application of inteins in plant transgenes, see, Yang, et al., (Transgene Res 15:583- 593 (2006)) and Evans, et al., (Annu. Rev. Plant Biol.56:375-392 (2005)).Attorney Docket: 212196-WO-SEC-1

[0050] In another embodiment fusion proteins are provided that include within its amino acid sequence an amino acid sequence comprising a IPD115, IPD119, IPD125, or IPD130 polypeptide of the disclosure. Methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art. Polynucleotides encoding a IPD115, IPD119, IPD125, or IPD130 polypeptide may be fused to signal sequences which will direct the localization of the IPD115, IPD119, IPD125, or IPD130 polypeptide to particular compartments of a prokaryotic or eukaryotic cell and / or direct the secretion of the IPD115, IPD119, IPD125, or IPD130 polypeptide of the embodiments from a prokaryotic or eukaryotic cell.

[0051] For example, in E. coli, one may wish to direct the expression of the protein to the periplasmic space through, but are not limited to, the use of a pelB signal sequence like the pelB pectate lyase signal sequence, the maltose binding protein (MBP) signal sequence, MBP, the ompA signal sequence, the signal sequence of the periplasmic E. coli heat-labile enterotoxin B-subunit and the signal sequence of alkaline phosphatase. See also the commercially available pMAL series of vectors (particularly the pMAL-p series) available from New England Biolabs (Ipswich, MA).

[0052] Plant plastid transit peptide / polypeptide fusions are known in the art. Apoplast transit peptides such as rice or barley alpha-amylase secretion signal are also known in the art. The plastid transit peptide is generally fused N-terminally to the polypeptide to be targeted (e.g., the fusion partner). The fusion protein may comprise, or alternatively consist essentially of, the plastid transit peptide and the IPD115, IPD119, IPD125, or IPD130 polypeptide to be targeted. In such embodiments, the plastid transit peptide is preferably at the N-terminus of the fusion protein but may include additional amino acid residues N-terminal to the plastid transit peptide. In a specific embodiment, the plastid transit peptide is in the N-terminal half, N-terminal third or N-terminal quarter of the fusion protein. Most or all of the plastid transit peptide is generally cleaved from the fusion protein upon insertion into the plastid. In one embodiment, the plastid transit peptide cleavage site may be homogenous or alternatively may vary by 1-10 amino acids. In some embodiments the IPD115, IPD119, IPD125, or IPD130 polypeptide can be recombinantly fused to a heterologous signal peptide or heterologous transit peptide.

[0053] In some embodiments fusion proteins are provided comprising a IPD115, IPD119, IPD125, or IPD130 polypeptide or chimeric Insecticidal toxin polypeptide of the disclosure represented by a formula selected from the group consisting of:Attorney Docket: 212196-WO-SEC-1 R1-L-R2, R2-L- R1, R1- R2or R2- R1wherein R1is a IPD115, IPD119, IPD125, or IPD130 polypeptide or chimeric IPD115, IPD119, IPD125, or IPD130 polypeptide of the disclosure and R2is a protein of interest. In some embodiments R1and R2are a IPD115, IPD119, IPD125, or IPD130 polypeptide or chimeric IPD115, IPD119, IPD125, or IPD130 polypeptide of the disclosure. The R1polypeptide is fused either directly or through a linker (L) segment to the R2polypeptide. The term "directly" defines fusions in which the polypeptides are joined without a peptide linker. Thus “L” represents a chemical bound or polypeptide segment to which both R1and R2are fused in frame, most commonly L is a linear peptide to which R1and R2are bound by amide bonds linking the carboxy terminus of R1to the amino terminus of L and carboxy terminus of L to the amino terminus of R2. By "fused in frame" is meant that there is no translation termination or disruption between the reading frames of R1and R2. The linking group (L) is generally a polypeptide of between 1 and 500 amino acids in length. The linkers joining the two molecules are preferably designed to (1) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered secondary structure which could interfere with the functional domains of the two proteins, (3) have minimal hydrophobic or charged characteristic which could interact with the functional protein domains and (4) provide steric separation of R1and R2such that R1and R2could interact simultaneously with their corresponding receptors on a single cell. Typically surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence. Other neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Additional amino acids may also be included in the linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the fusions.

[0054] In some embodiments the linkers comprise sequences selected from the group of formulas: (Gly3Ser)n, (Gly4Ser)n, (Gly5Ser)n, (GlynSer)nor (AlaGlySer)nwhere n is an integer. One example of a highly-flexible linker is the (GlySer)-rich spacer region present within the pIII protein of the filamentous bacteriophages, e.g. bacteriophages M13 or fd (Schaller, et al., 1975). Also included are linkers in which an endopeptidase recognition sequence is included, such as for example, Plasmin, Enterokinase, Kallikerin, Urokinase, Tissue Plasminogen activator, clostripain, Chymosin, Collagenase, Russell's Viper Venom Protease, Postproline cleavage enzyme, V8Attorney Docket: 212196-WO-SEC-1 protease, Thrombin and factor Xa. In some embodiments the linker comprises the amino acids from the multi-gene expression vehicle (MGEV), which is cleaved by vacuolar proteases. In other embodiments, peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptides. Especially useful are those hinge regions where the cysteines are replaced with serines. Linkers of the present disclosure include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines. The fusion proteins are not limited by the form, size or number of linker sequences employed and the only requirement of the linker is that functionally it does not interfere adversely with the folding and function of the individual molecules of the fusion. Methods for engineering IPD115, IPD119, IPD125, or IPD130 polypeptides

[0001] Methods for engineering IPD115, IPD119, IPD125, or IPD130 polypeptides are also encompassed by the disclosure. In some embodiments, the method for engineering IPD115, IPD119, IPD125, or IPD130 polypeptides uses rational protein design based on a secondary, tertiary or quaternary structure model of the IPD115, IPD119, IPD125, or IPD130 polypeptide. In silico modeling tools are well known to one skilled in the art and can be used in the methods of the disclosure. In some embodiments, the rational protein design uses an in-silico modeling tool selected from but not limited to PyMOL (PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC.), Maestro©, BioLuminate (Zhu, K.; et al., Proteins, 2014, 82(8), 1646–1655; Salam, N.K et al., Protein Eng. Des. Sel., 2014, 27(10), 365-74; Beard, H. et al. PLoS ONE, 2013, 8(12), e82849), MOE© (Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2015), Jmol, and Discovery Studio© (Accelrys Software Inc. Discovery Studio Modeling Environment, Release 3.5.0, San Diego: Accelrys Software Inc.2013). In some embodiments, the modeling uses Discovery Studio© software. In some embodiments, the method the structural coordinates can be determined by homology modeling. In some embodiments, the method the structural coordinates can be determined by X-ray crystallography or solution NMR.

[0002] In some embodiments, the IPD115, IPD119, IPD125, or IPD130 polypeptide is engineered by the method of the disclosure to have a modified physical property compared to the native IPD115, IPD119, IPD125, or IPD130 polypeptide. In some embodiments, the modified physicalAttorney Docket: 212196-WO-SEC-1 properties include, but are not limited to net surface charge and charge distribution on the protein surface, net hydrophobicity and hydrophobic residue distribution on the protein surface, surface charge density, surface hydrophobicity density, total count of surface ionizable groups, and protein size. In some embodiments, the modified physical in- properties include, but are not limited to solubility, folding, stability, protease stability, digestibility, planta expression, insecticidal potency, spectrum of insecticidal activity, ion channel activity of protomer pore, and receptor binding. In some embodiments, the modified physical property is improved protease stability, improved in-planta expression, improved solubility, improved potency, improved ion-channel activity of protomer pore, and / or improved receptor binding.

[0003] Using the methods of the disclosure, proteolytically-sensitive sites can be identified and may be modified or utilized to produce more stable or more biologically active IPD115, IPD119, IPD125, or IPD130 polypeptides.

[0004] Using methods of the disclosure, sites involved in receptor binding and / or pore formation can be identified and may be modified to create IPD115, IPD119, IPD125, or IPD130 polypeptides having enhanced insecticidal activity; enhanced ability to form channels; and reduced size.

[0005] Using methods of the disclosure, occupation of a site by a water molecule can be identified and can be modified to create IPD115, IPD119, IPD125, or IPD130 molecules having modified flexibility in a region or increasing the number of hydrophobic residues along that surface, which may be involved in receptor binding and / or pore formation.

[0006] Using methods of the disclosure, hydrogen bonding in a region can be identified and the amino acids may be substituted to modify the number of hydrogen bonds, including salt bridges, to create IPD115, IPD119, IPD125, or IPD130 polypeptides having a modified hydrophobic interaction surface facilitating pre-pore and pore formation and / or modified insecticidal activity.

[0007] Using methods of the disclosure, loop regions can be identified and may be modified to create IPD115, IPD119, IPD125, or IPD130 polypeptides having modified channel or pore formation, folding, and / or receptor binding.

[0008] Using methods of the disclosure, complex electrostatic surfaces and hydrophobic or hydrophilic interactions can be identified and modified to create IPD115, IPD119, IPD125, or IPD130 polypeptides having modified receptor interaction.

[0009] Using methods of the disclosure, metal binding sites can be identified and modified toAttorney Docket: 212196-WO-SEC-1 create IPD115, IPD119, IPD125, or IPD130 polypeptides having modified ion channel or pore activity.

[0010] Using methods of the disclosure, amino acids that may be buried or otherwise removed from the surface of the protein that hold in place the three-dimensional structure can be identified and modified to create IPD115, IPD119, IPD125, or IPD130 polypeptides having modified stability or flexibility.

[0011] Using methods of the disclosure, non-specific binding sites to other biomolecules can be identified and modified to create IPD115, IPD119, IPD125, or IPD130 polypeptides having modified receptor binding to the specific receptor and enhanced toxicity.

[0012] Appling various computational tools known to one skilled in the art, coupled with the understanding of saturated mutagenesis, and the structural / functional relationship for IPD115, IPD119, IPD125, or IPD130 polypeptides as disclosed herein, one skilled in the art can identify and modify various physical properties of IPD115, IPD119, IPD125, or IPD130 polypeptides for the better overall performance as an insecticidal protein against the desired targets. Combinatory mutagenesis at various regions can enhance specificity to the current active targets and potentially can also change activity spectrum against different targets. Such targeted combinatorial mutagenesis can be achieved with incorporation of mutagenic oligo nucleotides or generated by gene synthesis or the combination of both approaches. Mutagenesis on defined loop regions can also enhance physical properties of IPD115, IPD119, IPD125, or IPD130 polypeptides such as increasing protein stability by reducing protease degradation ability and increasing thermostability etc. In additional, combinatorial mutagenesis can be applied to the amino acid residues involved in hydrophobic interface surface. Enhancement of hydrophobic interface surface can potentially increase insecticidal activity, thermostability and other physical properties. Additional improvements can also be achieved through mutagenesis of other part of the molecule such as various beta-sheets and alpha helices to increase stability and activity. Nucleic Acid Molecules, and Variants and Fragments Thereof

[0013] Isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding Insecticidal toxin polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid moleculesAttorney Docket: 212196-WO-SEC-1 encoding proteins with regions of sequence homology are provided. As used herein, the term “nucleic acid molecule” refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0014] An "isolated" nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a heterologous recombinant bacterial or plant host cell. An isolated nucleic acid molecule, or biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is free of sequences (optimally protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. A “recombinant” nucleic acid molecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is in a recombinant bacterial or plant host cell. In some embodiments, an “isolated” or “recombinant” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecules encoding Insecticidal toxin polypeptides can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

[0015] In some embodiments an isolated nucleic acid molecule encoding Insecticidal toxin polypeptides has one or more change in the nucleic acid sequence compared to the native or genomic nucleic acid sequence. In some embodiments the change in the native or genomic nucleic acid sequence includes but is 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 the amino acid substitution, insertion, deletion and / or addition compared to the native or genomic sequence; removal of one or more intron; deletion of one or more upstream or downstream regulatoryAttorney Docket: 212196-WO-SEC-1 regions; and deletion of the 5’ and / or 3’ untranslated region associated with the genomic nucleic acid sequence. In some embodiments the nucleic acid molecule encoding a Insecticidal toxin polypeptide is a non-genomic sequence.

[0016] A variety of polynucleotides that encode Insecticidal toxin polypeptides or related proteins are contemplated. Such polynucleotides are useful for production of Insecticidal toxin polypeptides in host cells when operably linked to a suitable promoter, transcription termination and / or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode Insecticidal toxin polypeptides or related proteins. Polynucleotides encoding polypeptides

[0017] A variety of IPD115, IPD119, IPD125, or IPD130 polypeptides are contemplated. Sources of IPD115, IPD119, IPD125, or IPD130 polypeptides or related proteins include fern or other primitive plant species selected from, but not limited to, the Genus Pteris, Polypodium, Nephrolepis, Colysis, Tectaria, Davallia, Polystichum, Adiantum, Asplenium, Blechnum, Lygodium, Ophioglossum, Pyrrosia, Doryopteris, Dryopteris, Pellaea, Gymnocarpium, Cheilanthes, Pteridium, Christella, Lastreopsis, Campyloneurum, Hemionitis, Selliguea, and Arachniodes. In some embodiments, the polynucleotide encoding any one of IPD115, IPD119, IPD125, or IPD130 polypeptide is derived from a Selaginella species 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, 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 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, SelaginellaAttorney Docket: 212196-WO-SEC-1 substipitata, Selaginella tenella, Selaginella tortipila, Selaginella uliginosa, 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.

[0018] One source of polynucleotides that encode the IPD115, IPD119, IPD125, or IPD130 polypeptides or related proteins is a fern or other primitive plant species.

[0019] In some embodiments, the polynucleotide encoding the the IPD115, IPD119, IPD125, or IPD130 polypeptide is derived from a species in the Class Isoetopsida Order Selaginales.

[0020] In some embodiments, the polynucleotide encoding the the IPD115, IPD119, IPD125, or IPD130 polypeptide is derived from a fern species in the Class Isoetopsida, Order Selaginales, Family Selaginellaceae.

[0021] In some embodiments, the polynucleotide encoding the the IPD115, IPD119, IPD125, or IPD130 polypeptide is derived from a species in the Genus Selaginella.

[0022] In some embodiments, the polynucleotide encoding the the IPD115, IPD119, IPD125, or IPD130 polypeptide is derived from a Selaginella species comprising the IPD115, IPD119, IPD125, or IPD130 polynucleotide encoding any one of SEQ ID NOs: 1-104.

[0023] The polynucleotides of SEQ ID NOs: 1-104 can be used to express the IPD115, IPD119, IPD125, or IPD130 polypeptides in recombinant bacterial hosts that include but are not limited to Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas and Rhizobium bacterial host cells. The polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode the IPD115, IPD119, IPD125, or IPD130 polypeptides or related proteins. Such probes can be used to identify homologous or substantially homologous polynucleotides derived from fern or other primitive plant species selected from, but not limited to, the Genus Selaginella.

[0024] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a species in the Genus Pteris. In some embodiments, the IPD115, IPD119, IPD125, or IPD130 polypeptide is derived from a Pteris species selected from but not limted to Pteris aberrans, Pteris abyssinica, Pteris actiniopteroides, Pteris adscensionis, Pteris albersii, Pteris albertiae, Pteris altissima, Pteris amoena, Pteris angustata, Pteris angustipinna, Pteris angustipinnula, Pteris appendiculifera, Pteris arborea, Pteris argyraea, Pteris aspericaulis, PterisAttorney Docket: 212196-WO-SEC-1 asperula, Pteris atrovirens, Pteris auquieri, Pteris austrosinica, Pteris bahamensis, Pteris bakeri, Pteris baksaensis, Pteris balansae, Pteris bambusoides, Pteris barbigera, Pteris barombiensis, Pteris bavazzanoi, Pteris beecheyana, Pteris bella, Pteris berteroana, Pteris biaurita, Pteris biformis, Pteris blanchetiana, Pteris blumeana, Pteris boninensis, Pteris brassii, Pteris brevis, Pteris brooksiana, Pteris buchananii, Pteris buchtienii, Pteris burtonii, Pteris cadieri, Pteris caesia, Pteris caiyangheensis, Pteris calcarea, Pteris calocarpa, Pteris catoptera, Pteris chiapensis, Pteris chilensis, Pteris christensenii, Pteris chrysodioides, Pteris ciliaris, Pteris clemensiae, Pteris comans, Pteris commutata, Pteris concinna, Pteris confertinervia, Pteris confusa, Pteris congesta, Pteris consanguinea, Pteris coriacea, Pteris crassiuscula, Pteris cretica, Pteris croesus, Pteris cryptogrammoides, Pteris cumingii, Pteris dactylina, Pteris daguensis, Pteris dalhousiae, Pteris dataensis, Pteris dayakorum, Pteris decrescens, Pteris decurrens, Pteris deflexa, Pteris deltea, Pteris deltodon, Pteris deltoidea, Pteris dentata, Pteris denticulata, Pteris dispar, Pteris dissimilis, Pteris dissitifolia, Pteris distans, Pteris droogmaniana, Pteris edanyoi, Pteris ekmanii, Pteris elmeri, Pteris elongatiloba, Pteris endoneura, Pteris ensiformis, Pteris esquirolii, Pteris excelsa, Pteris famatinensis, Pteris fauriei, Pteris finotii, Pteris flava, Pteris formosana, Pteris fraseri, Pteris friesii, Pteris gallinopes, Pteris geminata, Pteris gigantea, Pteris glaucovirens, Pteris goeldii, Pteris gongalensis, Pteris grandifolia, Pteris grevilleana, Pteris griffithii, Pteris griseoviridis, Pteris guangdongensis, Pteris guizhouensis, Pteris haenkeana, Pteris hamulosa, Pteris hartiana, Pteris heteroclita, Pteris heteromorpha, Pteris heterophlebia, Pteris hillebrandii, Pteris hirsutissima, Pteris hirtula, Pteris hispaniolica, Pteris holttumii, Pteris hondurensis, Pteris hookeriana, Pteris hossei, Pteris hostmanniana, Pteris hui, Pteris humbertii, Pteris hunanensis, Pteris inaequalis, Pteris incompleta, Pteris inermis, Pteris insigni, Pteris intricata, Pteris intromissa, Pteris irregularis, Pteris iuzonensis, Pteris izuensis, Pteris johannis- winkleri, Pteris junghuhnii, Pteris kawabatae, Pteris keysseri, Pteris khasiana, Pteris kidoi, Pteris kinabaluensis, Pteris kingiana, Pteris kiuschiuensis, Pteris laevis, Pteris lanceifolia, Pteris lastii, Pteris laurea, Pteris laurisilvicola, Pteris lechleri, Pteris lepidopoda, Pteris leptophylla, Pteris liboensis, Pteris ligulata, Pteris limae, Pteris linearis, Pteris litoralis, Pteris livida, Pteris loheri, Pteris longifolia, Pteris longipes, Pteris longipetiolulata, Pteris longipinna, Pteris longipinnula, Pteris luederwaldtii, Pteris luschnathiana, Pteris luzonensis, Pteris lydgatei, Pteris macgregorii, Pteris macilenta, Pteris maclurei, Pteris maclurioides, Pteris macracantha, Pteris macrodon,Attorney Docket: 212196-WO-SEC-1 Pteris macrophylla, Pteris macroptera, Pteris madagascarica, Pteris majestica, Pteris malipoensis, Pteris manniana, Pteris melanocaulon, Pteris melanorhachis, Pteris menglaensis, Pteris mertensioides, Pteris mettenii, Pteris micracantha, Pteris microdictyon, Pteris microlepis, Pteris microptera, Pteris mildbraedii, Pteris moluccana, Pteris monghaiensis, Pteris montis- wilhelminae, Pteris morii, Pteris mucronulata, Pteris multiaurita, Pteris multifida, Pteris muricata, Pteris muricatopedata, Pteris muricella, Pteris mutilata, Pteris natiensis, Pteris navarrensis, Pteris nipponica, Pteris novae-caledoniae, Pteris obtusiloba, Pteris occidentalisinica, Pteris olivacea, Pteris opaca, Pteris oppositipinnata, Pteris orientalis, Pteris orizabae, Pteris oshimensis, Pteris otaria, Pteris pachysora, Pteris pacifica, Pteris paleacea, Pteris papuana, Pteris parhamii, Pteris paucinervata, Pteris paucipinnata, Pteris paulistana, Pteris pearcei, Pteris pedicellata, Pteris pediformis, Pteris pellucida, Pteris perrieriana, Pteris perrottetii, Pteris philippinensis, Pteris phuluangensis, Pteris pilosiuscula, Pteris plumbea, Pteris pluricaudata, Pteris podophylla, Pteris polita, Pteris polyphylla, Pteris porphyrophlebia, Pteris praetermissa, Pteris preussii, Pteris prolifera, Pteris propinqua, Pteris pseudolonchitis, Pteris pseudopellucida, Pteris pteridioides, Pteris puberula, Pteris pulchra, Pteris pungens, Pteris purdoniana, Pteris purpureorachis, Pteris quadriaurita, Pteris quinquefoliata, Pteris quinquepartita, Pteris radicans, Pteris ramosii, Pteris rangiferina, Pteris reducta, Pteris remotifolia, Pteris reptans, Pteris rigidula, Pteris rosenstockii, Pteris roseo-lilacina, Pteris ryukyuensis, Pteris satsumana, Pteris saxatilis, Pteris scabra, Pteris scabripes, Pteris schlechteri, Pteris schwackeana, Pteris semiadnata, Pteris semipinnata, Pteris sericea, Pteris setigera, Pteris setuloso-costulata, Pteris shimenensis, Pteris shimianensis, Pteris silvatica, Pteris similis, Pteris simplex, Pteris sintenensis, Pteris speciosa, Pteris splendens, Pteris splendida, Pteris squamaestipes, Pteris squamipes, Pteris stenophylla, Pteris stridens, Pteris striphnophylla, Pteris subindivisa, Pteris subquinata, Pteris subsimplex, Pteris sumatrana, Pteris swartziana, Pteris taiwanensis, Pteris talamauana, Pteris tapeinidiifolia, Pteris tarandus, Pteris tenuissima, Pteris togoensis, Pteris torricelliana, Pteris trachyrachis, Pteris transparens, Pteris tremula, Pteris treubii, Pteris tricolor, Pteris tripartita, Pteris tussaci, Pteris umbrosa, Pteris undulatipinna, Pteris usambarensis, Pteris vaupelii, Pteris venusta, Pteris verticillata, Pteris vieillardii, Pteris viridissima, Pteris vitiensis, Pteris vittata, Pteris wallichiana, Pteris wangiana, Pteris warburgii,Attorney Docket: 212196-WO-SEC-1 Pteris werneri, Pteris whitfordii, Pteris woodwardioides, Pteris wulaiensis, Pteris yakuinsularis, Pteris yamatensis, Pteris zahlbruckneriana, and Pteris zippelii.

[0025] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Polypodiaceae, Genus Polypodium L. In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Polypodiaceae, Genus Polypodium L. 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, Polypodium anetioides, Polypodium anfractuosum, Polypodium anguinum, Polypodium angustifolium f. remotifolia, Polypodium angustifolium var. amphostenon, Polypodium angustifolium var. heterolepis, Polypodium angustifolium var. monstrosa, Polypodium angustipaleatum, Polypodium angustissimum, Polypodium anisomeron var. pectinatum, Polypodium antioquianum, Polypodium aoristisorum, Polypodium apagolepis, Polypodium apicidens, Polypodium apiculatum, Polypodium apoense, Polypodium appalachianum, Polypodium appressum, Polypodium arenarium, Polypodium argentinum, Polypodium argutum, Polypodium armatum, Polypodium aromaticum, Polypodium aspersum, Polypodium assurgens, Polypodium atrum, Polypodium auriculatum, Polypodium balaonense, Polypodium balliviani, Polypodium bamleri, Polypodium bangii, Polypodium bartlettii, Polypodium basale, Polypodium bernoullii, Polypodium biauritum, Polypodium bifrons, Polypodium blepharodes, Polypodium bolivari, Polypodium bolivianum, Polypodium bolobense, Polypodium bombycinum, Polypodium bombycinum var. insularum, Polypodium bradeorum, Polypodium bryophilum, Polypodium bryopodum, Polypodium buchtienii, Polypodium buesii, Polypodium bulbotrichum, Polypodium caceresii, Polypodium californicum f. brauscombii, Polypodium californicum f. parsonsiae, Polypodium californicum, Polypodium calophlebium, Polypodium calvum, Polypodium camptophyllarium var. abbreviatum, Polypodium capitellatum, Polypodium carpinterae, Polypodium chachapoyense, Polypodium chartaceum, Polypodium chimantense, Polypodium chiricanum, Polypodium choquetangense, Polypodium christensenii, Polypodium christii, Polypodium chrysotrichum, Polypodium ciliolepis, Polypodium cinerascens,Attorney Docket: 212196-WO-SEC-1 Polypodium collinsii, Polypodium colysoides, Polypodium confluens, Polypodium conforme, Polypodium confusum, Polypodium congregatifolium, Polypodium connellii, Polypodium consimile var. bourgaeanum, Polypodium consimile var. minor, 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, Polypodium cucullatum var. 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 diaphanum, 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. 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, Polypodium formosanum, Polypodium fraxinifolium subsp. articulatum, Polypodium fraxinifolium subsp. 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 hancockii, Polypodium haplophlebicum, Polypodium harrisii, Polypodium hastatum var. simplex, Polypodium hawaiiense, Polypodium heanophyllum, Polypodium helleri, Polypodium hemionitidium, Polypodium henryi, Polypodium herzogii, Polypodium hesperium, Polypodium hessii, Polypodium hombersleyi, Polypodium hostmannii,Attorney Docket: 212196-WO-SEC-1 Polypodium humile, Polypodium hyalinum, Polypodium iboense, Polypodium induens var. subdentatum, Polypodium insidiosum, Polypodium insigne, Polypodium intermedium subsp. 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. trichophorum, Polypodium latevagans, Polypodium laxifrons, Polypodium laxifrons var. lividum, Polypodium lehmannianum, Polypodium leiorhizum, Polypodium leptopodon, Polypodium leuconeuron var. angustifolia, Polypodium leuconeuron var. latifolium, Polypodium leucosticta, Polypodium limulum, Polypodium lindigii, Polypodium lineatum, Polypodium lomarioides, Polypodium longifrons, Polypodium loretense, Polypodium loriceum var. 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 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 monoides, Polypodium monticola, Polypodium montigenum, Polypodium moritzianum, Polypodium moultonii, Polypodium multicaudatum, Polypodium multilineatum, Polypodium multisorum, Polypodium munchii, Polypodium muscoides, Polypodium myriolepis, Polypodium myriophyllum, Polypodium myriotrichum, Polypodium nematorhizon, Polypodium nemorale, Polypodium nesioticum, Polypodium nigrescentium, Polypodium nigripes, Polypodium nigrocinctum, Polypodium nimbatum, Polypodium nitidissimum, Polypodium nitidissimum var. latior, Polypodium nubrigenum, Polypodium oligolepis, Polypodium oligosorum, Polypodium oligosorum, Polypodium olivaceum, Polypodium olivaceum var. elatum, Polypodium oodes, Polypodium oosphaerum, PolypodiumAttorney Docket: 212196-WO-SEC-1 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 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 playfairii, Polypodium plebeium var. cooperi, Polypodium plectolepidioides, Polypodium pleolepis, Polypodium plesiosorum var.i, Polypodium podobasis, Polypodium podocarpum, Polypodium poloense, Polypodium polydatylon, Polypodium polypodioides var. aciculare, Polypodium polypodioides var. michauxianum, Polypodium praetermissum, Polypodium preslianum var. immersum, Polypodium procerum, Polypodium procerum, Polypodium productum, Polypodium productum, Polypodium prolongilobum, 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, Polypodium repens var. abruptum, Polypodium revolvens, Polypodium rhachipterygium, Polypodium rhomboideum, Polypodium rigens, Polypodium robustum, Polypodium roraimense, Polypodium roraimense, Polypodium rosei, Polypodium rosenstockii, Polypodium rubidum, Polypodium rudimentum, Polypodium rusbyi, Polypodium sablanianum, Polypodium sarmentosum, Polypodium saxicola, Polypodium schenckii, Polypodium schlechteri, Polypodium scolopendria, Polypodium scolopendria, Polypodium scolopendrium, Polypodium scouleri, Polypodium scutulatum, Polypodium segregatum, Polypodium semihirsutum, Polypodium semihirsutum var. fuscosetosum, Polypodium senile var. minor, Polypodium sericeolanatum, Polypodium serraeforme, Polypodium serricula, Polypodium sesquipedala, Polypodium sessilifolium, Polypodium setosum var. calvum, Polypodium setulosum, Polypodium shaferi, Polypodium sibomense, Polypodium siccum, Polypodium simacense, PolypodiumAttorney Docket: 212196-WO-SEC-1 simulans, Polypodium singeri, Polypodium sinicum, Polypodium sintenisii, Polypodium skutchii, Polypodium sloanei, Polypodium sodiroi, Polypodium sordidulum, Polypodium sordidum, Polypodium sphaeropteroides, Polypodium sphenodes, Polypodium sprucei, Polypodium sprucei var. furcativenosa, 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 var. 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 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, Polypodium tuberosum, Polypodium tunguraguae, Polypodium turquinum, Polypodium turrialbae, Polypodium ursipes, Polypodium vagans, Polypodium valdealatum, Polypodium versteegii, Polypodium villagranii, Polypodium virginianum f. cambroideum, Polypodium virginianum f. peraferens, Polypodium vittarioides, Polypodium vulgare, Polypodium vulgare L., Polypodium vulgare subsp. oreophilum, Polypodium vulgare var. acuminatum, Polypodium vulpinum, Polypodium williamsii, Polypodium wobbense, Polypodium x fallacissimum-guttatum, Polypodium xantholepis, Polypodium xiphopteris, Polypodium yarumalense, Polypodium yungense, and Polypodium zosteriforme.

[0026] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales.

[0027] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Nephrolepidaceae.

[0028] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Nephrolepidaceae, Genus Nephrolepis selected from, but not limited to, Nephrolepis abrupta, Nephrolepis acutifolia, Nephrolepis averyi,Attorney Docket: 212196-WO-SEC-1 Nephrolepis biserrata, Nephrolepis brownii, Nephrolepis copelandi, Nephrolepis cordifolia, Nephrolepis davalliae, Nephrolepis davallioides, Nephrolepis dicksonioides, Nephrolepis exaltata, Nephrolepis falcata, Nephrolepis falciformis, Nephrolepis hippocrepicis, Nephrolepis laurifolia, Nephrolepis lauterbachii, Nephrolepis medlerae, Nephrolepis obliterata, Nephrolepis pectinata, Nephrolepis pendula, Nephrolepis pseudobiserrata, Nephrolepis radicans, Nephrolepis rivularis, and Nephrolepis undulata.

[0029] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the order Polypodiales, Family Polypodiaceae, Genus Colysis selected from, but not limited to, Colysis ampla, Colysis digitata, Colysis diversifolia, Colysis elegans Colysis elliptica, Colysis flexiloba, Colysis hemionitidea, Colysis hemitoma, Colysis henryi, Colysis insignis, Colysis intermedia, Colysis leveillei, Colysis longipes, Colysis pedunculata, Colysis pentaphylla, Colysis pothifolia, Colysis pteropus, Colysis shintenensis, Colysis simplicifrons, Colysis triphylla, Colysis wrightii, and Colysis ×shintenensis.

[0030] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Tectariaceae.

[0031] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Tectariaceae, Genus Tectaria selected from, but not limited to, Tectaria acerifolia, Tectaria acrocarpa, Tectaria adenophora, Tectaria aequatoriensis, Tectaria amblyotis, Tectaria amphiblestra, Tectaria andersonii, Tectaria angelicifolia, Tectaria angulata, Tectaria antioquiana, Tectaria athyrioides, Tectaria athyriosora, Tectaria aurita, Tectaria balansae, Tectaria barberi, Tectaria barteri, Tectaria beccariana, Tectaria blumeana, Tectaria brachiata, Tectaria brauniana, Tectaria brevilobata, Tectaria brooksii, Tectaria buchtienii, Tectaria calcarea, Tectaria camerooniana, Tectaria chattagramica, Tectaria cherasica, Tectaria chimborazensis, Tectaria chinensis, Tectaria christii, Tectaria christovalensis, Tectaria cicutaria, Tectaria coadunata, Tectaria confluens, Tectaria consimilis, Tectaria cordulata, Tectaria coriandrifolia, Tectaria craspedocarpa, Tectaria crenata, Tectaria crinigera, Tectaria croftii, Tectaria curtisii, Tectaria danfuensis, Tectaria decaryana, Tectaria decastroi, Tectaria decurrens, Tectaria degeneri, Tectaria dolichosora, Tectaria draconoptera, Tectaria dubia, Tectaria durvillei, Tectaria ebenina, Tectaria estremerana, Tectaria exauriculata, Tectaria fauriei, Tectaria fengii, Tectaria fernandensis, Tectaria ferruginea, TectariaAttorney Docket: 212196-WO-SEC-1 filisquamata, Tectaria fimbriata, Tectaria fissa, Tectaria gaudichaudii, Tectaria gemmifera, Tectaria godeffroyi, Tectaria grandidentata, Tectaria griffithii var. singaporeana, Tectaria grossedentata, Tectaria hederifolia, Tectaria hekouensis, Tectaria heracleifolia, Tectaria herpetocaulos, Tectaria heterocarpa, Tectaria hilocarpa, Tectaria holttumii, Tectaria hookeri, Tectaria humbertiana, Tectaria hymenodes, Tectaria hymenophylla, Tectaria impressa, Tectaria incisa, Tectaria inopinata, Tectaria isomorpha, Tectaria jacobsii, Tectaria jardini, Tectaria johannis-winkleri, Tectaria keckii, Tectaria kehdingiana, Tectaria kingii, Tectaria kouniensis, Tectaria kweichowensis, Tectaria labrusca, Tectaria lacei, Tectaria laotica, Tectaria latifolia, Tectaria lawrenceana, Tectaria laxa, Tectaria leptophylla, Tectaria lifuensis, Tectaria lizarzaburui, Tectaria lobbii, Tectaria lombokensis, Tectaria macrosora, Tectaria macrota, Tectaria madagascarica, Tectaria magnifica, Tectaria manilensis, Tectaria marchionica, Tectaria media, Tectaria melanocaulis, Tectaria melanocauloides, Tectaria melanorachis, Tectaria menyanthidis, Tectaria mesodon, Tectaria mexicana, Tectaria microchlamys, Tectaria microlepis, Tectaria minuta, Tectaria moorei, Tectaria morlae, Tectaria moussetii, Tectaria murrayi, Tectaria nabirensis, Tectaria nausoriensis, Tectaria nebulosa, Tectaria nesiotica, Tectaria nicaraguensis, Tectaria nicotianifolia, Tectaria nitens, Tectaria novoguineensis, Tectaria organensis, Tectaria palmate, Tectaria pandurifolia, Tectaria pedata, Tectaria pentagonalis, Tectaria perdimorpha, Tectaria phaeocaulis, Tectaria pica, Tectaria pilosa, Tectaria plantaginea, Tectaria pleiosora, Tectaria pleiotoma, Tectaria poilanei, Tectaria polymorpha, Tectaria prolifera, Tectaria pseudosinuata, Tectaria x pteropus-minor, Tectaria pubens, Tectaria puberula, Tectaria pubescens, Tectaria quinquefida, Tectaria quitensis, Tectaria ramosii, Tectaria rara, Tectaria remotipinna, Tectaria repanda, Tectaria rheophytica, Tectaria rigida, Tectaria rivalis, Tectaria rockii, Tectaria rufescens, Tectaria rufovillosa, Tectaria sagenioides, Tectaria schmutzii, Tectaria schultzei, Tectaria seemannii, Tectaria semibipinnata, Tectaria semipinnata, Tectaria seramensis, Tectaria siifolia, Tectaria simaoensis, Tectaria simonsii, Tectaria simulans, Tectaria singaporeana, Tectaria sinuata, Tectaria squamipes, Tectaria stalactica, Tectaria stearnsii, Tectaria stenosemioides, Tectaria subcaudata, Tectaria subconfluens, Tectaria subcordata, Tectaria subdigitata, Tectaria subebenea, Tectaria subrepanda, Tectaria subsageniacea, Tectaria subtriloba, Tectaria subtriphylla, Tectaria sulitii, Tectaria suluensis, Tectaria sumatrana, Tectaria tabonensis, Tectaria taccifolia, Tectaria tahitensis, Tectaria tenerifrons, TectariaAttorney Docket: 212196-WO-SEC-1 tenuifolia, Tectaria teratocarpa, Tectaria ternata, Tectaria transiens, Tectaria translucens, Tectaria tricuspis, Tectaria trifida, Tectaria trifoliata, Tectaria triglossa, Tectaria triloba, Tectaria trimenii, Tectaria trinitensis, Tectaria tripartita, Tectaria variabilis, Tectaria vasta, Tectaria vieillardii, Tectaria villosa, Tectaria vitiensis, Tectaria vivipara, Tectaria waterlotii, Tectaria weberi, Tectaria wightii, Tectaria x amesiana, Tectaria x cynthiae, Tectaria yunnanensis, Tectaria zeylanica, and Tectaria zollingeri.

[0032] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Davalliaceae.

[0033] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Davalliaceae Genus Davallia selected from, but not limited to, Davallia adiantoides, Davallia amabilis, Davallia assamica, Davallia austrosinica, Davallia biflora, Davallia boryana, Davallia brachypoda, Davallia brevisora, Davallia bullata, Davallia bullata, Davallia calvescens, Davallia calvescens, Davallia canariensis, Davallia chaerophylla, Davallia chaerophylloide, Davallia chrysanthemifolia, Davallia clarkei, Davallia cumingii, Davallia cylindrica, Davallia divaricata, Davallia divaricata, Davallia divaricata var. orientale, Davallia domingensis, Davallia dubia, Davallia elmeri, Davallia falcata, Davallia falcinella, Davallia ferulacea, Davallia flaccida, Davallia formosana, Davallia fumarioides, Davallia goudotiana, Davallia gracilis, Davallia griffithiana, Davallia griffithiana, Davallia henryana, Davallia heterophylla, Davallia hookeriana, Davallia hymenophylloides, Davallia immersa, Davallia inaequalis var. minor, Davallia jamaicensis, Davallia khasiyana, Davallia kurzii, Davallia lepida, Davallia lepida, Davallia macraeana, Davallia magellanica, Davallia mariesii, Davallia membranulosa, Davallia membranulosa, Davallia millefolium, Davallia moorei, Davallia multidentata, Davallia nodosa, Davallia novae- guineae, Davallia orientalis, Davallia parallela, Davallia parkeri, Davallia parvipinnula, Davallia patens, Davallia pectinata, Davallia perdurans, Davallia pilosula, Davallia platylepis, Davallia polypodioides, Davallia polypodioides var. hispida, Davallia polypodioides var. pilosula, Davallia pseudocystopteris, Davallia puberula, Davallia pyramidata, Davallia pyxidata, Davallia repens, Davallia rhomboidea, Davallia rhomboidea, Davallia rhomboidea, Davallia sinensis, Davallia sloanei, Davallia solida, Davallia solida, Davallia stipellata, Davallia strigosa, Davallia strigosa, Davallia strigosa var. rhomboidea, Davallia subalpina, Davallia subsolida,Attorney Docket: 212196-WO-SEC-1 Davallia teyermannii, Davallia triangularis, Davallia tripinnata, Davallia truncata, Davallia tyermanni, Davallia tyermannii, Davallia uncinella, Davallia urophylla, Davallia vestita, Davallia wilfordii var. contracta, and Davallia yunnanensis.

[0034] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Dryopteridaceae.

[0035] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Dryopteridaceae, 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 ariticulatipilosum, Polystichum assurgentipinnum, Polystichum atkinsonii, Polystichum attenuatum, Polystichum auriculum, Polystichum bakerianum, Polystichum baoxingense, Polystichum biaristatum, Polystichum bifidum, Polystichum bigemmatum, Polystichum bissectum, Polystichum bomiense, Polystichum brachypterum, Polystichum braunii, Polystichum capillipes, Polystichum castaneum, Polystichum chingiae, Polystichum christii, Polystichum chunii, Polystichum consimile, Polystichum costularisorum, Polystichum craspedosorum, Polystichum crassinervium, Polystichum cringerum, Polystichum cuneatiforme, Polystichum cyclolobum, Polystichum daguanense, Polystichum dangii, Polystichum delavayi, Polystichum deltodon, Polystichum dielsii, Polystichum diffundens, Polystichum discretum, Polystichum disjunctum, Polystichum duthiei, Polystichum elevatovenusum, Polystichum erosum, Polystichum 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 huae, Polystichum ichangense, Polystichum inaense, 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 lonchitis, Polystichum longiaristatum, Polystichum longidens,Attorney Docket: 212196-WO-SEC-1 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, Polystichum morii, Polystichum moupinense, Polystichum muscicola, Polystichum nayongense, Polystichum neoliuii, Polystichum neolobatum, Polystichum nepalense, Polystichum nigrum, Polystichum ningshenense, Polystichum nudisorum, Polystichum obliquum, Polystichum oblongum, Polystichum oligocarpum, Polystichum omeiense, Polystichum oreodoxa, Polystichum orientalitibeticum, 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, Polystichum punctiferum, 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, Polystichum sinense, Polystichum sinotsus-simense, Polystichum sozanense, Polystichum speluncicola, Polystichum squarrosum, Polystichum stenophyllum, Polystichum stimulans, Polystichum subacutidens, Polystichum subdeltodon, Polystichum subfimbriatum, Polystichum submarginale, Polystichum submite, Polystichum subulatum, Polystichum tacticopterum, Polystichum taizhongense, Polystichum tangmaiense, 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.

[0036] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Pteridaceae, Genus Adiantaceae selected from, but not limited to, Adiantum aethiopicum, Adiantum aleuticum, Adiantum bonatianum,Attorney Docket: 212196-WO-SEC-1 Adiantum cajennense, Adiantum capillus-junonis, Adiantum capillus-veneris, Adiantum caudatum, Adiantum chienii, Adiantum chilense, Adiantum cuneatum, Adiantum cunninghamii, Adiantum davidii, Adiantum diaphanum, Adiantum edentulum, Adiantum edgeworthii, Adiantum excisum, Adiantum fengianum, Adiantum fimbriatum, Adiantum flabellulatum, Adiantum formosanum, Adiantum formosum, Adiantum fulvum, Adiantum gravesii, Adiantum hispidulum, Adiantum induratum, Adiantum jordanii, Adiantum juxtapositum, Adiantum latifolium, Adiantum leveillei, Adiantum lianxianense, Adiantum malesianum, Adiantum mariesii, Adiantum monochlamys, Adiantum myriosorum, Adiantum obliquum, Adiantum ogasawarense, Adiantum pedatum, Adiantum pentadactylon, Adiantum peruvianum, Adiantum philippense, Adiantum princeps, Adiantum pubescens, Adiantum raddianum, Adiantum reniforme, Adiantum roborowskii, Adiantum serratodentatum, Adiantum sinicum, Adiantum soboliferum, Adiantum subcordatum, Adiantum tenerum, Adiantum terminatum, Adiantum tetraphyllum, Adiantum venustum, Adiantum viridescens, and Adiantum viridimontanum.

[0037] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Aspleniaceae, Genus Asplenium. In some embodiments, the nucleic acid molecule encoding the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Aspleniaceae, Genus Asplenium L selected from, but not limited to, Asplenium abbreviatum, Asplenium abrotanoides, Asplenium abscissum var. subaequilaterale, Asplenium abscissum, Asplenium achilleifolium, Asplenium acuminatum, Asplenium adiantifrons, Asplenium adiantoides, Asplenium adiantoides var. squamulosum, Asplenium adiantum-nigrum L., Asplenium adiantum-nigrum var. adiantum- nigrum, Asplenium adiantum-nigrum var. yuanum, Asplenium adnatum, Asplenium aethiopicum, Asplenium affine, Asplenium affine var. affine, Asplenium affine var. gilpinae, Asplenium affine var. mettenii, Asplenium affine var. pecten, Asplenium africanum, Asplenium afzelii, Asplenium aitchisonii, Asplenium alatulum, Asplenium alatum, Asplenium alfredii, Asplenium altajense, Asplenium amabile, Asplenium ambohitantelense, Asplenium anceps var. proliferum, Asplenium andapense, Asplenium andersonii, Asplenium angustatum, Asplenium angustum, Asplenium anisophyllum, Asplenium annetii, Asplenium antiquum, Asplenium antrophyoides, Asplenium apertum, Asplenium apogamum, Asplenium aquaticum, Asplenium arboreum, Asplenium arcanum, Asplenium arcuatum, Asplenium argentinum, Asplenium argutum, AspleniumAttorney Docket: 212196-WO-SEC-1 aspidiiforme, Asplenium aspidioides, Asplenium asterolepis, Asplenium auricularium var. acutidens, Asplenium auricularium var. subintegerrimum, Asplenium auriculatum, Asplenium auriculatum var. aequilaterale, Asplenium auritum, Asplenium auritum var. auriculatum, Asplenium auritum var. auritum, Asplenium auritum var. bipinnatifidum, Asplenium auritum var. bipinnatisectum, Asplenium auritum var. davallioides, Asplenium auritum var. macilentum, Asplenium auritum var. rigidum, Asplenium auritum var. subsimplex, Asplenium austrochinense, Asplenium ayopayense, Asplenium badinii, Asplenium balense, Asplenium ballivianii, Asplenium bangii, Asplenium bangii, Asplenium barbaense, Asplenium barclayanum, Asplenium barkamense, Asplenium barteri, Asplenium basiscopicum, Asplenium bicrenatum, Asplenium bifrons, Asplenium bipartitum, Asplenium blastophorum, Asplenium blepharodes, Asplenium blepharophorum, Asplenium boiteaui, Asplenium bolivianum, Asplenium boltonii, Asplenium borealichinense, Asplenium bradei, Asplenium bradeorum, Asplenium bradleyi, Asplenium brausei, Asplenium breedlovei, Asplenium buettneri, Asplenium buettneri var. hildebrandtii, Asplenium bulbiferum, Asplenium bullatum var. bullatum, Asplenium bullatum var. shikokianum, Asplenium bullatum, Asplenium cancellatum, Asplenium capillipes, Asplenium cardiophyllum (Hance), Asplenium caripense, Asplenium carvalhoanum, Asplenium castaneoviride, Asplenium castaneum, Asplenium caudatum, Asplenium celtidifolium (Kunze), Asplenium ceratolepis, Asplenium changputungense, Asplenium chaseanum, Asplenium cheilosorum, Asplenium chengkouense, Asplenium chihuahuense, Asplenium chimantae, Asplenium chimborazense, Asplenium chingianum, Asplenium chlorophyllum, Asplenium chondrophyllum, Asplenium cicutarium, Asplenium cicutarium var. paleaceum, Asplenium cirrhatum, Asplenium cladolepton, Asplenium claussenii, Asplenium coenobiale, Asplenium commutatum, Asplenium congestum, Asplenium conquisitum, Asplenium consimile, Asplenium contiguum, Asplenium contiguum var. hirtulum, Asplenium corderoi, Asplenium cordovense, Asplenium coriaceum, Asplenium coriifolium, Asplenium correardii, Asplenium costale, Asplenium costale var. robustum, Asplenium cowanii, Asplenium crenulatoserrulatum, Asplenium crenulatum, Asplenium crinicaule, Asplenium crinulosum, Asplenium cristatum, Asplenium cryptolepis Fernald, Asplenium cultrifolium L., Asplenium cuneatiforme, Asplenium cuneatum, Asplenium curvatum, Asplenium cuspidatum, Asplenium cuspidatum var cuspidatum, Asplenium cuspidatum var. foeniculaceum, Asplenium cuspidatum var. triculum, Asplenium cuspidatum var. tripinnatum,Attorney Docket: 212196-WO-SEC-1 Asplenium dalhousiae, Asplenium dareoides, Asplenium davallioides, Asplenium davisii, Asplenium debile, Asplenium debile, Asplenium decussatum, Asplenium delavayi, Asplenium delicatulum, Asplenium delicatulum var. cocosensis, Asplenium delitescens, Asplenium delitescens X laetum, Asplenium densum, Asplenium dentatum L., Asplenium dentatum L., Asplenium depauperatum, Asplenium deqenense, Asplenium dianae, Asplenium difforme, Asplenium dilatatum, Asplenium dimidiatum, Asplenium dimidiatum var. boliviense, Asplenium diplazisorum, Asplenium dissectum, Asplenium distans, Asplenium divaricatum, Asplenium divergens, Asplenium divisissimum, Asplenium doederleinii, Asplenium donnell-smithii, Asplenium dregeanum, Asplenium dulongjiangense, Asplenium duplicatoserratum, Asplenium eatonii, Asplenium ebeneum, Asplenium ebenoides, Asplenium ecuadorense, Asplenium eggersii, Asplenium emarginatum, Asplenium enatum, Asplenium ensiforme fo. bicuspe, Asplenium ensiforme fo. ensiforme, Asplenium ensiforme fo. stenophyllum, Asplenium ensiforme, Asplenium erectum var. erectum, Asplenium erectum var. gracile, Asplenium erectum var. usambarense, Asplenium erectum var. zeyheri, &, Asplenium erosum L., Asplenium escaleroense, Asplenium esculentum, Asplenium eutecnum, Asplenium excelsum, Asplenium excisum, Asplenium exiguum, Asplenium extensum, Asplenium falcatum, Asplenium falcinellum, Asplenium faurei, Asplenium feei, Asplenium fengyangshanense, Asplenium ferulaceum, Asplenium fibrillosum, Asplenium filix- femina, Asplenium finckii, Asplenium finlaysonianum, Asplenium flabellulatum, Asplenium flabellulatum var flabellulatum, Asplenium flabellulatum var. partitum, Asplenium flaccidum, Asplenium flavescens, Asplenium flavidum, Asplenium flexuosum, Asplenium fluminense, Asplenium foeniculaceum, Asplenium formosanum, Asplenium formosum var. carolinum, Asplenium formosum var. incultum, Asplenium formosum, Asplenium fournieri, Asplenium fragile, Asplenium fragile var. lomense, Asplenium fragrans, Asplenium fragrans var. foeniculaceum, Asplenium franconis var. gracile, Asplenium fraxinifolium, Asplenium friesiorum, Asplenium friesiorum var. nesophilum, Asplenium fugax, Asplenium fujianense, Asplenium furcatum, Asplenium furfuraceum, Asplenium fuscipes, Asplenium fuscopubescens, Asplenium galeottii, Asplenium gautieri, Asplenium gemmiferum, Asplenium gentryi, Asplenium geppii, Asplenium ghiesbreghtii, Asplenium gilliesii, Asplenium gilpinae, Asplenium glanduliserratum, Asplenium glenniei, Asplenium goldmannii, Asplenium gomezianum, Asplenium grande, Asplenium grandifolium, Asplenium grandifrons, Asplenium gregoriae, Asplenium griffithianum, AspleniumAttorney Docket: 212196-WO-SEC-1 gulingense, Asplenium hainanense, Asplenium hallbergii, Asplenium hallei, Asplenium hallii, Asplenium hangzhouense, Asplenium haplophyllum, Asplenium harpeodes, Asplenium harpeodes var. glaucovirens, Asplenium harpeodes var. incisum, Asplenium harrisii Jenman, Asplenium harrisonii, Asplenium hastatum, Asplenium hebeiense, Asplenium hemionitideum, Asplenium hemitomum, Asplenium henryi, Asplenium herpetopteris, Asplenium herpetopteris var herpetopteris, Asplenium herpetopteris var. acutipinnata, Asplenium herpetopteris var. masoulae, Asplenium herpetopteris var. villosum, Asplenium hesperium, Asplenium heterochroum, Asplenium hians, Asplenium hians var. pallescens, Asplenium hoffmannii, Asplenium holophlebium, Asplenium hondoense, Asplenium horridum, Asplenium hostmannii, Asplenium humistratum, Asplenium hypomelas, Asplenium inaequilaterale, Asplenium incisum, Asplenium incurvatum, Asplenium indicum, Asplenium indicum var. indicum, Asplenium indicum var. yoshingagae, Asplenium induratum, Asplenium indusiatum, Asplenium inexpectatum, Asplenium insigne, Asplenium insiticium, Asplenium insolitum, Asplenium integerrimum, Asplenium interjectum, Asplenium jamesonii, Asplenium jaundeense, Asplenium juglandifolium, Asplenium kangdingense, Asplenium kansuense, Asplenium kassneri, Asplenium kaulfussii, Asplenium kellermanii, Asplenium kentuckiense, Asplenium khullarii, Asplenium kiangsuense, Asplenium kunzeanum, Asplenium lacerum, Asplenium laciniatum, Asplenium laciniatum var. acutipinna, Asplenium laciniatum var. laciniatum, Asplenium laetum fo. minor, Asplenium laetum, Asplenium laetum var. incisoserratum, Asplenium lamprocaulon, Asplenium laserpitiifolium var. morrisonense, Asplenium lastii, Asplenium latedens, Asplenium latifolium, Asplenium laui, Asplenium laurentii, Asplenium leandrianum, Asplenium lechleri, Asplenium leiboense, Asplenium lepidorachis, Asplenium leptochlamys, Asplenium leptophyllum, Asplenium levyi, Asplenium lindbergii, Asplenium lindeni, Asplenium lineatum, Asplenium lividum, Asplenium lobatum, Asplenium lobulatum, Asplenium lokohoense, Asplenium longicauda, Asplenium longicaudatum, Asplenium longifolium, Asplenium longisorum, Asplenium longjinense, Asplenium lorentzii, Asplenium loriceum, Asplenium loxogrammoides, Asplenium lugubre, Asplenium lunulatum, Asplenium lunulatum var. pteropus, Asplenium lushanense, Asplenium lydgatei, Asplenium macilentum, Asplenium macraei, Asplenium macrodictyon, Asplenium macrophlebium, Asplenium macrophyllum, Asplenium macropterum, Asplenium macrosorum, Asplenium macrotis, Asplenium macrurum, Asplenium mainlingense, Asplenium mangindranense, Asplenium mannii, AspleniumAttorney Docket: 212196-WO-SEC-1 marginatum L., Asplenium marojejyense, Asplenium martianum, Asplenium matsumurae, Asplenium mauritiensis Lorence, Asplenium maximum, Asplenium, ii, Asplenium megalura, Asplenium megaphyllum, Asplenium meiotomum, Asplenium melanopus, Asplenium membranifolium, Asplenium meniscioides, Asplenium mesosorum, Asplenium mexicanum, Asplenium micropaleatum, Asplenium microtum, Asplenium mildbraedii, Asplenium mildei, Asplenium minimum, Asplenium minutum, Asplenium miradorense, Asplenium miyunense, Asplenium moccenianum, Asplenium mocquerysii, Asplenium modestum, Asplenium monanthemum var. menziesii, Asplenium monanthes L., Asplenium monanthes var monanthes, Asplenium monanthes var. castaneum, Asplenium monanthes var. wagneri, Asplenium monanthes var. yungense, Asplenium monodon, Asplenium montanum, Asplenium mosetenense, Asplenium moupinense, Asplenium mucronatum, Asplenium munchii, Asplenium muticum, Asplenium myapteron, Asplenium myriophyllu, Asplenium nakanoanum, Asplenium nanchuanense, Asplenium nemorale, Asplenium neolaserpitiifolium, Asplenium neomutijugum, Asplenium neovarians, Asplenium nesii, Asplenium nesioticum, Asplenium nidus L., Asplenium nigricans, Asplenium niponicum, Asplenium normale, Asplenium normale var. angustum, Asplenium obesum, Asplenium oblongatum, Asplenium oblongifolium, Asplenium obovatum, Asplenium obscurum, Asplenium obscurum var. angustum, Asplenium obtusatum var. obtusatum, Asplenium obtusatum var. sphenoides, Asplenium obtusifolium L., Asplenium obtusissimum, Asplenium obversum, Asplenium ochraceum, Asplenium oellgaardii, Asplenium ofeliae, Asplenium oldhami, Asplenium oligosorum, Asplenium olivaceum, Asplenium onopteris L., Asplenium onustum, Asplenium ortegae, Asplenium otites, Asplenium palaciosii, Asplenium palmeri, Asplenium partitum, Asplenium parvisorum, Asplenium parviusculum, Asplenium parvulum, Asplenium patens, Asplenium paucifolium, Asplenium paucijugum, Asplenium paucivenosum, Asplenium pearcei, Asplenium pekinense, Asplenium pellucidum, Asplenium pendulum, Asplenium petiolulatum, Asplenium phyllitidis, Asplenium pimpinellifolium, Asplenium pinnatifidum, Asplenium pinnatum, Asplenium platyneuron, Asplenium platyneuron var. bacculum-rubrum, Asplenium platyneuron var. incisum, Asplenium platyphyllum, Asplenium plumbeum, Asplenium poloense, Asplenium polymeris, Asplenium polymorphum, Asplenium polyodon, Asplenium polyodon var. knudsenii, Asplenium polyodon var. nitidulum, Asplenium polyodon var. sectum, Asplenium polyodon var. subcaudatum, Asplenium polyphyllum, Asplenium poolii, Asplenium poolii fo. simplex, AspleniumAttorney Docket: 212196-WO-SEC-1 poolii var. linearipinnatum, Asplenium potosinum, Asplenium potosinum var. incisum, Asplenium praegracile, Asplenium praemorsum, Asplenium preussii, Asplenium pringleanum, Asplenium pringlei, Asplenium prionitis, Asplenium procerum, Asplenium progrediens, Asplenium projectum, Asplenium prolongatum, Asplenium propinquum, Asplenium protensum, Asplenium pseudoangustum, Asplenium pseudoerectum, Asplenium pseudofontanum, Asplenium pseudolaserpitiifolium, Asplenium pseudonormale, Asplenium pseudopellucidum, Asplenium pseudopraemorsum, Asplenium pseudovarians, Asplenium pseudowilfordii, Asplenium pseudowrightii, Asplenium psilacrum, Asplenium pteropus, Asplenium pubirhizoma, Asplenium pulchellum, Asplenium pulchellum var. subhorizontale, Asplenium pulcherrimum, Asplenium pulicosum, Asplenium pulicosum var. maius, Asplenium pululahuae, Asplenium pumilum, Asplenium pumilum var. hymenophylloides, Asplenium pumilum var. laciniatum, Asplenium purdieanum, Asplenium purpurascens, Asplenium pyramidatum, Asplenium qiujiangense, Asplenium quercicola, Asplenium quitense, Asplenium raddianum, Asplenium radiatum, Asplenium radicans L., Asplenium radicans, Asplenium radicans var. costaricense, Asplenium radicans var. partitum, Asplenium radicans var. radicans, Asplenium radicans var. uniseriale, Asplenium recumbens, Asplenium reflexum, Asplenium regulare var. latior, Asplenium repandulum, Asplenium repens, Asplenium repente, Asplenium resiliens, Asplenium retusulum, Asplenium rhipidoneuron, Asplenium rhizophorum L., Asplenium rhizophyllum, Asplenium rhizophyllum L., Asplenium rhizophyllum var. proliferum, Asplenium rhomboideum, Asplenium rigidum, Asplenium riparium, Asplenium rivale, Asplenium rockii, Asplenium roemerianum, Asplenium roemerianum var. mindensis, Asplenium rosenstockianum, Asplenium rubinum, Asplenium ruizianum, Asplenium rusbyanum, Asplenium ruta-muraria L., Asplenium ruta-muraria var. cryptolepis, Asplenium rutaceum, Asplenium rutaceum var. disculiferum, Asplenium rutaefolium, Asplenium rutifolium, Asplenium salicifolium L., Asplenium salicifolium var. aequilaterale, Asplenium salicifolium var. salicifolium, Asplenium sampsoni, Asplenium sanchezii, Asplenium sanderi, Asplenium sandersonii, Asplenium sanguinolentum, Asplenium sarelii, Asplenium sarelii var. magnum, Asplenium sarelii var. sarelii, Asplenium saxicola, Asplenium scalifolium, Asplenium scandicinum, Asplenium schizophyllum, Asplenium schkuhrii, Asplenium sciadophilum, Asplenium scolopendrium L., Asplenium scortechinii, Asplenium seileri, Asplenium semipinnatum, Asplenium septentrionale, Asplenium serra, Asplenium serra var.Attorney Docket: 212196-WO-SEC-1 imrayanum, Asplenium serratissimum, Asplenium serratum L., Asplenium serratum var. caudatum, Asplenium serricula, Asplenium sessilifolium, Asplenium sessilifolium var. guatemalense, Asplenium sessilifolium var. minus, Asplenium sessilifolium var. occidentale, Asplenium sessilipinnum, Asplenium setosum, Asplenium shepherdii, Asplenium shepherdii var. bipinnatum, Asplenium shepherdii var. flagelliferum, Asplenium shikokianum, Asplenium simii, Asplenium simonsianum, Asplenium sintenisii, Asplenium skinneri, Asplenium skinneri, Asplenium sodiroi, Asplenium soleirolioides, Asplenium solidum var. stenophyllum, Asplenium solmsii, Asplenium sp.-N.-Halle-2234, Asplenium spathulinum, Asplenium spectabile, Asplenium speluncae, Asplenium sphaerosporum, Asplenium sphenotomum, Asplenium spinescens, Asplenium splendens, Asplenium sprucei, Asplenium squamosum L., Asplenium standleyi, Asplenium stellatum, Asplenium stenocarpum, Asplenium stoloniferum, Asplenium stolonipes, Asplenium striatum L., Asplenium stuebelianum, Asplenium stuhlmannii, Asplenium suave, Asplenium subalatum, Asplenium subcrenatum, Asplenium subdigitatum, Asplenium subdimidiatum, Asplenium subintegrum, Asplenium sublaserpitiifolium, Asplenium sublongum, Asplenium subnudum, Asplenium suborbiculare, Asplenium subtenuifolium, Asplenium subtile, Asplenium subtoramanum, Asplenium subtrapezoideum, Asplenium subvarians, Asplenium sulcatum, Asplenium sylvaticum, Asplenium szechuanense, Asplenium taiwanense, Asplenium tenerrimum, Asplenium tenerum, Asplenium tenuicaule, Asplenium tenuifolium, Asplenium tenuifolium var. minor, Asplenium tenuifolium var. tenuifolium, Asplenium tenuissimum, Asplenium ternatum, Asplenium theciferum, Asplenium theciferum var. concinnum, Asplenium thunbergii, Asplenium tianmushanense, Asplenium tianshanense, Asplenium tibeticum, Asplenium tocoraniense, Asplenium toramanum, Asplenium trapezoideum, Asplenium tricholepis, Asplenium trichomanes L., Asplenium trichomanes subsp. inexpectans, Asplenium trichomanes subsp. quadrivalens, Asplenium trichomanes subsp. trichomanes, Asplenium trichomanes var. harovii, Asplenium trichomanes var. herbaceum, Asplenium trichomanes var. repens, Asplenium trichomanes var. viridissimum, Asplenium trichomanes-dentatum L., Asplenium trigonopterum, Asplenium trilobatum, Asplenium trilobum, Asplenium triphyllum, Asplenium triphyllum var. compactum, Asplenium triphyllum var. gracillimum, Asplenium triphyllum var. herbaceum, Asplenium tripteropus, Asplenium triquetrum, Asplenium truncorum, Asplenium tsaratananense, Asplenium tucumanense, Asplenium tuerckheimii, Asplenium tunquiniense, Asplenium ulbrichtii,Attorney Docket: 212196-WO-SEC-1 Asplenium ultimum, Asplenium unilaterale, Asplenium unilaterale var. decurrens, Asplenium unilaterale var. udum, Asplenium unilaterale var. unilaterale, Asplenium uniseriale, Asplenium uropteron, Asplenium vagans, Asplenium vareschianum, Asplenium variabile var. paucijugum, Asplenium variabile var. variabile, Asplenium varians subsp. fimbriatum, Asplenium varians, Asplenium vastum, Asplenium venturae, Asplenium venulosum, Asplenium verapax, Asplenium vesiculosum, Asplenium vespertinum, Asplenium villosum, Asplenium virens, Asplenium viride, Asplenium viridifrons, Asplenium virillae, Asplenium viviparioides, Asplenium viviparum, Asplenium viviparum var viviparum, Asplenium viviparum var. lineatu, Asplenium volubile, Asplenium vulcanicum, Asplenium wacketii, Asplenium wagneri, Asplenium wallichianum, Asplenium warneckei, Asplenium wilfordii, Asplenium williamsii, Asplenium wrightii, Asplenium wrightioides, Asplenium wuliangshanense, Asplenium xianqianense, Asplenium xinjiangense, Asplenium xinyiense, Asplenium yelagagense, Asplenium yoshinagae, Asplenium yunnanense, Asplenium zamiifolium, Asplenium zanzibaricum, Asplenium biscayneanum, Asplenium curtissii, Asplenium ebenoides, Asplenium herb-wagneri, Asplenium heteroresiliens, Asplenium kenzoi, Asplenium plenum, Asplenium wangii, and Asplenium ×clermontiae, Asplenium ×gravesii.

[0038] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Blechnaceae, Genus Blechnum L. In some embodiments, the nucleic acid molecule encoding the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Polypodiales, Family Blechnaceae, Genus Blechnum L. selected from, but not limited to, Blechnum amabile, Blechnum appendiculatum, Blechnum articulatum, Blechnum australe, Blechnum austrobrasilianum, Blechnum binervatum, Blechnum blechnoides, Blechnum brasiliense, Blechnum capense, Blechnum cartilagineum, Blechnum castaneum, Blechnum chambersii, Blechnum chilense, Blechnum colensoi, Blechnum contiguum, Blechnum cordatum, Blechnum coriaceum, Blechnum discolor, Blechnum doodioides, Blechnum durum, Blechnum eburneum, Blechnum ensiforme, Blechnum filiforme, Blechnum fluviatile, Blechnum fragile, Blechnum fraseri, Blechnum fullagari, Blechnum gibbum, Blechnum glandulosum, Blechnum gracile, Blechnum hancockii, Blechnum hastatum, Blechnum howeanum, Blechnum indicum, Blechnum kunthianum, Blechnum laevigatum, Blechnum loxense, Blechnum magellanicum, Blechnum membranaceum, Blechnum microbasis, Blechnum microphyllum, Blechnum milnei, Blechnum minus, Blechnum mochaenum, Blechnum montanum, BlechnumAttorney Docket: 212196-WO-SEC-1 moorei, Blechnum moritzianum, Blechnum nigrum, Blechnum niponicum, Blechnum norfolkianum, Blechnum novae-zelandiae, Blechnum nudum, Blechnum obtusatum, Blechnum occidentale, Blechnum oceanicum, Blechnum orientale, Blechnum patersonii, Blechnum penna- marina, Blechnum polypodioides, Blechnum procerum, Blechnum punctulatum, Blechnum sampaioanum, Blechnum schiedeanum, Blechnum schomburgkii, Blechnum serrulatum, Blechnum simillimum, Blechnum spicant, Blechnum stipitellatum, Blechnum tabulare, Blechnum triangularifolium, Blechnum vieillardii, Blechnum vulcanicum, Blechnum wattsii, Blechnum whelanii, and Blechnum wurunuran.

[0039] In some embodiments, the nucleic acid encoding the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Order Schizaeales; Family Schizaeaceae, Genus Lygodium selected from, but not limited to, Lygodium articulatum, Lygodium circinatum, Lygodium conforme, Lygodium cubense, Lygodium digitatum, Lygodium flexuosum, Lygodium heterodoxum, Lygodium japonicum, Lygodium kerstenii, Lygodium lanceolatum, Lygodium longifolium, Lygodium merrilii, Lygodium micans, Lygodium microphyllum, Lygodium microstachyum, Lygodium oligostachyum, Lygodium palmatum, Lygodium polystachyum, Lygodium radiatum, Lygodium reticulatum, Lygodium salicifolium, Lygodium scandens, Lygodium smithianum, Lygodium subareolatum, Lygodium trifurcatum, Lygodium venustum, Lygodium versteeghii, Lygodium volubile, and Lygodium yunnanense.

[0040] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Genus Ophioglossum L., Botrychium, Botrypus, Helminthostachys, Ophioderma, Cheiroglossa, Sceptridium or Mankyua. In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Ophioglossum Genus is selected from, but not limited to, Ophioglossum californicum, Ophioglossum coriaceum, Ophioglossum costatum, Ophioglossum crotalophoroides, Ophioglossum engelmannii, Ophioglossum falcatum, Ophioglossum gomezianum, Ophioglossum gramineum, Ophioglossum kawamurae, Ophioglossum lusitanicum, Ophioglossum namegatae, Ophioglossum nudicaule, Ophioglossum palmatum, Ophioglossum parvum, Ophioglossum pedunculosum, OphioglossumAttorney Docket: 212196-WO-SEC-1 pendulum, Ophioglossum petiolatum, Ophioglossum pusillum, Ophioglossum reticulatum, Ophioglossum richardsiae, Ophioglossum thermale, and Ophioglossum vulgatum.

[0041] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Pyrrosia Genus selected from, but not limited to, Pyrrosia abbreviata, Pyrrosia angustata, Pyrrosia angustissima, Pyrrosia assimilis, Pyrrosia asterosora, Pyrrosia blepharolepis, Pyrrosia boothii, Pyrrosia borneensis, Pyrrosia brassii, Pyrrosia christii, Pyrrosia confluens, Pyrrosia costata, Pyrrosia dimorpha, Pyrrosia dispar, Pyrrosia distichocarpa, Pyrrosia drakeana, Pyrrosia eleagnifolia, Pyrrosia fengiana, Pyrrosia flocculosa, Pyrrosia foveolata, Pyrrosia fuohaiensis, Pyrrosia gardneri, Pyrrosia hastata, Pyrrosia heterophylla, Pyrrosia intermedia, Pyrrosia laevis, Pyrrosia lanceolata, Pyrrosia liebuschii, Pyrrosia linearifolia, Pyrrosia lingua, Pyrrosia longifolia, Pyrrosia macrocarpa, Pyrrosia madagascariensis, Pyrrosia mannii, Pyrrosia matsudai, Pyrrosia mechowii, Pyrrosia micraster, Pyrrosia mollis, Pyrrosia novo-guineae, Pyrrosia nummulariifolia, Pyrrosia oblanceolata, Pyrrosia obovata, Pyrrosia pannosa, Pyrrosia petiolosa, Pyrrosia piloselloides, Pyrrosia polydactyla, Pyrrosia porosa, Pyrrosia princeps, Pyrrosia pseudodrakeana, Pyrrosia rasamalae, Pyrrosia rhodesiana, Pyrrosia rupestris, Pyrrosia samarensis, Pyrrosia scolopendrina, Pyrrosia sheareri, Pyrrosia shennongensis, Pyrrosia similis, Pyrrosia sphaerosticha, Pyrrosia stigmosa, Pyrrosia stolzii, Pyrrosia subfurfuracea, Pyrrosia transmorrisonensis, and Pyrrosia tricholepis.

[0042] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Doryopteris Genus selected from, but not limited to, Doryopteris collina, Doryopteris concolor, Doryopteris conformis, Doryopteris cordata, Doryopteris cordifolia, Doryopteris crenulans, Doryopteris cyclophylla, Doryopteris davidsei, Doryopteris decipiens, Doryopteris decora, Doryopteris effusa, Doryopteris humbertii, Doryopteris kirkii, Doryopteris kitchingii, Doryopteris latiloba, Doryopteris lomariacea, Doryopteris lorentzii, Doryopteris ludens, Doryopteris madagascariensis, Doryopteris michelii, Doryopteris nobilis, Doryopteris ornithopus, Doryopteris patens, Doryopteris patula, Doryopteris patula, Doryopteris pedata, Doryopteris pedata, Doryopteris pedatoides, Doryopteris pilosa, Doryopteris rediviva, Doryopteris sagittifolia, Doryopteris triphylla, and Doryopteris tryonii.

[0043] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Dryopteris Genus selected from, but not limited to, DryopterisAttorney Docket: 212196-WO-SEC-1 abbreviata, Dryopteris acuminata, Dryopteris aemula, Dryopteris affinis, Dryopteris aitoniana, Dryopteris alpestris, Dryopteris amurensis, Dryopteris anadroma, Dryopteris antarctica, Dryopteris anthracinisquama, Dryopteris aquilinoides, Dryopteris ardechensis, Dryopteris arguta, Dryopteris assimilis, Dryopteris athamantica, Dryopteris atrata, Dryopteris austriaca, Dryopteris azorica, Dryopteris barbigera, Dryopteris basisora, Dryopteris bernieri, Dryopteris bissetiana, Dryopteris bodinieri, Dryopteris borreri, Dryopteris campyloptera, Dryopteris carthusiana, Dryopteris caucasica, Dryopteris caudifrons, Dryopteris caudipinna, Dryopteris celsa, Dryopteris championii, Dryopteris chinensis, Dryopteris chrysocoma, Dryopteris cinnamomea, Dryopteris clintoniana, Dryopteris cochleata, Dryopteris commixta, Dryopteris conjugata, Dryopteris coreanomontana, Dryopteris corleyi, Dryopteris costalisora, Dryopteris crassirhizoma, Dryopteris crinalis, Dryopteris crispifolia, Dryopteris cristata, Dryopteris cycadina, Dryopteris cyclopeltidiformis, Dryopteris cystolepidota, Dryopteris decipiens, Dryopteris dehuaensis, Dryopteris dickinsii, Dryopteris diffracta, Dryopteris dilatata, Dryopteris erythrosora, Dryopteris expansa, Dryopteris fatuhivensis, Dryopteris filix-mas, Dryopteris flaccisquama, Dryopteris formosana, Dryopteris fragrans, Dryopteris fuscipes, Dryopteris fuscoatra, Dryopteris futura, Dryopteris gamblei, Dryopteris glabra, Dryopteris goeringiana, Dryopteris goldieana, Dryopteris guanchica, Dryopteris gushanica, Dryopteris gymnophylla, Dryopteris gymnosora, Dryopteris hadanoi, Dryopteris handeliana, Dryopteris hangchowensis, Dryopteris hasseltii, Dryopteris hawaiiensis, Dryopteris hayatae, Dryopteris hendersonii, Dryopteris himachalensis, Dryopteris hondoensis, Dryopteris huberi, Dryopteris hwangii, Dryopteris inaequalis, Dryopteris indusiata, Dryopteris insularis, Dryopteris integriloba, Dryopteris intermedia, Dryopteris juxtaposita, Dryopteris karwinskyana, Dryopteris kinkiensis, Dryopteris kinokuniensis, Dryopteris knoblochii, Dryopteris koidzumiana, Dryopteris komarovii, Dryopteris labordei, Dryopteris lacera, Dryopteris lachoongensis, Dryopteris laeta, Dryopteris lepidopoda, Dryopteris lepidorachis, Dryopteris liankwangensis, Dryopteris ludoviciana, Dryopteris lunanensis, Dryopteris marginalis, Dryopteris marginata, Dryopteris mauiensis, Dryopteris maximowiczii, Dryopteris maxonii, Dryopteris medioxima, Dryopteris melanocarpa, Dryopteris monticola, Dryopteris munchii, Dryopteris namegatae, Dryopteris neolacera, Dryopteris nipponensis, Dryopteris nubigena, Dryopteris odontoloma, Dryopteris oligodonta, Dryopteris oreades, Dryopteris pacifica, Dryopteris pallida, Dryopteris panda, Dryopteris paraerythrosora, Dryopteris parafuscipes, Dryopteris patula,Attorney Docket: 212196-WO-SEC-1 Dryopteris pentheri, Dryopteris podophylla, Dryopteris polita, Dryopteris polylepis, Dryopteris pseudofilix-mas, Dryopteris pseudosparsa, Dryopteris pseudovaria, Dryopteris pulcherrima, Dryopteris pycnopteroides, Dryopteris redactopinnata, Dryopteris reflexosquamata, Dryopteris remota, Dryopteris rosea, Dryopteris rossii, Dryopteris rosthornii, Dryopteris rubiginosa, Dryopteris rubrobrunnea, Dryopteris ryo-itoana, Dryopteris sabae, Dryopteris sacrosancta, Dryopteris saffordii, Dryopteris salvinii, Dryopteris sandwicensis, Dryopteris saxifraga, Dryopteris saxifragivaria, Dryopteris scottii, Dryopteris setosa, Dryopteris shibipedis, Dryopteris shikokiana, Dryopteris shiroumensis, Dryopteris sichotensis, Dryopteris sieboldii, Dryopteris silaensis, Dryopteris simasakii, Dryopteris simplicior, Dryopteris sinofibrillosa, Dryopteris sinosparsa, Dryopteris sordidipes, Dryopteris sororia, Dryopteris sparsa, Dryopteris spinosa, Dryopteris squamifera, Dryopteris squamiseta, Dryopteris stenolepis, Dryopteris stewartii, Dryopteris subbipinnata, Dryopteris subexaltata, Dryopteris sublacera, Dryopteris submarginata, Dryopteris submontana, Dryopteris subpycnopteroides, Dryopteris subreflexipinna, Dryopteris subtriangularis, Dryopteris tetrapinnata, Dryopteris tokyoensis, Dryopteris triangularis, Dryopteris tsoongii, Dryopteris tsugiwoi, Dryopteris tsutsuiana, Dryopteris unidentata, Dryopteris uniformis, Dryopteris varia, Dryopteris wallichiana, Dryopteris wattsii, Dryopteris x benedictii, Dryopteris x ebinoensis, Dryopteris x triploidea, and Dryopteris yakusilvicola.

[0044] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Pellaea Genus selected from, but not limited to, Pellaea andromedifolia, Pellaea angulosa, Pellaea atropurpurea, Pellaea boivinii, Pellaea brachyptera, Pellaea breweri, Pellaea bridgesii, Pellaea calidirupium, Pellaea calomelanos, Pellaea cordifolia, Pellaea crenata, Pellaea cymbiformis, Pellaea doniana, Pellaea dura, Pellaea falcata, Pellaea flavescens, Pellaea glabella, Pellaea gleichenioides, Pellaea intermedia, Pellaea longipilosa, Pellaea lyngholmii, Pellaea maxima, Pellaea mucronata, Pellaea notabilis, Pellaea ovata, Pellaea paradoxa, Pellaea patula, Pellaea paupercula, Pellaea pectiniformis, Pellaea pinnata, Pellaea pringlei, Pellaea pteroides, Pellaea riedelii, Pellaea rotundifolia, Pellaea rufa, Pellaea sagittata, Pellaea sp. UC 1795070, Pellaea sp. UC1788706, Pellaea sp. Wen 9479, Pellaea sp. Wen 9490, Pellaea ternifolia, Pellaea trichophylla, Pellaea truncata, Pellaea viridis, Pellaea wrightiana, and Pellaea glaciogena.

[0045] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Gymnocarpium Genus selected from, but not limited to, GymnocarpiumAttorney Docket: 212196-WO-SEC-1 appalachianum, Gymnocarpium brittonianum, Gymnocarpium disjunctum, Gymnocarpium Dryopteris, Gymnocarpium jessoense, Gymnocarpium oyamense, Gymnocarpium remotepinnatum, Gymnocarpium robertianum, and Gymnocarpium sp. TH2007-996.

[0046] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Cheilanthes Genus selected from, but not limited to, Cheilanthes acrostica, Cheilanthes adiantoides, Cheilanthes aemula, Cheilanthes alabamensis, Cheilanthes austrotenuifolia, Cheilanthes bonariensis, Cheilanthes brownii, Cheilanthes catanensis, Cheilanthes caudata, Cheilanthes cavernicola, Cheilanthes clevelandii, Cheilanthes contigua, Cheilanthes cooperae, Cheilanthes covillei, Cheilanthes distans, Cheilanthes eatonii, Cheilanthes feei, Cheilanthes fendleri, Cheilanthes fragillima, Cheilanthes glauca, Cheilanthes gracillima, Cheilanthes guanchica, Cheilanthes hispanica, Cheilanthes horridula, Cheilanthes humilis, Cheilanthes intertexta, Cheilanthes intramarginalis, Cheilanthes lanosa, Cheilanthes lasiophylla, Cheilanthes lendigera, Cheilanthes leucopoda, Cheilanthes lindheimeri, Cheilanthes maderensis, Cheilanthes microphylla, Cheilanthes micropteris, Cheilanthes myriophylla, Cheilanthes newberryi, Cheilanthes nitida, Cheilanthes nudiuscula, Cheilanthes parryi, Cheilanthes paucijuga, Cheilanthes peninsularis, Cheilanthes persica, Cheilanthes pinnatifida, Cheilanthes praetermissa, Cheilanthes prenticei, Cheilanthes pringlei, Cheilanthes pseudovellea, Cheilanthes pteroides, Cheilanthes pulchella, Cheilanthes pumilio, Cheilanthes sciadioides, Cheilanthes sieberi Kunze, Cheilanthes tenuifolia, Cheilanthes tinaei, Cheilanthes tomentosa, Cheilanthes vellea, Cheilanthes villosa, Cheilanthes viscida, Cheilanthes wootonii, Cheilanthes wrightii, and Cheilanthes yavapensis.

[0047] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Pteridium Genus selected from, but not limited to, Pteridium aquilinum, Pteridium arachnoideum, Pteridium brownseyi, Pteridium campestris, Pteridium capense, Pteridium caudatum, Pteridium ceheginense, Pteridium centrali-africanum, Pteridium esculentum, Pteridium falcatum, Pteridium feei, Pteridium heredia, Pteridium lanuginosum, Pteridium latiusculum, Pteridium linea, Pteridium pinetorum, Pteridium psittacinum, Pteridium revolutum, Pteridium semihastatum, Pteridium tauricum, Pteridium yarrabense, and Pteridium yunnanense.

[0048] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Christella Genus selected from, but not limited to, Christella arida,Attorney Docket: 212196-WO-SEC-1 Christella augescens, Christella calvescens, Christella crinipes, Christella dentata, Christella hispidula, Christella latipinna, Christella molliuscula, Christella papilio, Christella parasitica, Christella procurrens, Christella scaberula, Christella sp. 097, Christella sp.2257, and Christella subulata.

[0049] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Lastreopsis Genus selected from, but not limited to, Lastreopsis acuminata, Lastreopsis acuta, Lastreopsis amplissima, Lastreopsis barteriana, Lastreopsis boivinii, Lastreopsis currori, Lastreopsis decomposita, Lastreopsis effusa, Lastreopsis exculta, Lastreopsis glabella, Lastreopsis hispida, Lastreopsis killipii, Lastreopsis marginans, Lastreopsis microsora, Lastreopsis munita, Lastreopsis nigritiana, Lastreopsis perrieriana, Lastreopsis pseudoperrieriana, Lastreopsis rufescens, Lastreopsis silvestris, Lastreopsis smithiana, Lastreopsis sp. Kessler 1434, Lastreopsis subrecedens, Lastreopsis subsericea, Lastreopsis subsimilis, Lastreopsis tenera, Lastreopsis tinarooensis, Lastreopsis vogelii, Lastreopsis walleri, Lastreopsis windsorensis, and Lastreopsis wurunuran.

[0050] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Campyloneurum Genus selected from, but not limited to, Campyloneurum abruptum, Campyloneurum aglaolepis, Campyloneurum amphostemon, Campyloneurum anetioides, Campyloneurum angustifolium, Campyloneurum angustipaleatum, Campyloneurum aphanophlebium, Campyloneurum asplundii, Campyloneurum austrobrasilianum, Campyloneurum brevifolium, Campyloneurum centrobrasilianum, Campyloneurum chlorolepis, Campyloneurum coarctatum, Campyloneurum cochense, Campyloneurum costatum, Campyloneurum decurrens, Campyloneurum densifolium, Campyloneurum falcoideum, Campyloneurum fasciale, Campyloneurum fuscosquamatum, Campyloneurum herbaceum, Campyloneurum inflatum, Campyloneurum lapathifolium, Campyloneurum lorentzii, Campyloneurum magnificum Moore, Campyloneurum major, Campyloneurum nitidissimum, Campyloneurum oellgaardi, Campyloneurum ophiocaulon, Campyloneurum oxypholis, Campyloneurum pascoense, Campyloneurum phyllitidis, Campyloneurum repens, Campyloneurum rigidum, Campyloneurum solutum, Campyloneurum sphenodes, Campyloneurum sublucidum, Campyloneurum tenuipes, CampyloneurumAttorney Docket: 212196-WO-SEC-1 tucumanense, Campyloneurum vexatum, Campyloneurum vulpinum, Campyloneurum wacketii, Campyloneurum wurdackii, and Campyloneurum xalapense.

[0051] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Hemionitis Genus selected from, but not limited to, Hemionitis acrosticha, Hemionitis acrostichoides, Hemionitis alismifolia, Hemionitis argentea, Hemionitis arifolia, Hemionitis asplenioides, Hemionitis aurea, Hemionitis aureo-nitens, Hemionitis bipinnata, Hemionitis blumeana, Hemionitis boryanum, Hemionitis brasiliana, Hemionitis cajenensis, Hemionitis callifolia, Hemionitis chaerophylla, Hemionitis citrifolia, Hemionitis concava, Hemionitis cordata, Hemionitis cordifolia, Hemionitis coriacea, Hemionitis cumingiana, Hemionitis dealbata, Hemionitis discolor, Hemionitis elegans, Hemionitis elongata, Hemionitis esculenta, Hemionitis falcata, Hemionitis gigantea, Hemionitis grandifolia, Hemionitis griffithii, Hemionitis gymnopteroidea, Hemionitis hastata, Hemionitis hederifolia, Hemionitis hookeriana, Hemionitis hosei, Hemionitis humilis, Hemionitis immersa, Hemionitis incisa, Hemionitis intermedia, Hemionitis japonica, Hemionitis lanceolata, Hemionitis latifolia, Hemionitis leptophylla, Hemionitis lessonii, Hemionitis levyi, Hemionitis lineata, Hemionitis maingayi, Hemionitis muelleri, Hemionitis obtusa, Hemionitis opaca, Hemionitis otonis, Hemionitis palmata, Hemionitis parasitica, Hemionitis parvula, Hemionitis pedata, Hemionitis pedatifida, Hemionitis pinnata, Hemionitis pinnatifida, Hemionitis plantaginea, Hemionitis podophylla, Hemionitis polypodioides, Hemionitis pothifolia, Hemionitis pozoi, Hemionitis prolifera, Hemionitis reinwardtiana, Hemionitis reticulata, Hemionitis rigida, Hemionitis rufa, Hemionitis sagittata, Hemionitis semicostata, Hemionitis sessilifolia, Hemionitis × smithii, Hemionitis spatulata, Hemionitis stipitata, Hemionitis subcordata, Hemionitis tomentosa, Hemionitis toxotis, Hemionitis triloba, Hemionitis trinervis, Hemionitis vestita, Hemionitis vittaeformis, Hemionitis wilfordii, and Hemionitis zollingeri.

[0052] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Selliguea Genus selected from, but not limited to, Selliguea albicaula, Selliguea albidopaleata, Selliguea albidosquamata, Selliguea albopes, Selliguea archboldii, Selliguea bakeri, Selliguea balbi, Selliguea banaensis, Selliguea bellisquamata, Selliguea bisulcata, Selliguea brooksii, Selliguea caudiformis, Selliguea ceratophylla, Selliguea chenkouensis, Selliguea chinensis, Selliguea chrysotricha, Selliguea conjuncta, Selliguea connexa,Attorney Docket: 212196-WO-SEC-1 Selliguea costulata, Selliguea craspedosora, Selliguea crenatopinnata, Selliguea cretifera, Selliguea cruciformis, Selliguea cunea, Selliguea dactylina, Selliguea dekockii, Selliguea digitata, Selliguea ebenipes, Selliguea echinospora, Selliguea elmeri, Selliguea enervis, Selliguea engleri, Selliguea erythrocarpa, Selliguea feei, Selliguea feeoides, Selliguea ferrea, Selliguea fukienensis, Selliguea glauca, Selliguea glaucopsis, Selliguea gracilipes, Selliguea griffithiana, Selliguea hainanensis, Selliguea hastata, Selliguea hellwigii, Selliguea heterocarpa, Selliguea hirsuta, Selliguea hirtella, Selliguea hunyaensis, Selliguea integerrima, Selliguea katuii, Selliguea kingpingensis, Selliguea kwangtungensis, Selliguea laciniata, Selliguea lagunensis, Selliguea laipoensis, Selliguea lancea, Selliguea lanceola, Selliguea lateritia, Selliguea lauterbachii, Selliguea likiangensis, Selliguea majoensis, Selliguea malacodon, Selliguea metacoela, Selliguea montana, Selliguea murudensis, Selliguea neglecta, Selliguea nigropaleacea, Selliguea nigrovenia, Selliguea oblongifolia, Selliguea obtusa, Selliguea omeiensis, Selliguea oodes, Selliguea oxyloba, Selliguea palmatifida, Selliguea pampylocarpa, Selliguea pellucidifolia, Selliguea pianmaensis, Selliguea pingpienensis, Selliguea plantaginea, Selliguea platyphylla, Selliguea pseudoacrosticha, Selliguea pyrolifolia, Selliguea quasidivaricata, Selliguea rhynchophylla, Selliguea rigida, Selliguea roseomarginata, Selliguea rotunda, Selliguea setacea, Selliguea shandongensis, Selliguea shensiensis, Selliguea similis, Selliguea simplicifolia, Selliguea simplicissima, Selliguea soridens, Selliguea sri-ratu, Selliguea stenophylla, Selliguea stenosquamis, Selliguea stewartii, Selliguea suboxyloba, Selliguea subsparsa, Selliguea subtaeniata, Selliguea taeniata, Selliguea tafana, Selliguea taiwanensis, Selliguea tamdaoensis, Selliguea tarningensis, Selliguea tenuipes, Selliguea tibetana, Selliguea triloba, Selliguea triquetra, Selliguea violascens, Selliguea waltonii, Selliguea whitfordii, Selliguea wuliangshanensis, Selliguea wuyishanica, Selliguea yakuinsularis, and Selliguea yakushimensis.

[0053] In some embodiments, the IPD115, IPD119, IPD125, OR IPD130 polypeptide is derived from a fern species in the Arachniodes Genus selected from, but not limited to, Arachniodes abrupta, Arachniodes acuminata, Arachniodes ailaoshanensis, Arachniodes amabilis, Arachniodes amoena, Arachniodes anshunensis, Arachniodes argillicola, Arachniodes arisanica, Arachniodes aristata, Arachniodes aristatissima, Arachniodes aspidioides, Arachniodes assamica, Arachniodes attenuata, Arachniodes australis, Arachniodes austro-yunnanensis, Arachniodes × azuminoensis, Arachniodes baiseensis, Arachniodes basipinnata, Arachniodes bella, Arachniodes bipinnata,Attorney Docket: 212196-WO-SEC-1 Arachniodes blinii, Arachniodes borealis, Arachniodes calcarata, Arachniodes cantilenae, Arachniodes carvifolia, Arachniodes caudata, Arachniodes caudifolia, Arachniodes cavalerii, Arachniodes centrochinensis, Arachniodes chaerophylloides, Arachniodes chinensis, Arachniodes ii, Arachniodes clivorum, Arachniodes coadnata, Arachniodes coniifolia, Arachniodes cornopteris, Arachniodes cornucervi, Arachniodes costulisora, Arachniodes cyrtomifolia, Arachniodes damiaoshanensis, Arachniodes davalliaeformis, Arachniodes dayaoensis, Arachniodes decomposita, Arachniodes denticulata, Arachniodes denticulata, Arachniodes denticulatabarbensis, Arachniodes denticulatajucunda, Arachniodes diffracta, Arachniodes dimorphophyllum, Arachniodes duplicatoserrata, Arachniodes elevatas, Arachniodes emeiensis, Arachniodes erythrosora, Arachniodes exilis, Arachniodes falcata, Arachniodes fengii, Arachniodes fengyangshanensis, Arachniodes festina, Arachniodes foeniculacea, Arachniodes foliosa, Arachniodes formosa, Arachniodes formosissima, Arachniodes fujianensis, Arachniodes futeshanensis, Arachniodes gansuensis, Arachniodes gigantea, Arachniodes gijiangensis, Arachniodes gizushanensis, Arachniodes globisora, Arachniodes gongshanensis, Arachniodes gradata, Arachniodes grossa, Arachniodes guangnanensis, Arachniodes guangtongensis, Arachniodes guangxiensis, Arachniodes guanxianensis, Arachniodes hainanensis, Arachniodes haniffii, Arachniodes hasseltii, Arachniodes hekiana, Arachniodes hekouensis, Arachniodes henryi, Arachniodes heyuanensis, Arachniodes hiugana, Arachniodes holttumii, Arachniodes huapingensis, Arachniodes hunanensis, Arachniodes hupingshanensis, Arachniodes × ikeminensis, Arachniodes insularis, Arachniodes intermedia, Arachniodes ishingensis, Arachniodes japonica, Arachniodes jiangxiensis, Arachniodes jinfoshanensis, Arachniodes jingdongensis, Arachniodes jinpingensis, Arachniodes jiulongshanensis, Arachniodes kansuensis, Arachniodes kenzo-satakei, Arachniodes kurosawae, Arachniodes kweichowensis, Arachniodes lanceolata, Arachniodes leuconeura, Arachniodes leucostegioides, Arachniodes liyangensis, Arachniodes longipinna, Arachniodes lurida, Arachniodes lushanensis, Arachniodes lushuiensis, Arachniodes macrocarpa, Arachniodes macrostegia, Arachniodes macrostegia, Arachniodes maguanensis, Arachniodes maoshanensis, Arachniodes masakii, Arachniodes maxima, Arachniodes maximowiczii, Arachniodes maximowiczii, Arachniodes menglianensis, Arachniodes mengziensis, Arachniodes michelii, Arachniodes minamitanii, Arachniodes miqueliana, Arachniodes mirabilis, Arachniodes × mitsuyoshiana, Arachniodes multifida,Attorney Docket: 212196-WO-SEC-1 Arachniodes mutica, Arachniodes nanchuanensis, Arachniodes nanqingensis, Arachniodes neoaristata, Arachniodes neobipinnata, Arachniodes neofalcata, Arachniodes neohunanensis, Arachniodes neopodophylla, Arachniodes nibashanensis, Arachniodes nigrospinosa, Arachniodes nipponica, Arachniodes nitidula, Arachniodes obtusiloba, Arachniodes obtusipinnula, Arachniodes obtusissima, Arachniodes ochropteroides, Arachniodes okinawensis, Arachniodes oohorae, Arachniodes palmipes, Arachniodes parasimplicior, Arachniodes pianmaensis, Arachniodes pinnatifida, Arachniodes pseudo-assamica, Arachniodes pseudo-longipinna, Arachniodes pseudo-repens, Arachniodes pseudo-simplicior, Arachniodes pseudoaristata, Arachniodes pseudocavalerii, Arachniodes × pseudohekiana, Arachniodes pubescens, Arachniodes puncticulata, Arachniodes quadripinnata, Arachniodes reducta, Arachniodes repens, Arachniodes respiciens, Arachniodes × respiciens, Arachniodes rhomboidea, Arachniodes rhomboidearhomboidea, Arachniodes rigidissima, Arachniodes sarasiniorum, Arachniodes sasamotoi, Arachniodes semifertilis, Arachniodes setifera, Arachniodes shuangbaiensis, Arachniodes sichuanensis, Arachniodes similis, Arachniodes simplicior, Arachniodes simulans, Arachniodes sino-aristata, Arachniodes sino-rhomboidea, Arachniodes sinomiqueliana, Arachniodes sledgei, Arachniodes sparsa, Arachniodes speciosa, Arachniodes spectabilis, Arachniodes sphaerosora, Arachniodes spino-serrulata, Arachniodes sporadosora, Arachniodes squamulosa, Arachniodes standishii, Arachniodes subamabilis, Arachniodes subamoena, Arachniodes subaristata, Arachniodes subreflexipinna, Arachniodes suijiangensis, Arachniodes superba, Arachniodes × takayamensis, Arachniodes tibetana, Arachniodes tiendongensis, Arachniodes tomitae, Arachniodes tonkinensis, Arachniodes triangularis, Arachniodes tripinnata, Arachniodes tsiangiana, Arachniodes valida, Arachniodes walkerae, Arachniodes webbiana, Arachniodes wulingshanensis, Arachniodes xinpingensis, Arachniodes yakusimensis, Arachniodes yandangshanensis, Arachniodes yaomashanensis, Arachniodes yaoshanensis, Arachniodes yasu-inouei, Arachniodes yinjiangensis, Arachniodes yixinensis, Arachniodes yoshinagae, Arachniodes yunnanensis, Arachniodes yunqiensis, Arachniodes zeylanica, and Arachniodes ziyunshanensis.

[0054] Polynucleotides encoding Insecticidal toxin polypeptides can also be synthesized de novo from a Insecticidal toxin polypeptide sequence. The sequence of the polynucleotide geneAttorney Docket: 212196-WO-SEC-1 can be deduced from a Insecticidal toxin polypeptide sequence through use of the genetic code. Computer programs such as “BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can be used to convert a peptide sequence to the corresponding nucleotide sequence encoding the peptide. Examples of Insecticidal toxin polypeptide sequences that can be used to obtain corresponding nucleotide encoding sequences include, but are not limited to the Insecticidal toxin polypeptides of SEQ ID NOs: 1-104. Furthermore, synthetic Insecticidal toxin polynucleotide sequences of the disclosure can be designed so that they will be expressed in plants.

[0055] In some embodiments the nucleic acid molecule encoding a toxin polypeptide is a polynucleotide having the sequence set forth in any one of SEQ ID NOs: 1-104, and variants, fragments and complements thereof. “Complement” is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. “Polynucleotide sequence variants” is used herein to refer to a nucleic acid sequence that except for the degeneracy of the genetic code encodes the same polypeptide.

[0056] In some embodiments the nucleic acid molecule encoding the Insecticidal toxin 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 that has one or more change in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some embodiments the change to a native or genomic nucleic acid molecule includes but is 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 to introduce at least one amino acid substitution, insertion, deletion and / or addition compared to the native or genomic sequence; removal of one or more intron 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 the 5’ and / or 3’ untranslated region associated with the genomic nucleic acid sequence; insertion of a heterologous 5’ and / or 3’ untranslated region; and modification of a polyadenylation site. In some embodiments the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.Attorney Docket: 212196-WO-SEC-1

[0057] In some embodiments the nucleic acid molecule encoding na Insecticidal toxin polypeptide disclosed herein is a non-genomic polynucleotide having a nucleotide sequence encoding an amino acid sequence 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, to the amino acid sequence of any one of SEQ ID NOs: 1-104, wherein the Insecticidal toxin polypeptide has insecticidal activity.

[0058] In some embodiments the nucleic acid molecule encodes a Insecticidal toxin polypeptide variant comprising one or more amino acid substitutions to the amino acid sequence of any one of SEQ ID NOs: 1-104.

[0059] Also provided are nucleic acid molecules that encode transcription and / or translation products that are subsequently spliced to ultimately produce functional Insecticidal toxin polypeptides. Splicing can be accomplished in vitro or in vivo, and can involve cis- or trans- splicing. The substrate for splicing can be polynucleotides (e.g., RNA transcripts) or polypeptides. An example of cis-splicing of a polynucleotide is where an intron inserted into a coding sequence is removed and the two flanking exon regions are spliced to generate a Insecticidal toxin polypeptide encoding sequence. An example of trans-splicing would be where a polynucleotide is encrypted by separating the coding sequence into two or more fragments that can be separately transcribed and then spliced to form the full-length pesticidal encoding sequence. The use of a splicing enhancer sequence, which can be introduced into a construct, can facilitate splicing either in cis or trans-splicing of polypeptides (US Patent Numbers 6,365,377 and 6,531,316). Thus, in some embodiments the polynucleotides do not directly encode a full-length Insecticidal toxin polypeptide, but rather encode a fragment or fragments of a Insecticidal toxin polypeptide. These polynucleotides can be used to express a functional Insecticidal toxin polypeptide through a mechanism involving splicing, where splicing may occur at the level of polynucleotide (e.g., intron / exon) and / or polypeptide (e.g., intein / extein). This may be useful, for example, in controlling expression of pesticidal activity, since a functional pesticidal polypeptide will only be expressed if all required fragments are expressed in an environment that permits splicing processes to generate functional product. In another example, introduction of one or more insertion sequences into a polynucleotide can facilitate recombination with a low homology polynucleotide;Attorney Docket: 212196-WO-SEC-1 use of an intron or intein for the insertion sequence facilitates the removal of the intervening sequence, thereby restoring function of the encoded variant.

[0060] Nucleic acid molecules that are fragments of these nucleic acid sequences encoding Insecticidal toxin polypeptides are also encompassed by the embodiments. “Nucleotide fragment” as used herein refers to a portion of the nucleic acid sequence encoding a Insecticidal toxin polypeptide. A nucleotide fragment of a nucleic acid sequence may encode a biologically active portion of a Insecticidal toxin polypeptide or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Nucleic acid molecules that are fragments of a nucleic acid sequence encoding a Insecticidal toxin polypeptide comprise at least about 150, 180, 210, 240, 270, 300, 330, 360, 400, 450, or 500 contiguous nucleotides or up to the number of nucleotides present in a full-length nucleic acid sequence encoding a Insecticidal toxin polypeptide disclosed herein, depending upon the intended use. “Contiguous nucleotides” is used herein to refer to nucleotide residues that are immediately adjacent to one another. Fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the Insecticidal toxin polypeptide and, hence, retain insecticidal activity. “Retains insecticidal activity” is used herein to refer to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the insecticidal activity of any one of the full-length Insecticidal toxin polypeptides set forth in SEQ ID NOs: 1-104. In some embodiments, the insecticidal activity is against a Lepidopteran species. In one embodiment, the insecticidal activity is against a Lepidopteran pest selected from European corn borer (Ostrinia nubilalis; ECB), corn earworm (Helicoverpa zea; CEW), black cutworm (Agrotis ipsilon; BCW), fall armyworm (Spodoptera frugiperda; FAW), Soybean looper (Pseudoplusia includens; SBL) and Velvetbean caterpillar (Anticarsia gemmatalis; VBC)).

[0061] In some embodiments the Insecticidal toxin polypeptide is encoded by a nucleic acid sequence sufficiently homologous to any one of the nucleic acid sequences of SEQ ID NOs: 1- 104.

[0062] "Percent (%) sequence identity" with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentAttorney Docket: 212196-WO-SEC-1 sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence ×100).

[0063] In some embodiments a Insecticidal toxin polynucleotide encodes a Insecticidal toxin 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 sequence identity across the entire length of the amino acid sequence of any one of SEQ ID NOs: 1-104.

[0064] In some embodiments polynucleotides are provided encoding chimeric polypeptides comprising regions of at least two different Insecticidal toxin polypeptides of the disclosure.

[0065] In some embodiments polynucleotides are provided encoding chimeric polypeptides comprising an N-terminal Region of a first Insecticidal toxin polypeptide of the disclosure operably fused to a C-terminal Region of a second Insecticidal toxin polypeptide of the disclosure.

[0066] The embodiments also encompass nucleic acid molecules encoding Insecticidal toxin polypeptide variants. “Variants” of the Insecticidal toxin polypeptide encoding nucleic acid sequences include those sequences that encode the Insecticidal toxin polypeptides disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that have been generated, for example, by using site- directed mutagenesis but which still encode the Insecticidal toxin polypeptides disclosed as discussed below.

[0067] The present disclosure provides isolated or recombinant polynucleotides that encode anyAttorney Docket: 212196-WO-SEC-1 of the Insecticidal toxin polypeptides disclosed herein. Those having ordinary skill in the art will readily appreciate that due to the degeneracy of the genetic code, a multitude of nucleotide sequences encoding Insecticidal toxin polypeptides of the present disclosure exist.

[0068] The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleic acid sequences, such as for example by introducing one or more nucleotide substitutions, additions and / or deletions into the corresponding nucleic acid sequence disclosed herein, thereby leading to changes in the amino acid sequence of the encoded Insecticidal toxin polypeptides, without altering the biological activity of the proteins. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.

[0069] Alternatively, variant nucleic acid sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutant polypeptides can be screened for ability to confer pesticidal activity to identify mutants that retain activity using standard assay techniques.

[0070] The polynucleotides of the disclosure and fragments thereof are optionally used as substrates for a variety of recombination and recursive recombination reactions, in addition to standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., to produce additional pesticidal polypeptide homologues and fragments thereof with desired properties. Libraries of variant polynucleotides so produced, cells comprising said libraries, and any recombinant polynucleotide produced by such methods are also embodiments of the disclosure. Additionally, such methods optionally comprise selecting a variant polynucleotide from such libraries based on pesticidal activity of the encoded polypeptide.

[0071] A variety of diversity generating protocols, including nucleic acid recursive recombination protocols are available and fully described in the art. The procedures can be used separately and / or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well as variants of encoded proteins. The encoded protein may be screened for a desired activity or property, e.g. pesticidal activity, for example, in an automated or automatable format, in series or in parallel, by any of the assays known in the art.

[0072]

[0073] Descriptions of a variety of diversity generating procedures for generating modified nucleicAttorney Docket: 212196-WO-SEC-1 acid sequences, e.g., those coding for polypeptides having pesticidal activity or fragments thereof, are found in the following publications and the 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 3:284-290; Christians, et al., (1999) Nat Biotechnol 17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al., (1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA 94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724- 733; Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) Nat Biotechnol 14:315- 319; Gates, et al., (1996) J Mol Biol 255:373-386; Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457; Crameri and Stemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene, 164:49- 53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995) Bio / Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 and Stemmer, (1994) PNAS USA 91:10747-10751.

[0074] Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling, et al., (1997) Anal Biochem 254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369- 374; Smith, (1985) 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 (Eckstein and Lilley, eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA 82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass, et al., (1988) Science 242:240-245); oligonucleotide-directed mutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500; Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller and Smith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modified DNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764; Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye and Eckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988) Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res 16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol 154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, et al., (1988) Nucl Acids Res 16:6987-6999).

[0075] Additional suitable methods include point mismatch repair (Kramer, et al., (1984) CellAttorney Docket: 212196-WO-SEC-1 38:879-887), mutagenesis using repair-deficient host strains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter, (1987) Methods in Enzymol 154:382-403), deletion mutagenesis (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115), restriction-selection and restriction-purification (Wells, et al., (1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total gene synthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar and Khorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene 34:315-323 and Grundström, et al., (1985) Nucl Acids Res 13:3305-3316), double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech 4:450-455). Additional details on many of the above methods can be found in Methods Enzymol Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.

[0076] The nucleotide sequences of the embodiments can also be used to isolate corresponding sequences from a fern source. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences that are selected based on their sequence identity to the entire sequences set forth herein or to fragments thereof are encompassed by the embodiments and include sequences that are orthologs. The term "orthologs" refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation and share substantial sequence identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species.

[0077] In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter "Sambrook". See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.Attorney Docket: 212196-WO-SEC-1

[0078] To identify potential Insecticidal toxin polypeptides from plant collections, the plant cell lysates can be screened with antibodies generated against Insecticidal toxin polypeptides using Western blotting and / or ELISA methods. Positive samples can be further analyzed by various techniques such as antibody based protein purification and identification. Methods of generating antibodies are well known in the art as discussed infra.

[0079] Alternatively, mass spectrometry based protein identification method can be used to identify homologs of Insecticidal toxin polypeptides using protocols in the literatures (Scott Patterson, (1998), 10.22, 1-24, Current Protocol in Molecular Biology published by John Wiley & Son Inc). Additional techniques (protein purification and molecular biology) can be used to isolate the protein and identify the sequences of the homologs.

[0080] In hybridization methods, all or part of the pesticidal nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra. The hybridization probes may be genomic DNA fragments, synthetic oligonucleotides, cDNA fragments, RNA fragments or other oligonucleotides and may be labeled with a detectable group such as 32P or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme or an enzyme co-factor. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleic acid sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutive nucleotides of nucleic acid sequences encoding Insecticidal toxin polypeptides of the disclosure or a fragment or variant thereof. Methods for the preparation of probes for hybridization and stringency conditions are generally known in the art and are disclosed in Sambrook and Russell, (2001), supra, herein incorporated by reference. AlphaFold

[0081] In another embodiment, portions of the polypeptides disclosed herein including, but not limited to, domains, structurally significant regions, minimal active core polypeptide, receptor binding domains, active sites, and protease cleavage sites may be identified using the AlphaFold computational program.Attorney Docket: 212196-WO-SEC-1

[0082] AlphaFold is a computational method that can regularly predict protein structures with atomic accuracy even in cases in which no similar structure is known. The AlphaFold network directly predicts the 3D coordinates of all heavy atoms for a given protein using the primary amino acid sequence and aligned sequences of homologues as inputs. The AlphaFold methods are scalable to very long proteins with accurate domains and domain-packing, and the model is able to provide precise, per-residue estimates of its reliability that should enable confident use of its structure predictions. (Jumper, J., Evans, R., Pritzel, A. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). Antibodies

[0083] Antibodies to a Insecticidal toxin polypeptide of the embodiments or to variants or fragments thereof are also encompassed. The antibodies of the disclosure include polyclonal and monoclonal antibodies as well as fragments thereof which retain their ability to bind to a Insecticidal toxin polypeptide. An antibody, monoclonal antibody or fragment thereof is said to be capable of binding a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody, monoclonal antibody or fragment thereof. The term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as fragments or binding regions or domains thereof (such as, for example, Fab and F(ab).sub.2 fragments) which are capable of binding hapten. Such fragments are typically produced by proteolytic cleavage, such as papain or pepsin. Alternatively, hapten-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry. Methods for the preparation of the antibodies of the present disclosure are generally known in the art. For example, see, Antibodies, A Laboratory Manual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory, N.Y. (1988), as well as the references cited therein. Standard reference works setting forth the general principles of immunology include: Klein, J. Immunology: The Science of Cell- Noncell Discrimination, John Wiley & Sons, N.Y. (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, N.Y. (1980) and Campbell, "Monoclonal Antibody Technology," In Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon, et al., (eds.), Elsevier, Amsterdam (1984). . Antibodies against Insecticidal toxin polypeptides or antigen-binding portions thereof can be produced by a varietyAttorney Docket: 212196-WO-SEC-1 of techniques, including conventional monoclonal antibody methodology, for example the standard somatic cell hybridization technique of Kohler and Milstein, (1975) Nature 256:495. Other techniques for producing monoclonal antibody can also be employed such as viral or oncogenic transformation of B lymphocytes. An animal system for preparing hybridomas is a murine system. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. The antibody and monoclonal antibodies of the disclosure can be prepared by utilizing a Insecticidal toxin polypeptide as antigens.

[0084] A kit for detecting the presence of a Insecticidal toxin polypeptide or detecting the presence of a nucleotide sequence encoding a Insecticidal toxin polypeptide in a sample is provided. In one embodiment, the kit provides antibody-based reagents for detecting the presence of a Insecticidal toxin polypeptide in a tissue sample. In another embodiment, the kit provides labeled nucleic acid probes useful for detecting the presence of one or more polynucleotides encoding a Insecticidal toxin polypeptide. The kit is provided along with appropriate reagents and controls for carrying out a detection method, as well as instructions for use of the kit. Receptor identification and isolation

[0085] Receptors to the Insecticidal toxin polypeptides of the embodiments or to variants or fragments thereof are also encompassed. Methods for identifying receptors are known in the art (see, Hofmann, et. al., (1988) Eur. J. Biochem.173:85-91; Gill, et al., (1995) J. Biol. Chem.27277- 27282) and can be employed to identify and isolate the receptor that recognizes the Insecticidal toxin polypeptide using the brush-border membrane vesicles from susceptible insects. In addition to the radioactive labeling method listed in the cited literatures, a Insecticidal toxin polypeptide can be labeled with fluorescent dye and other common labels such as streptavidin. Brush-border membrane vesicles (BBMV) of susceptible insects such as soybean looper and stink bugs can be prepared according to the protocols listed in the references of Hofmann and Gill above and separated on SDS-PAGE gel and blotted on suitable membrane. Labeled Insecticidal toxin polypeptide can be incubated with blotted membrane of BBMV and labeled Insecticidal toxin polypeptide can be identified with the labeled reporters. Identification of protein band(s) that interact with the Insecticidal toxin polypeptide can be detected by N-terminal amino acid gas phaseAttorney Docket: 212196-WO-SEC-1 sequencing or mass spectrometry based protein identification method (Patterson, (1998) 10.22, 1- 24, Current Protocol in Molecular Biology published by John Wiley & Son Inc). Once the protein is identified, the corresponding gene can be cloned from genomic DNA or cDNA library of the susceptible insects and binding affinity can be measured directly with the Insecticidal toxin polypeptide. Receptor function for insecticidal activity by the Insecticidal toxin polypeptide can be verified by RNAi type of gene knock out method (Rajagopal, et al., (2002) J. Biol. Chem. 277:46849–46851). Nucleotide Constructs, Expression Cassettes and Vectors

[0086] The use of the term "nucleotide constructs" herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotide constructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and- loop structures and the like.

[0087] A further embodiment relates to a transformed organism such as an organism selected from plant and insect cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises a DNA molecule of the embodiments, an expression cassette comprising the DNA molecule or a vector comprising the expression cassette, which may be stably incorporated into the genome of the transformed organism.

[0088] The sequences of the embodiments are provided in DNA constructs for expression in theAttorney Docket: 212196-WO-SEC-1 organism of interest. The construct will include 5' and 3' regulatory sequences operably linked to a sequence of the embodiments. The term "operably linked" as used herein refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. Optionally, operably linked may also mean that the nucleic acid sequences being linked are contiguous and where necessary to join two protein coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.

[0089] Such a DNA construct is provided with a plurality of restriction sites for insertion of the Insecticidal toxin polypeptide gene sequence of the disclosure to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes.

[0090] The DNA construct will generally include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the embodiments, and a transcriptional and translational termination region (i.e., termination region) functional in the organism serving as a host. The transcriptional initiation region (i.e., the promoter) may be native, analogous, foreign or heterologous to the host organism and / or to the sequence of the embodiments. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The term "foreign" as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. Where the promoter or any other nucleotide or amino acid sequence is "foreign" or "heterologous" in reference to a sequence is a sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same / analogous species, one or both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.Attorney Docket: 212196-WO-SEC-1 As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.

[0091] In some embodiments the DNA construct comprises a polynucleotide encoding a Insecticidal toxin polypeptide of the embodiments. In some embodiments the DNA construct comprises a polynucleotide encoding a fusion protein comprising a Insecticidal toxin polypeptide of the embodiments.

[0092] In some embodiments the DNA construct may also include a transcriptional enhancer sequence. As used herein, the term an “enhancer” refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants (, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9:1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments.

[0093] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host or any combination thereof).

[0094] Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau, et al., (1991) Mol. Gen. Genet.262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) Genes Dev.5:141-149; Mogen, et al., (1990) Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al., (1989) Nucleic Acids Res.17:7891-7903 and Joshi, et al., (1987) Nucleic Acid Res.15:9627-9639.

[0095] Where appropriate, a nucleic acid may be optimized for increased expression in the hostAttorney Docket: 212196-WO-SEC-1 organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred usage. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res.17:477-498). Thus, the maize-preferred for a particular amino acid may be derived from known gene sequences from maize. Maize usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra. Methods are available in the art for synthesizing plant-preferred genes. See, for example, Murray, et al., (1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep 37:677-684, 2010, herein incorporated by reference. A Zea maize usage table can be also found at kazusa.or.jp / / cgi- bin / show.cgi?species=4577, which can be accessed using the www prefix. A Glycine max usage table 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.

[0096] In some embodiments the recombinant nucleic acid molecule encoding a Insecticidal toxin polypeptide has maize optimized codons.

[0097] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term "host cell" as used herein refers to a cell which contains a vector and supports the replication and / or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E. 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 maize host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0098] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include:Attorney Docket: 212196-WO-SEC-1 picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and maize chlorotic mottle virus leader (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 co-translational or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum or Golgi apparatus.

[0099] “Signal sequence” as used herein refers to a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. Insecticidal toxins of bacteria are often synthesized as protoxins, which are proteolytically activated in the gut of the target pest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments, the signal sequence is located in the native sequence or may be derived from a sequence of the embodiments. “Leader sequence” as used herein refers to any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a subcellular organelle. Thus, this includes leader sequences targeting transport and / or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. Research in proteomics of the higher plant chloroplast has identified numerous nuclear-encoded thylakoid lumen proteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al. Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta 1503:350-356, 2001), the lumen targeting signal peptide of which can potentially be used in accordance with the present disclosure. (See Kieselbach et al., Photosynthesis Research, 78:249-264, 2003, in particular, Table 2 of this publication disclosing 85 proteins from the chloroplast lumen, which is incorporated herein by reference.

[0100] Suitable chloroplast transit peptides (CTP) are well known to one skilled in the art alsoAttorney Docket: 212196-WO-SEC-1 include chimeric CT’s comprising but not limited to, an N-terminal domain, a central domain or a C-terminal domain from a CTP from Oryza sativa 1-decoy-D xylose-5-Phosphate Synthase Oryza sativa-Superoxide dismutase Oryza sativa-soluble starch synthase Oryza sativa-NADP-dependent Malic acid enzyme Oryza sativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 Oryza sativa- L-Ascorbate peroxidase 5 Oryza sativa-Phosphoglucan water dikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malate dehydrogenase, Zea Mays Thioredoxin M-type (See US Patent Application Publication 2012 / 0304336).

[0101] The Insecticidal toxin polypeptide gene to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast- preferred sequences.

[0102] In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0103] A number of promoters can be used in the practice of the embodiments. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 1999 / 43838 and US Patent Number 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.18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723- 2730); ALS promoter (US Patent Number 5,659,026) and the like. Other constitutive promoters include, for example, those discussed in US Patent Number6,177,611.

[0104] Depending on the desired outcome, it may be beneficial to express the gene from anAttorney Docket: 212196-WO-SEC-1 inducible promoter. Of particular interest for regulating the expression of the nucleotide sequences of the embodiments in plants are wound-inducible promoters. Such wound-inducible promoters, may respond to damage caused by insect feeding, and include potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath.28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1 and wun2; win1 and win2 (Stanford, et al., (1989) Mol. Gen. Genet.215:200- 208); systemin (McGurl, et al., (1992) Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol. 22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene (Corderok, et al., (1994) Plant J.6(2):141-150) and the like.

[0105] Additionally, pathogen-inducible promoters may be employed in the methods and nucleotide constructs of the embodiments. Such pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. Plant Pathol.89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and Van Loon, (1985) Plant Mol. Virol.4:111-116. See also, WO 1999 / 43819.

[0106] Of interest are promoters that are expressed locally at or near the site of pathogen infection. See, for example, Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-Microbe Interactions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen, et al., (1996) Plant J.10:955-966; Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J.3:191-201; Siebertz, et al., (1989) Plant Cell 1:961-968. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero, et al., (1992) Physiol. Mol. Plant Path.41:189-200).

[0107] Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-Attorney Docket: 212196-WO-SEC-1 emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237).

[0108] Tissue-preferred promoters can be utilized to target enhanced Insecticidal toxin polypeptide expression within a particular plant tissue. Tissue-preferred promoters include those discussed in Yamamoto, et al., (1997) Plant J.12(2)255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, et al., (1996) Plant Physiol.112(2):525-535; Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Lam, (1994) Results Probl. Cell Differ.20:181-196; Orozco, et al., (1993) Plant Mol Biol.23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

[0109] Leaf-preferred promoters are known in the art. See, for example, Yamamoto, et al., (1997) Plant J.12(2):255-265; Kwon, et al., (1994) Plant Physiol.105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J.3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0110] Root-preferred or root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire, et al., (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. Biol.14(3):433-443 (root- specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobinAttorney Docket: 212196-WO-SEC-1 genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen- fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a b-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. 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 enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri, et al., (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1' gene fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772) and rolB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691. See also, US Patent Number5,401,836.

[0111] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seed- germinating" promoters (those promoters active during seed germination). See, Thompson, et al., (1989) BioEssays 10:108. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1-phosphate synthase) (see, US Patent Number 6,225,529). Gamma-zein and Glb-1 are endosperm-specific promoters. For dicots, seed-specific promoters include, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku and Goldberg, (1989) Plant Cell 1:1079-1101), bean b-phaseolin, napin, b-conglycinin, glycinin 1, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also, WO 2000 / 12733, where seed-preferred promoters from end1 and end2 genes are disclosed. In dicots, seed specific promoters include but are not limited to seed coat promoter from Arabidopsis, pBAN; and the early seed promoters from Arabidopsis, p26, p63, and p63tr. A promoter that has “preferred” expression in a particular tissueAttorney Docket: 212196-WO-SEC-1 is expressed in that tissue to a greater degree than in at least one other plant tissue. Some tissue- preferred promoters show expression almost exclusively in the particular tissue.

[0112] Where low level expression is desired, weak promoters will be used. Generally, the term "weak promoter" as used herein refers to a promoter that drives expression of a coding sequence at a low level. By low level expression at levels of between about 1 / 1000 transcripts to about 1 / 100,000 transcripts to about 1 / 500,000 transcripts is intended. Alternatively, it is recognized that the term “weak promoters” also encompasses promoters that drive expression in only a few cells and not in others to give a total low level of expression. Where a promoter drives expression at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels.

[0113] Such weak constitutive promoters include, for example the core promoter of the Rsyn7 promoter (WO 1999 / 43838 and US Patent Number 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, those disclosed in US Patent Number 6,177,611.

[0114] The above list of promoters is not meant to be limiting. Any appropriate promoter can be used in the embodiments.

[0115] Generally, the expression cassette will comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature 303:209- 213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820); streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res. 5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419- 423); glyphosate (Shaw, et al., (1986) Science 233:478-481 and US Patent Application Serial Numbers 10 / 004,357 and 10 / 427,692); phosphinothricin (DeBlock, et al., (1987) EMBO J.Attorney Docket: 212196-WO-SEC-1 6:2513-2518). See generally, Yarranton, (1992) Curr. Opin. Biotech.3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. Microbiol.6:2419-2422; Barkley, et al., (1980) in The Operon, pp.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; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol.10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol.78 (Springer-Verlag, Berlin) and Gill, et al., (1988) Nature 334:721-724.

[0116] The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the embodiments. Plant Transformation

[0117] The methods of the embodiments involve introducing a polypeptide or polynucleotide into a plant. "Introducing" as used herein means presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the embodiments do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide(s) or polypeptide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide(s) or polypeptide(s) into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0118] "Stable transformation" is a transformation in which the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" as used herein means that a polynucleotide isAttorney Docket: 212196-WO-SEC-1 introduced into the plant and does not integrate into the genome of the plant. “Plant” as used herein generically includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds and progeny of the same. The plant is a monocot or dicot. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. A “plant element" is intended to reference either a whole plant or a plant component, which may comprise differentiated and / or undifferentiated tissues, for example but not limited to plant tissues, parts, and cell types. In one embodiment, a plant element is one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb, tuber, corm, keiki, shoot, bud, tumor tissue, and various forms of cells and culture (e.g., single cells, protoplasts, embryos, callus tissue). It should be noted that a protoplast is not technically "intact" plant cell (as naturally found with all components), as protoplasts lack a cell wall. plant organ" refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. A plant element "is synonymous to a portion" of a plant, and refers to any part of the plant, and can include distinct tissues and / or organs, and may be used interchangeably with tissue" throughout. Similarly, a plant reproductive element" is intended to generically reference any part of a plant that is able to initiate other plants via either sexual or asexual reproduction of that plant, for example but not limited to: seed, seedling, root, shoot, cutting, scion, graft, stolon, bulb, tuber, corm, keiki, or bud. The plant element may be in plant or in a plant organ, tissue culture, or cell culture.

[0119] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (US Patent Numbers 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 Numbers 5,879,918; 5,886,244 and 5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al., (1988) Biotechnology 6:923-Attorney Docket: 212196-WO-SEC-1 926) 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. Additional transformation procedures can be found in Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol.87:671-674 (soybean); McCabe, et al., (1988) Bio / Technology 6:923- 926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol. 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 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); US Patent Numbers 5,240,855; 5,322,783 and 5,324,646; Klein, et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 311:763-764; US Patent Number 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, New York), pp.197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992) Theor. Appl. Genet.84:560-566 (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).

[0120] In specific embodiments, the sequences of the embodiments can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the Insecticidal toxin polynucleotide or variants and fragments thereof directly into the plant or the introduction of the Insecticidal toxin polypeptide transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet.202:179-185; Nomura, et al., (1986) Plant Sci.44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci.91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science 107:775-784. Alternatively, the Insecticidal toxin polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, transcription from the particle-bound DNAAttorney Docket: 212196-WO-SEC-1 can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma #P3143).

[0121] Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO 1999 / 25821, WO 1999 / 25854, WO 1999 / 25840, WO 1999 / 25855 and WO 1999 / 25853. Briefly, the polynucleotide of the embodiments can be contained in transfer cassette flanked by two non- identical recombination sites. The transfer cassette is introduced into a plant have stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.

[0122] Plant transformation vectors may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as “binary vectors”. Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a “gene of interest” (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the pesticidal gene are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediatedAttorney Docket: 212196-WO-SEC-1 DNA transfer, as is understood in the art (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. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.

[0123] In general, 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 a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Following integration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of appropriate selection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this selection treatment by transferring regularly to a fresh medium. By continuous passage and challenge with appropriate selection, one identifies and proliferates the cells that are transformed with the plasmid vector. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.

[0124] Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent. The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g., Hiei, et al., (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating 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. Since the transformed material contains many cells; both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants.Attorney Docket: 212196-WO-SEC-1

[0125] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive or inducible expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved.

[0126] The nucleotide sequences of the embodiments may be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest within a viral DNA or RNA molecule. It is recognized that the recombinant proteins of the embodiments may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired Insecticidal toxin polypeptide. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a Insecticidal toxin polypeptide of the embodiments, may have the desired pesticidal activity. Such viral polyproteins and the nucleotide sequences that encode for them are encompassed by the embodiments. Methods for providing plants with nucleotide constructs and producing the encoded proteins in the plants, which involve viral DNA or RNA molecules, are known in the art.

[0127] Methods for transformation of chloroplasts are known in the art. See, for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride, et al., (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0128] The embodiments further relate to plant-propagating material of a transformed plant of the embodiments including, but not limited to, seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.Attorney Docket: 212196-WO-SEC-1

[0129] The embodiments may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources 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 italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), sugarcane (Saccharum spp.), oats, barley, and vegetables.

[0130] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Plants of the embodiments include crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

[0131] Plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mung bean, lima bean, fava bean, lentils, chickpea, etc.

[0132] Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.

[0133] PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell, (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.Attorney Docket: 212196-WO-SEC-1

[0134] Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, (2001) supra). In Northern blot analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, (2001) supra). Expression of RNA encoded by the pesticidal gene is then tested by hybridizing the filter to a radioactive probe derived from a pesticidal gene, by methods known in the art (Sambrook and Russell, (2001) supra). Western blot, biochemical assays and the like may be carried out on the transgenic plants to confirm the presence of protein encoded by the pesticidal gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the Insecticidal toxin polypeptide. Methods to Introduce Genome Editing Technologies into Plants

[0135] In some embodiments, the disclosed Insecticidal toxin polynucleotide compositions can be introduced into the genome of a plant using genome editing technologies, or previously introduced Insecticidal toxin polynucleotides in the genome of a plant may be edited using genome editing technologies. For example, the disclosed polynucleotides can be introduced into a desired location in the genome of a plant through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. For example, the disclosed polynucleotides can be introduced into a desired location in a genome using a CRISPR-Cas system, for the purpose of site-specific insertion. The desired location in a plant genome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with an existing trait of interest. Existing traits of interest could be either an endogenous trait or a previously introduced trait.

[0136] In some embodiments, where the disclosed Insecticidal toxin polynucleotide has previously been introduced into a genome, genome editing technologies may be used to alter or modify the introduced polynucleotide sequence. Site specific modifications that can be introduced into the disclosed Insecticidal toxin polynucleotide compositions include those produced using any method for introducing site specific modification, including, but not limited to, through the use of gene repair oligonucleotides, or through the use of double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. Such technologies can be used to modify the previously introduced polynucleotide through the insertion, deletion orAttorney Docket: 212196-WO-SEC-1 substitution of nucleotides within the introduced polynucleotide. Alternatively, double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that may be added include, additional expression elements, such as enhancer and promoter sequences. In another embodiment, genome editing technologies may be used to position additional insecticidally-active proteins in close proximity to the disclosed Insecticidal toxin polynucleotide compositions disclosed herein within the genome of a plant, in order to generate molecular stacks of insecticidally-active proteins.

[0137] An “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 comprises at least one alteration when compared to non-altered target sequence. Such "alterations" include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii). CRISPR System Components Cas Endonucleases and Effectors

[0138] Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain, and include restriction endonucleases that cleave DNA at specific sites without damaging the bases. Examples of endonucleases include restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas (CRISPR-associated) effector endonucleases.

[0139] Cas endonucleases, either as single effector proteins or in an effector complex with other components, unwind the DNA duplex at the target sequence and optionally cleave at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, a crRNA or guide RNA) that is in complex with the Cas effector protein. Such recognition and cutting of a target sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Alternatively, a Cas endonuclease herein may lack DNA cleavage or nicking activity, but can still specifically bind to a DNA target sequence when complexed with a suitable RNA component. (See also U.S. Patent Application US20150082478 published 19 March 2015 andAttorney Docket: 212196-WO-SEC-1 US20150059010 published 26 February 2015).

[0140] Cas endonucleases may occur as individual effectors (Class 2 CRISPR systems) or as part of larger effector complexes (Class I CRISPR systems).

[0141] Cas endonucleases that have been described include, but are not limited to, for example:Cas3 (a feature of Class 1 type I systems), Cas9 (a feature of Class 2 type II systems) and Cas12 (Cpf1) (a feature of Class 2 type V systems).

[0142] Cas endonucleases and effector proteins can be used for targeted genome editing (via simplex and multiplex double-strand breaks and nicks) and targeted genome regulation (via tethering of epigenetic effector domains to either the Cas protein or sgRNA. A Cas endonuclease can also be engineered to function as an RNA-guided recombinase, and via RNA tethers could serve as a scaffold for the assembly of multiprotein and nucleic acid complexes (Mali et al., 2013, Nature Methods Vol.10:957-963).

[0143] Cas endonucleases, when complexed with a cognate guide RNA, recognize, bind to, and optionally nick or cleave a target polynucleotide.

[0144] A Cas endonuclease, effector protein, or functional fragment thereof, for use in the disclosed methods, can be isolated from a native source, or from, a recombinant source where the genetically modified host cell is modified to express the nucleic acid sequence encoding the protein. Alternatively, the Cas protein can be produced using cell free protein expression systems, or be synthetically produced. Effector Cas nucleases may be isolated and introduced into a heterologous cell, or may be modified from its native form to exhibit a different type or magnitude of activity than what it would exhibit in its native source. Such modifications include but are not limited to: fragments, variants, substitutions, deletions, and insertions.

[0145] Fragments and variants of Cas endonucleases and Cas effector proteins can be obtained via methods such as site-directed mutagenesis and synthetic construction. Methods for measuring endonuclease activity are well known in the art such as, but not limiting to, WO2013166113 published 07 November 2013, WO2016186953 published 24 November 2016, and WO2016186946 published 24 November 2016.

[0146] The Cas endonuclease can comprise a modified form of the Cas polypeptide. The modified form of the Cas polypeptide can include an amino acid change (e.g., deletion, insertion, or substitution) that reduces the naturally-occurring nuclease activity of the Cas protein. For example,Attorney Docket: 212196-WO-SEC-1 in some instances, the modified form of the Cas protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas polypeptide (US20140068797 published 06 March 2014). In some cases, the modified form of the Cas polypeptide has no substantial nuclease activity and is referred to as catalytically “inactivated Cas” or “deactivated Cas (dCas).” An inactivated Cas / deactivated Cas includes a deactivated Cas endonuclease (dCas). A catalytically inactive Cas effector protein can be fused to a heterologous sequence to induce or modify activity.

[0147] A Cas endonuclease can be part of a fusion protein comprising one or more heterologous protein domains (e.g., 1, 2, 3, or more domains in addition to the Cas protein). Such a fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains, such as between Cas and a first heterologous domain. Examples of protein domains that may be fused to a Cas protein herein include, without limitation, epitope tags (e.g., histidine [His], V5, FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]), reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], beta-galactosidase, beta-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]), and domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity (e.g., VP16 or VP64), transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. A Cas protein can also be in fusion with a protein that binds DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16.

[0148] A catalytically active and / or inactive Cas endonuclease can be fused to a heterologous sequence (US20140068797 published 06 March 2014). Suitable fusion partners include, but are not limited to, a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide (e.g., a histone or other DNA-binding protein) associated with the target DNA. Additional suitable fusion partners include, but are not limited to, a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitinAttorney Docket: 212196-WO-SEC-1 ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. Further suitable fusion partners include, but are not limited to, a polypeptide that directly provides for increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule / drug-responsive transcription regulator, etc.). A partially active or catalytically inactive Cas-alpha endonuclease can also be fused to another protein or domain, for example Clo51 or FokI nuclease, to generate double-strand breaks (Guilinger et al. Nature Biotechnology, volume 32, number 6, June 2014).

[0149] A catalytically active or inactive Cas protein, such as the Cas-alpha protein described herein, can also be in fusion with a molecule that directs editing of single or multiple bases in a polynucleotide sequence, for example a site-specific deaminase that can change the identity of a nucleotide, for example from C•G to T•A or an A•T to G•C (Gaudelli et al., Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage." Nature (2017); Nishida et al. “Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.” Science 353 (6305) (2016); Komor et al. “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533 (7603) (2016):420-4. A base editing fusion protein may comprise, for example, an active (double strand break creating), partially active (nickase) or deactivated (catalytically inactive) Cas-alpha endonuclease and a deaminase (such as, but not limited to, a cytidine deaminase, an adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABEs, or the like). Base edit repair inhibitors and glycosylase inhibitors (e.g., uracil glycosylase inhibitor (to prevent uracil removal)) are contemplated as other components of a base editing system, in some embodiments.

[0150] Cas endonucleases can be expressed and purified by methods known in the art, for example as described in WO / 2016 / 186953 published 24 November 2016.

[0151] Guide Polynucleotides

[0152] The guide polynucleotide enables target recognition, binding, and optionally cleavage by the Cas endonuclease, and can be a single molecule or a double molecule. The guide polynucleotide sequence can be a RNA sequence, a DNA sequence, or a combination thereof (a RNA-DNA combination sequence). Optionally, the guide polynucleotide can comprise at least oneAttorney Docket: 212196-WO-SEC-1 nucleotide, phosphodiester bond or linkage modification such as, but not limited, to Locked Nucleic Acid (LNA), 5-methyl dC, 2,6-Diaminopurine, 2’-Fluoro A, 2’-Fluoro U, 2'-O-Methyl RNA, phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule, or 5’ to 3’ covalent linkage resulting in circularization. A guide polynucleotide that solely comprises ribonucleic acids is also referred to as a “guide RNA” or “gRNA” (US20150082478 published 19 March 2015 and US20150059010 published 26 February 2015). A guide polynucleotide may be engineered or synthetic.

[0153] The guide polynucleotide includes a chimeric non-naturally occurring guide RNA comprising regions that are not found together in nature (i.e., they are heterologous with each other). For example, a chimeric non-naturally occurring guide RNA comprising a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA, linked to a second nucleotide sequence that can recognize the Cas endonuclease, such that the first and second nucleotide sequence are not found linked together in nature.

[0154] The guide polynucleotide can be a double molecule (also referred to as duplex guide polynucleotide) comprising a crNucleotide sequence (such as a crRNA) and a tracrNucleotide (such as a tracrRNA) sequence. In some cases, there is a linker polynucleotide that connects the crRNA and tracrRNA to form a single guide, for example an sgRNA.

[0155] The crNucleotide includes a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA and a second nucleotide sequence (also referred to as a tracr mate sequence) that is part of a Cas endonuclease recognition (CER) domain. The tracr mate sequence can hybridized to a tracrNucleotide along a region of complementarity and together form the Cas endonuclease recognition domain or CER domain. The CER domain is capable of interacting with a Cas endonuclease polypeptide. The crNucleotide and the tracrNucleotide of the duplex guide polynucleotide can be RNA, DNA, and / or RNA-DNA- combination sequences. In some embodiments, the crNucleotide molecule of the duplex guide polynucleotide is referred to as “crDNA” (when composed of a contiguous stretch of DNA nucleotides) or “crRNA” (when composed of a contiguous stretch of RNA nucleotides), or “crDNA-RNA” (when composed of aAttorney Docket: 212196-WO-SEC-1 combination of DNA and RNA nucleotides). The crNucleotide can comprise a fragment of the crRNA naturally occurring in Bacteria and Archaea. The size of the fragment of the crRNA naturally occurring in Bacteria and Archaea that can be present in a crNucleotide disclosed herein can range from, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, a crRNA molecule is selected from the group consisting of: SEQID NOs: 57, 58, and 59.

[0156] In some embodiments the tracrNucleotide is referred to as “tracrRNA” (when composed of a contiguous stretch of RNA nucleotides) or “tracrDNA” (when composed of a contiguous stretch of DNA nucleotides) or “tracrDNA-RNA” (when composed of a combination of DNA and RNA nucleotides. In one embodiment, the RNA that guides the RNA / Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA. The tracrRNA (trans-activating CRISPR RNA) comprises, in the 5’-to-3’ direction, (i) a sequence that anneals with the repeat region of CRISPR type II crRNA and (ii) a stem loop-comprising portion (Deltcheva et al., Nature 471:602-607). The duplex guide polynucleotide can form a complex with a Cas endonuclease, wherein said guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) can direct the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double-strand break) into the target site. (US20150082478 published 19 March 2015 and US20150059010 published 26 February 2015).

[0157] The guide polynucleotide can also be a single molecule (also referred to as single guide polynucleotide) comprising a crNucleotide sequence linked to a tracrNucleotide sequence. The single guide polynucleotide comprises a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA and a Cas endonuclease recognition domain (CER domain), that interacts with a Cas endonuclease polypeptide.

[0158] Protospacer Adjacent Motif (PAM)

[0159] A “protospacer adjacent motif” (PAM) herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that can be recognized (targeted) by a guide polynucleotide / Cas endonuclease system. The Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not followed by a PAM sequence. TheAttorney Docket: 212196-WO-SEC-1 sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used. The PAM sequence can be of any length but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long.

[0160] A “randomized PAM” and “randomized protospacer adjacent motif” are used interchangeably herein, and refer to a random DNA sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide / Cas endonuclease system. The randomized PAM sequence can be of any length but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long. A randomized nucleotide includes anyone of the nucleotides A, C, G or T.

[0161]

[0162] Guide Polynucleotide / Cas Endonuclease Complexes

[0163] A guide polynucleotide / Cas endonuclease complex described herein is capable of recognizing, binding to, and optionally nicking, unwinding, or cleaving all or part of a target sequence.

[0164] A guide polynucleotide / Cas endonuclease complex that can cleave both strands of a DNA target sequence typically comprises a Cas protein that has all of its endonuclease domains in a functional state (e.g., wild type endonuclease domains or variants thereof retaining some or all activity in each endonuclease domain). Thus, a wild type Cas protein (e.g., a Cas protein disclosed herein), or a variant thereof retaining some or all activity in each endonuclease domain of the Cas protein, is a suitable example of a Cas endonuclease that can cleave both strands of a DNA target sequence.

[0165] A guide polynucleotide / Cas endonuclease complex that can cleave one strand of a DNA target sequence can be characterized herein as having nickase activity (e.g., partial cleaving capability). A Cas nickase typically comprises one functional endonuclease domain that allows the Cas to cleave only one strand (i.e., make a nick) of a DNA target sequence. For example, a Cas9 nickase may comprise (i) a mutant, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., wild type HNH domain). As another example, a Cas9 nickase may comprise (i) a functional RuvC domain (e.g., wild type RuvC domain) and (ii) a mutant, dysfunctional HNH domain. A pair of Cas nickases can be used to increase the specificity of DNA targeting. In general, this can be done by providing two Cas nickases that, by virtue of being associated with RNA componentsAttorney Docket: 212196-WO-SEC-1 with different guide sequences, target and nick nearby DNA sequences on opposite strands in the region for desired targeting. Such nearby cleavage of each DNA strand creates a double-strand break (i.e., a DSB with single-stranded overhangs), which is then recognized as a substrate for non-homologous-end-joining, NHEJ (prone to imperfect repair leading to mutations) or homologous recombination, HR. Each nick in these embodiments can be at least about 5, between 5 and 10, at least 10, between 10 and 15, at least15, between 15 and 20, at least 20, between 20 and 30, at least 30, between 30 and 40, at least 40, between 40 and 50, at least 50, between 50 and 60, at least 60, between 60 and 70, at least 70, between 70 and 80, at least 80, between 80 and 90, at least 90, between 90 and 100, or 100 or greater (or any integer between 5 and 100) bases apart from each other, for example. One or two Cas nickase proteins herein can be used in a Cas nickase pair. For example, a Cas9 nickase with a mutant RuvC domain, but functioning HNH domain (i.e., Cas9 HNH+ / RuvC-), can be used (e.g., Streptococcus pyogenes Cas9 HNH+ / RuvC-). Each Cas9 nickase (e.g., Cas9 HNH+ / RuvC-) can be directed to specific DNA sites nearby each other (up to 100 base pairs apart) by using suitable RNA components herein with guide RNA sequences targeting each nickase to each specific DNA site.

[0166] A guide polynucleotide / Cas endonuclease complex in certain embodiments can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence. Such a complex may comprise a Cas protein in which all of its nuclease domains are mutant, dysfunctional. For example, a Cas9 protein that can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence, may comprise both a mutant, dysfunctional RuvC domain and a mutant, dysfunctional HNH domain. A Cas protein herein that binds, but does not cleave, a target DNA sequence can be used to modulate gene expression, for example, in which case the Cas protein could be fused with a transcription factor (or portion thereof) (e.g., a repressor or activator, such as any of those disclosed herein). Modification of Genomes with Novel CRISPR-Cas System Components

[0167] As described herein, a guided Cas endonuclease can recognize, bind to a DNA target sequence and introduce a single strand (nick) or double-strand break. Once a single or double- strand break is induced in the DNA, the cell’s DNA repair mechanism is activated to repair the break. Error-prone DNA repair mechanisms can produce mutations at double-strand break sites.Attorney Docket: 212196-WO-SEC-1 The most common repair mechanism to bring the broken ends together is the nonhomologous end- joining (NHEJ) pathway (Bleuyard et al., (2006) DNA Repair 5:1-12). The structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements (such as chromosomal translocations) are possible (Siebert and Puchta, 2002, Plant Cell 14:1121- 31; Pacher et al., 2007, Genetics 175:21-9).

[0168] DNA double-strand breaks appear to be an effective factor to stimulate homologous recombination pathways (Puchta et al., (1995) Plant Mol Biol 28:281-92; Tzfira and White, (2005) Trends Biotechnol 23:567-9; Puchta, (2005) J Exp Bot 56:1-14). Using DNA-breaking agents, a two- to nine-fold increase of homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al., (1995) Plant Mol Biol 28:281-92). In maize protoplasts, experiments with linear DNA molecules demonstrated enhanced homologous recombination between plasmids (Lyznik et al., (1991) Mol Gen Genet 230:209-18).

[0169] Homology-directed repair (HDR) is a mechanism in cells to repair double-stranded and single stranded DNA breaks. Homology-directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. 79:181-211). The most common form of HDR is called homologous recombination (HR), which has the longest sequence homology requirements between the donor and acceptor DNA. Other forms of HDR include single-stranded annealing (SSA) and breakage-induced replication, and these require shorter sequence homology relative to HR. Homology-directed repair at nicks (single-stranded breaks) can occur via a mechanism distinct from HDR at double-strand breaks (Davis and Maizels. PNAS (0027-8424), 111 (10), p. E924-E932).

[0170] Alteration of the genome of a prokaryotic and eukaryotic cell or organism cell, for example, through homologous recombination (HR), is a powerful tool for genetic engineering. Homologous recombination has been demonstrated in plants (Halfter et al., (1992) Mol Gen Genet 231:186-93) and insects (Dray and Gloor, 1997, Genetics 147:689-99). Homologous recombination has also been accomplished in other organisms. For example, at least 150-200 bp of homology was required for homologous recombination in the parasitic protozoan Leishmania (Papadopoulou and Dumas, (1997) Nucleic Acids Res 25:4278-86). In the filamentous fungus Aspergillus nidulans, gene replacement has been accomplished with as little as 50 bp flanking homology (Chaveroche et al., (2000) Nucleic Acids Res 28:e97). Targeted gene replacement has also been demonstrated in theAttorney Docket: 212196-WO-SEC-1 ciliate Tetrahymena thermophila (Gaertig et al., (1994) Nucleic Acids Res 22:5391-8). In mammals, homologous recombination has been most successful in the mouse using pluripotent embryonic stem cell lines (ES) that can be grown in culture, transformed, selected and introduced into a mouse embryo (Watson et al., 1992, Recombinant DNA, 2nd Ed., Scientific American Books distributed by WH Freeman & Co.).

[0171] Gene Targeting

[0172] The guide polynucleotide / Cas systems described herein can be used for gene targeting.

[0173] In general, DNA targeting can be performed by cleaving one or both strands at a specific polynucleotide sequence in a cell with a Cas protein associated with a suitable polynucleotide component. Once a single or double-strand break is induced in the DNA, the cell’s DNA repair mechanism is activated to repair the break via nonhomologous end-joining (NHEJ) or Homology- Directed Repair (HDR) processes which can lead to modifications at the target site.

[0174] The length of the DNA sequence at the target site can vary, and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. It is further possible that the target site can be palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary strand. The nick / cleavage site can be within the target sequence or the nick / cleavage site could be outside of the target sequence. In another variation, the cleavage could occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other cases, the incisions could be staggered to produce single-stranded overhangs, also called “sticky ends”, which can be either 5' overhangs, or 3' overhangs. Active variants of genomic target sites can also be used. Such active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the given target site, wherein the active variants retain biological activity and hence are capable of being recognized and cleaved by an Cas endonuclease.

[0175] Assays to measure the single or double-strand break of a target site by an endonuclease are known in the art and generally measure the overall activity and specificity of the agent on DNA substrates comprising recognition sites.

[0176] A targeting method herein can be performed in such a way that two or more DNA target sites are targeted in the method, for example. Such a method can optionally be characterized as aAttorney Docket: 212196-WO-SEC-1 multiplex method. Two, three, four, five, six, seven, eight, nine, ten, or more target sites can be targeted at the same time in certain embodiments. A multiplex method is typically performed by a targeting method herein in which multiple different RNA components are provided, each designed to guide a guide polynucleotide / Cas endonuclease complex to a unique DNA target site.

[0177]

[0178] Gene Editing

[0179] The process for editing a genomic sequence combining DSB and modification templates generally comprises: introducing into a host cell a DSB-inducing agent, or a nucleic acid encoding a DSB-inducing agent, that recognizes a target sequence in the chromosomal sequence and is able to induce a DSB in the genomic sequence, and at least one polynucleotide modification template comprising at least one nucleotide alteration when compared to the nucleotide sequence to be edited. The polynucleotide modification template can further comprise nucleotide sequences flanking the at least one nucleotide alteration, in which the flanking sequences are substantially homologous to the chromosomal region flanking the DSB. Genome editing using DSB-inducing agents, such as Cas-gRNA complexes, has been described, for example in US20150082478 published on 19 March 2015, WO2015026886 published on 26 February 2015, WO2016007347 published 14 January 2016, and WO / 2016 / 025131 published on 18 February 2016.

[0180] Some uses for guide RNA / Cas endonuclease systems have been described (see for example:US20150082478 A1 published 19 March 2015, WO2015026886 published 26 February 2015, and US20150059010 published 26 February 2015) and include but are not limited to modifying or replacing nucleotide sequences of interest (such as a regulatory elements), insertion of polynucleotides of interest, gene knock-out, gene-knock in, modification of splicing sites and / or introducing alternate splicing sites, modifications of nucleotide sequences encoding a protein of interest, amino acid and / or protein fusions, and gene silencing by expressing an inverted repeat into a gene of interest.

[0181] Proteins may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of the protein(s) can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel et al., (1987) Meth Enzymol 154:367-82; U.S. Patent No.Attorney Docket: 212196-WO-SEC-1 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance regarding amino acid substitutions not likely to affect biological activity of the protein is found, for example, in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, D.C.). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable. Conservative deletions, insertions, and amino acid substitutions are not expected to produce radical changes in the characteristics of the protein, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by routine screening assays. Assays for double-strand-break-inducing activity are known and generally measure the overall activity and specificity of the agent on DNA substrates comprising target sites.

[0182] Described herein are methods for genome editing with a Cas endonuclease and complexes with a Cas endonuclease and a guide polynucleotide. Following characterization of the guide RNA and PAM sequence, components of the endonuclease and associated CRISPR RNA (crRNA) may be utilized to modify chromosomal DNA in other organisms including plants. To facilitate optimal expression and nuclear localization (for eukaryotic cells), the genes comprising the complex may be optimized as described in WO2016186953 published 24 November 2016, and then delivered into cells as DNA expression cassettes by methods known in the art. The components necessary to comprise an active complex may also be delivered as RNA with or without modifications that protect the RNA from degradation or as mRNA capped or uncapped (Zhang, Y. et al., 2016, Nat. Commun. 7:12617) or Cas protein guide polynucleotide complexes (WO2017070032 published 27 April 2017), or any combination thereof. Additionally, a part or part(s) of the complex and crRNA may be expressed from a DNA construct while other components are delivered as RNA with or without modifications that protect the RNA from degradation or as mRNA capped or uncapped (Zhang et al. 2016 Nat. Commun. 7:12617) or Cas protein guide polynucleotide complexes (WO2017070032 published 27 April 2017) or any combination thereof. To produce crRNAs in-vivo, tRNA derived elements may also be used to recruit endogenous RNAses to cleave crRNA transcripts into mature forms capable of guiding the complex to its DNA target site, as described, for example, in WO2017105991 published 22 June 2017. Nickase complexes may be utilized separately or concertedly to generate a single or multiple DNA nicks on one or both DNAAttorney Docket: 212196-WO-SEC-1 strands. Furthermore, the cleavage activity of the Cas endonuclease may be deactivated by altering key catalytic residues in its cleavage domain (Sinkunas, T. et al., 2013, EMBO J. 32:385-394) resulting in a RNA guided helicase that may be used to enhance homology directed repair, induce transcriptional activation, or remodel local DNA structures. Moreover, the activity of the Cas cleavage and helicase domains may both be knocked-out and used in combination with other DNA cutting, DNA nicking, DNA binding, transcriptional activation, transcriptional repression, DNA remodeling, DNA deamination, DNA unwinding, DNA recombination enhancing, DNA integration, DNA inversion, and DNA repair agents.

[0183] The transcriptional direction of the tracrRNA for the CRISPR-Cas system (if present) and other components of the CRISPR-Cas system (such as variable targeting domain, crRNA repeat, loop, anti-repeat) can be deduced as described in WO2016186946 published 24 November 2016, and WO2016186953 published 24 November 2016.

[0184] As described herein, once the appropriate guide RNA requirement is established, the PAM preferences for each new system disclosed herein may be examined. If the cleavage complex results in degradation of the randomized PAM library, the complex can be converted into a nickase by disabling the ATPase dependent helicase activity either through mutagenesis of critical residues or by assembling the reaction in the absence of ATP as described previously (Sinkunas, T. et al., 2013, EMBO J. 32:385-394). Two regions of PAM randomization separated by two protospacer targets may be utilized to generate a double-stranded DNA break which may be captured and sequenced to examine the PAM sequences that support cleavage by the respective complex.

[0185] In one embodiment, the invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing into a cell at least one PGEN described herein, and identifying at least one cell that has a modification at said target, wherein the modification at said target site is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, the chemical alteration of at least one nucleotide, and (v) any combination of (i) – (iv).

[0186] The nucleotide to be edited can be located within or outside a target site recognized and cleaved by a Cas endonuclease. In one embodiment, the at least one nucleotide modification is not a modification at a target site recognized and cleaved by a Cas endonuclease. In another embodiment, there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,Attorney Docket: 212196-WO-SEC-1 22, 23, 24, 25, 26, 27, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900 or 1000 nucleotides between the at least one nucleotide to be edited and the genomic target site.

[0187] A knock-out may be produced by an indel (insertion or deletion of nucleotide bases in a target DNA sequence through NHEJ), or by specific removal of sequence that reduces or completely destroys the function of sequence at or near the targeting site.

[0188] A guide polynucleotide / Cas endonuclease induced targeted mutation can occur in a nucleotide sequence that is located within or outside a genomic target site that is recognized and cleaved by the Cas endonuclease.

[0189] The method for editing a nucleotide sequence in the genome of a cell can be a method without the use of an exogenous selectable marker by restoring function to a non-functional gene product.

[0190] In one embodiment, the invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing into a cell at least one PGEN described herein and at least one donor DNA, wherein said donor DNA comprises a polynucleotide of interest, and optionally, further comprising identifying at least one cell that said polynucleotide of interest integrated in or near said target site.

[0191] In one aspect, the methods disclosed herein may employ homologous recombination (HR) to provide integration of the polynucleotide of interest at the target site.

[0192] Various methods and compositions can be employed to produce a cell or organism having a polynucleotide of interest inserted in a target site via activity of a CRISPR-Cas system component described herein. In one method described herein, a polynucleotide of interest is introduced into the organism cell via a donor DNA construct. As used herein, “donor DNA” is a DNA construct that comprises a polynucleotide of interest to be inserted into the target site of a Cas endonuclease. The donor DNA construct further comprises a first and a second region of homology that flank the polynucleotide of interest. The first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome.

[0193] The donor DNA can be tethered to the guide polynucleotide. Tethered donor DNAs can allow for co-localizing target and donor DNA, useful in genome editing, gene insertion, and targeted genome regulation, and can also be useful in targeting post-mitotic cells where functionAttorney Docket: 212196-WO-SEC-1 of endogenous HR machinery is expected to be highly diminished (Mali et al., 2013, Nature Methods Vol.10:957-963).

[0194] The amount of homology or sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and / or regions having unit integral values in the ranges of about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-1000 bp, 600-1250 bp, 700- 1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5–3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to and including the total length of the target site. These ranges include every integer within the range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bps. The amount of homology can also be described by percent sequence identity over the full aligned length of the two polynucleotides which includes percent sequence identity of about at least 50%, 55%, 60%, 65%, 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 100%. Sufficient homology includes any combination of polynucleotide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus. Sufficient homology can also be described by the predicted ability of two polynucleotides to specifically hybridize under high stringency conditions, see, for example, Sambrook et al., (1989) Molecular Cloning:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0195] Episomal DNA molecules can also be ligated into the double-strand break, for example, integration of T-DNAs into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta, (1998) EMBO J. 17:6086-95). Once the sequence around the double-strand breaks is altered, for example, by exonuclease activities involved in the maturation of double-strand breaks, gene conversion pathways can restore the original structure if a homologous sequence is available, such as a homologous chromosome in non-dividing somaticAttorney Docket: 212196-WO-SEC-1 cells, or a sister chromatid after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenic DNA sequences may also serve as a DNA repair template for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

[0196] In one embodiment, the disclosure comprises a method for editing a nucleotide sequence in the genome of a cell, the method comprising introducing into at least one PGEN described herein, and a polynucleotide modification template, wherein said polynucleotide modification template comprises at least one nucleotide modification of said nucleotide sequence, and optionally further comprising selecting at least one cell that comprises the edited nucleotide sequence.

[0197] The guide polynucleotide / Cas endonuclease system can be used in combination with at least one polynucleotide modification template to allow for editing (modification) of a genomic nucleotide sequence of interest. (See also US20150082478, published 19 March 2015 and WO2015026886 published 26 February 2015).

[0198] Polynucleotides of interest and / or traits can be stacked together in a complex trait locus as described in WO2012129373 published 27 September 2012, and in WO2013112686, published 01 August 2013. The guide polynucleotide / Cas9 endonuclease system described herein provides for an efficient system to generate double-strand breaks and allows for traits to be stacked in a complex trait locus.

[0199] A guide polynucleotide / Cas system as described herein, mediating gene targeting, can be used in methods for directing heterologous gene insertion and / or for producing complex trait loci comprising multiple heterologous genes in a fashion similar as disclosed in WO2012129373 published 27 September 2012, where instead of using a double-strand break inducing agent to introduce a gene of interest, a guide polynucleotide / Cas system as disclosed herein is used. By inserting independent transgenes within 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2, or even 5 centimorgans (cM) from each other, the transgenes can be bred as a single genetic locus (see, for example, US20130263324 published 03 October 2013 or WO2012129373 published 14 March 2013). After selecting a plant comprising a transgene, plants comprising (at least) one transgenes can be crossed to form an F1 that comprises both transgenes. In progeny from these F1 (F2 or BC1) 1 / 500 progeny would have the two different transgenes recombined onto the same chromosome. The complex locus can then be bred as single genetic locus with both transgene traits. This process can beAttorney Docket: 212196-WO-SEC-1 repeated to stack as many traits as desired.

[0200] Further uses for guide RNA / Cas endonuclease systems have been described (See for example:US20150082478 published 19 March 2015, WO2015026886 published 26 February 2015, US20150059010 published 26 February 2015, WO2016007347 published 14 January 2016, and PCT application WO2016025131 published 18 February 2016) and include but are not limited to modifying or replacing nucleotide sequences of interest (such as a regulatory elements), insertion of polynucleotides of interest, gene knock-out, gene-knock in, modification of splicing sites and / or introducing alternate splicing sites, modifications of nucleotide sequences encoding a protein of interest, amino acid and / or protein fusions, and gene silencing by expressing an inverted repeat into a gene of interest.

[0201] Resulting characteristics from the gene editing compositions and methods described herein may be evaluated. Chromosomal intervals that correlate with a phenotype or trait of interest can be identified. A variety of methods well known in the art are available for identifying chromosomal intervals. The boundaries of such chromosomal intervals are drawn to encompass markers that will be linked to the gene controlling the trait of interest. In other words, the chromosomal interval is drawn such that any marker that lies within that interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for a particular trait. In one embodiment, the chromosomal interval comprises at least one QTL, and furthermore, may indeed comprise more than one QTL. Close proximity of multiple QTLs in the same interval may obfuscate the correlation of a particular marker with a particular QTL, as one marker may demonstrate linkage to more than one QTL. Conversely, e.g., if two markers in close proximity show co-segregation with the desired phenotypic trait, it is sometimes unclear if each of those markers identifies the same QTL or two different QTL. The term “quantitative trait locus” or “QTL” refers to a region of DNA that is associated with the differential expression of a quantitative phenotypic trait in at least one genetic background, e.g., in at least one breeding population. The region of the QTL encompasses or is closely linked to the gene or genes that affect the trait in question. An “allele of a QTL” can comprise multiple genes or other genetic factors within a contiguous genomic region or linkage group, such as a haplotype. An allele of a QTL can denote a haplotype within a specified window wherein said window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers. A haplotype can be defined by the uniqueAttorney Docket: 212196-WO-SEC-1 fingerprint of alleles at each marker within the specified window. Introduction of CRISPR-Cas System Components into a Cell

[0202] The methods and compositions described herein do not depend on a particular method for introducing a sequence into an organism or cell, only that the polynucleotide or polypeptide gains access to the interior of at least one cell of the organism. Introducing includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient (direct) provision of a nucleic acid, protein or polynucleotide-protein complex (PGEN, RGEN) to the cell.

[0203] Methods for introducing polynucleotides or polypeptides or a polynucleotide-protein complex into cells or organisms are known in the art including, but not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods, ballistic particle acceleration (particle bombardment), whiskers mediated transformation, Agrobacterium-mediated transformation, direct gene transfer, viral-mediated introduction, transfection, transduction, cell- penetrating peptides, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, topical applications, sexual crossing , sexual breeding, and any combination thereof.

[0204] For example, the guide polynucleotide (guide RNA, crNucleotide + tracrNucleotide, guide DNA and / or guide RNA-DNA molecule) can be introduced into a cell directly (transiently) as a single stranded or double stranded polynucleotide molecule. The guide RNA (or crRNA + tracrRNA) can also be introduced into a cell indirectly by introducing a recombinant DNA molecule comprising a heterologous nucleic acid fragment encoding the guide RNA (or crRNA + tracrRNA), operably linked to a specific promoter that is capable of transcribing the guide RNA (crRNA+tracrRNA molecules) in said cell. The specific promoter can be, but is not limited to, a RNA polymerase III promoter, which allow for transcription of RNA with precisely defined, unmodified, 5’- and 3’-ends (Ma et al., 2014, Mol. Ther. Nucleic Acids 3:e161; DiCarlo et al., 2013, Nucleic Acids Res. 41:4336-4343; WO2015026887, published 26 February 2015). Any promoter capable of transcribing the guide RNA in a cell can be used and includes a heat shock / heat inducible promoter operably linked to a nucleotide sequence encoding the guide RNA.

[0205] Plant cells differ from animal cells (such as human cells), fungal cells (such as yeast cells) and protoplasts, including for example plant cells comprise a plant cell wall which may act as a barrier to the delivery of components.Attorney Docket: 212196-WO-SEC-1

[0206] Delivery of the Cas endonuclease, and / or the guide RNA, and / or a ribonucleoprotein complex, and / or a polynucleotide encoding any one or more of the preceding, into plant cells can be achieved through methods known in the art, for example but not limited to: Rhizobiales- mediated transformation (e.g., Agrobacterium, Ochrobactrum), particle mediated delivery (particle bombardment), polyethylene glycol (PEG)-mediated transfection (for example to protoplasts), electroporation, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)- mediated direct protein delivery.

[0207] The Cas endonuclease, such as the Cas endonuclease described herein, can be introduced into a cell by directly introducing the Cas polypeptide itself (referred to as direct delivery of Cas endonuclease), the mRNA encoding the Cas protein, and / or the guide polynucleotide / Cas endonuclease complex itself, using any method known in the art. The Cas endonuclease can also be introduced into a cell indirectly by introducing a recombinant DNA molecule that encodes the Cas endonuclease. The endonuclease can be introduced into a cell transiently or can be incorporated into the genome of the host cell using any method known in the art. Uptake of the endonuclease and / or the guided polynucleotide into the cell can be facilitated with a Cell Penetrating Peptide (CPP) as described in WO2016073433 published 12 May 2016. Any promoter capable of expressing the Cas endonuclease in a cell can be used and includes a heat shock / heat inducible promoter operably linked to a nucleotide sequence encoding the Cas endonuclease.

[0208] Direct delivery of a polynucleotide modification template into plant cells can be achieved through particle mediated delivery, and any other direct method of delivery, such as but not limiting to, polyethylene glycol (PEG)-mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery can be successfully used for delivering a polynucleotide modification template in eukaryotic cells, such as plant cells.

[0209] The donor DNA can be introduced by any means known in the art. The donor DNA may be provided by any transformation method known in the art including, for example, Agrobacterium-mediated transformation or biolistic particle bombardment. The donor DNA may be present transiently in the cell or it could be introduced via a viral replicon. In the presence of the Cas endonuclease and the target site, the donor DNA is inserted into the transformed plant’s genome.Attorney Docket: 212196-WO-SEC-1

[0210] Direct delivery of any one of the guided Cas system components can be accompanied by direct delivery (co-delivery) of other mRNAs that can promote the enrichment and / or visualization of cells receiving the guide polynucleotide / Cas endonuclease complex components. For example, direct co-delivery of the guide polynucleotide / Cas endonuclease components (and / or guide polynucleotide / Cas endonuclease complex itself) together with mRNA encoding phenotypic markers (such as but not limiting to transcriptional activators such as CRC (Bruce et al.2000 The Plant Cell 12:65-79) can enable the selection and enrichment of cells without the use of an exogenous selectable marker by restoring function to a non-functional gene product as described in WO2017070032 published 27 April 2017.

[0211] Introducing a guide RNA / Cas endonuclease complex described herein, (representing the cleavage ready complex described herein) into a cell includes introducing the individual components of said complex either separately or combined into the cell, and either directly (direct delivery as RNA for the guide and protein for the Cas endonuclease and protein subunits, or functional fragments thereof) or via recombination constructs expressing the components (guide RNA, Cas endonuclease, protein subunits, or functional fragments thereof). Introducing a guide RNA / Cas endonuclease complex (RGEN) into a cell includes introducing the guide RNA / Cas endonuclease complex as a ribonucleotide-protein into the cell. The ribonucleotide-protein can be assembled prior to being introduced into the cell as described herein. The components comprising the guide RNA / Cas endonuclease ribonucleotide protein (at least one Cas endonuclease, at least one guide RNA, at least one protein subunit) can be assembled in vitro or assembled by any means known in the art prior to being introduced into a cell (targeted for genome modification as described herein).

[0212] Direct delivery of the RGEN ribonucleoprotein, allows for genome editing at a target site in the genome of a cell which can be followed by rapid degradation of the complex, and only a transient presence of the complex in the cell. This transient presence of the RGEN complex may lead to reduced off-target effects. In contrast, delivery of RGEN components (guide RNA, Cas9 endonuclease) via plasmid DNA sequences can result in constant expression of RGENs from these plasmids which can intensify off target effects (Cradick, T. J. et al. (2013) Nucleic Acids Res 41:9584-9592; Fu, Y et al. (2014) Nat. Biotechnol.31:822-826).

[0213] Direct delivery can be achieved by combining any one component of the guide RNA / CasAttorney Docket: 212196-WO-SEC-1 endonuclease complex (RGEN), representing the cleavage ready complex described herein, (such as at least one guide RNA, at least one Cas protein, and optionally one additional protein), with a delivery matrix comprising a microparticle (such as but not limited to of a gold particle, tungsten particle, and silicon carbide whisker particle) (see also WO2017070032 published 27 April 2017). The delivery matrix may comprise any one of the components, such as the Cas endonuclease, that is attached to a solid matrix (e.g., a particle for bombardment).

[0214] In one aspect the guide polynucleotide / Cas endonuclease complex, is a complex wherein the guide RNA and Cas endonuclease protein forming the guide RNA / Cas endonuclease complex are introduced into the cell as RNA and protein, respectively.

[0215] In one aspect the guide polynucleotide / Cas endonuclease complex, is a complex wherein the guide RNA and Cas endonuclease protein and the at least one protein subunit of a complex forming the guide RNA / Cas endonuclease complex are introduced into the cell as RNA and proteins, respectively.

[0216] In one aspect the guide polynucleotide / Cas endonuclease complex, is a complex wherein the guide RNA and Cas endonuclease protein and the at least one protein subunit of a complex forming the guide RNA / Cas endonuclease complex (cleavage ready complex) are preassembled in vitro and introduced into the cell as a ribonucleotide-protein complex.

[0217] Protocols for introducing polynucleotides, polypeptides or polynucleotide-protein complexes (PGEN, RGEN) into eukaryotic cells, such as plants or plant cells are known and include microinjection (Crossway et al., (1986) Biotechniques 4:320-34 and U.S. Patent No. 6,300,543), meristem transformation (U.S. Patent No. 5,736,369), electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-6, Agrobacterium-mediated transformation (U.S. Patent Nos.5,563,055 and 5,981,840), whiskers mediated transformation (Ainley et al.2013, Plant Biotechnology Journal 11:1126-1134; Shaheen A. and M. Arshad 2011 Properties and Applications of Silicon Carbide (2011), 345-358 Editor(s):Gerhardt, Rosario. Publisher:InTech, Rijeka, Croatia. CODEN:69PQBP; ISBN:978-953-307-201-2), direct gene transfer (Paszkowski et al., (1984) EMBO J 3:2717-22), and ballistic particle acceleration (U.S. Patent Nos.5,879,918; 5,886,244; 5,932,782; Tomes et al., (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment" in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg & Phillips (Springer-Verlag, Berlin); McCabe et al., (1988) Biotechnology 6:923-6;Attorney Docket: 212196-WO-SEC-1 Weissinger et al., (1988) Ann Rev Genet 22:421-77; Sanford et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al., (1988) Plant Physiol 87:671-4 (soybean); Finer and McMullen, (1991) In vitro Cell Dev Biol 27P:175-82 (soybean); Singh et al., (1998) Theor Appl Genet 96:319-24 (soybean); Datta et al., (1990) Biotechnology 8:736-40 (rice); Klein et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-9 (maize); Klein et al., (1988) Biotechnology 6:559-63 (maize); U.S. Patent Nos.5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol 91:440-4 (maize); Fromm et al., (1990) Biotechnology 8:833-9 (maize); Hooykaas-Van Slogteren et al., (1984) Nature 311:763-4; U.S. Patent No.5,736,369 (cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-9 (Liliaceae); De Wet et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al., (Longman, New York), pp.197-209 (pollen); Kaeppler et al., (1990) Plant Cell Rep 9:415-8) and Kaeppler et al., (1992) Theor Appl Genet 84:560-6 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4:1495-505 (electroporation); Li et al., (1993) Plant Cell Rep 12:250-5; Christou and Ford (1995) Annals Botany 75:407-13 (rice) and Osjoda et al., (1996) Nat Biotechnol 14:745-50 (maize via Agrobacterium tumefaciens).

[0218] Alternatively, polynucleotides may be introduced into plant or plant cells by contacting cells or organisms with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide within a viral DNA or RNA molecule. In some examples a polypeptide of interest may be initially synthesized as part of a viral polyprotein, which is later processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known.

[0219] The polynucleotide or recombinant DNA construct can be provided to or introduced into a prokaryotic and eukaryotic cell or organism using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the polynucleotide construct directly into the plant.

[0220] Nucleic acids and proteins can be provided to a cell by any method including methods using molecules to facilitate the uptake of anyone or all components of a guided Cas system (protein and / or nucleic acids), such as cell-penetrating peptides and nanocarriers.

[0221] Other methods of introducing polynucleotides into a prokaryotic and eukaryotic cell orAttorney Docket: 212196-WO-SEC-1 organism or plant part can be used, including plastid transformation methods, and the methods for introducing polynucleotides into tissues from seedlings or mature seeds.

[0222] Stable transformation is intended to mean that the nucleotide construct introduced into an organism integrates into a genome of the organism and is capable of being inherited by the progeny thereof. Transient transformation is intended to mean that a polynucleotide is introduced into the organism and does not integrate into a genome of the organism or a polypeptide is introduced into an organism. Transient transformation indicates that the introduced composition is only temporarily expressed or present in the organism.

[0223] A variety of methods are available to identify those cells having an altered genome at or near a target site without using a screenable marker phenotype. Such methods can be viewed as directly analyzing a target sequence to detect any change in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blots, and any combination thereof. Stacking of traits in transgenic plant

[0224] Transgenic plants may comprise a stack of one or more insecticidal polynucleotides disclosed herein with one or more additional polynucleotides resulting in the production or suppression of multiple polypeptide sequences. Transgenic plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene and co- transformation of genes into a single plant cell. As used herein, the term “stacked” includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid or both traits are incorporated into the genome of a plastid). In one non-limiting example, “stacked traits” comprise a molecular stack where the sequences are physically adjacent to each other. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. Co- transformation of genes can be carried out using single transformation vectors comprising multiple genes or genes carried separately on multiple vectors. If the sequences are stacked by geneticallyAttorney Docket: 212196-WO-SEC-1 transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO 1999 / 25821, WO 1999 / 25854, WO 1999 / 25840, WO 1999 / 25855 and WO 1999 / 25853, all of which are herein incorporated by reference.

[0225] In some embodiments, one or more of the polynucleotides encoding the Insecticidal toxin polypeptide(s) disclosed herein, alone or stacked with one or more additional insect resistance traits can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like). Thus, the polynucleotide embodiments can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests.

[0226] Transgenes useful for stacking include but are not limited to: transgenes that confer resistance to an herbicide; transgenes that confer or contribute to an altered grain characteristic; genes that control male-sterility; genes that create a site for site specific DNA integration; genes that affect abiotic stress resistance; genes that confer increased yield, genes that confer plant digestibility; and transgenes that confer resistance to insects or disease.

[0227] Examples of transgenes that confer resistance to insects include genes encoding a Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser, et al., (1986) Gene 48:109, who disclose the cloning and nucleotide sequence ofAttorney Docket: 212196-WO-SEC-1 a Bt delta-endotoxin gene. Moreover, DNA molecules encoding delta-endotoxin genes can be purchased from American Type Culture Collection (Rockville, Md.), for example, under ATCC®Accession Numbers 40098, 67136, 31995 and 31998. Other non-limiting examples of Bacillus thuringiensis transgenes being genetically engineered are given in the following patents and patent applications: US Patent Numbers 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, 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.

[0228] Genes encoding pesticidal proteins may also be stacked including but are not limited to: insecticidal proteins from Pseudomonas sp. such as PSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonas protegens strain CHA0 and Pf-5 (previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology 10:2368-2386: GenBank Accession No. EU400157); from Pseudomonas taiwanensis (Liu, et al., (2010) J. Agric. Food Chem.58:12343- 12349) and from Pseudomonas 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 Photorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxinology Journal 3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro. 67:2062-2069), US Patent Number 6,048,838, and US Patent Number 6,379,946; a PIP-1 polypeptide of US Patent Number 9,688,730; an AfIP-1A and / or AfIP-1B polypeptide of US Patent Number 9,475,847; a PIP-47 polypeptide of US Patent Number 10,006,045; an IPD045 polypeptide, an IPD064 polypeptide, an IPD074 polypeptide, an IPD075 polypeptide, and an IPD077 polypeptide of PCT Publication Number WO 2016 / 114973; an IPD080 polypeptide of International Patent Application Publication Number WO2018 / 075350; an IPD078 polypeptide, an IPD084 polypeptide, an IPD085 polypeptide, an IPD086 polypeptide, an IPD087 polypeptide, an IPD088 polypeptide, and an IPD089 polypeptide of International Patent Application Publication Number WO2018 / 084936; PIP-72 polypeptide of US Patent Publication Number US20160366891; a PtIP-50 polypeptide and a PtIP-65 polypeptide of US Patent Application Publication Number US20170166921; an IPD098 polypeptide, an IPD059 polypeptide, an IPD108 polypeptide, an IPD109 polypeptide ofAttorney Docket: 212196-WO-SEC-1 International Patent Application Publication Number WO2018 / 232072; a PtIP-83 polypeptide of US Publication Number US20160347799; a PtIP-96 polypeptide of US Publication Number US20170233440; an IPD079 polypeptide of PCT Publication Number WO2017 / 23486; an IPD082 polypeptide of International Patent Application Publication Number WO 2017 / 105987, an IPD090 polypeptide of International Patent Application Publication Number WO2017 / 192560, an IPD093 polypeptide of International Patent Application Publication Number WO2018 / 111551; an IPD103 polypeptide of International Patent Application Publication Number WO2018 / 005411; an IPD101 polypeptide of International Patent Application Publication Number WO2018 / 118811; an IPD121 polypeptide of International Patent Application Publication Number WO2018 / 208882, and δ- endotoxins including, but not limited to, the 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, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34, Cry35,Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry50, Cry51, Cry52, Cry53, Cry 54, Cry55, Cry56, Cry57, Cry58, Cry59, Cry60, Cry61, Cry62, Cry63, Cry64, Cry65, Cry66, Cry67, Cry68, Cry69, Cry70, Cry71, and Cry 72 classes of δ-endotoxin genes and the B. thuringiensis cytolytic Cyt1 and Cyt2 genes.

[0229] Examples of δ-endotoxins also include but are not limited to Cry1A proteins of US Patent Numbers 5,880,275 and 7,858,849; a DIG-3 or DIG-11 toxin (N-terminal deletion of α-helix 1 and / or α-helix 2 variants of Cry proteins such as Cry1A) of US Patent Numbers 8,304,604 and 8.304,605, Cry1B of US Patent Application Serial Number 10 / 525,318, US Patent Application Publication Number US20160194364, and US Patent Numbers 9,404,121 and 8,772,577; Cry1B variants of PCT Publication Number WO2016 / 61197 and Serial Number PCT / US17 / 27160; Cry1C of US Patent Number 6,033,874; Cry1D protein of US20170233759; a Cry1E protein of PCT Publication Number WO2018 / 075197; Cry1F of US Patent Numbers 5,188,960, 6,218,188; Cry1A / F chimeras of US Patent Numbers 7,070,982; 6,962,705 and 6,713,063; a Cry1J variant of US Publication US20170240603; a Cry2 protein such as Cry2Ab protein of US Patent Number 7,064,249; a Cry3A protein including but not limited to an engineered hybrid insecticidal protein (eHIP) created by fusing unique combinations of variable regions and conserved blocks of at least two different Cry proteins (US Patent Application Publication Number 2010 / 0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein; Cry8 proteins of US Patent Numbers 7,329,736, 7,449,552,Attorney Docket: 212196-WO-SEC-1 7,803,943, 7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such as such as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E, and Cry9F families; a Cry15 protein of Naimov, et al., (2008) Applied and Environmental Microbiology 74:7145–7151; a Cry22, a Cry34Ab1 protein of US Patent Numbers 6,127,180, 6,624,145 and 6,340,593; a CryET33 and CryET34 protein of US Patent Numbers 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 and CryET34 homologs of US Patent Publication Number 2006 / 0191034, 2012 / 0278954, and PCT Publication Number WO 2012 / 139004; a Cry35Ab1 protein of US Patent Numbers 6,083,499, 6,548,291 and 6,340,593; a Cry46 protein of US Patent Number 9,403,881, a Cry 51 protein, a Cry binary toxin; a TIC901 or related toxin; TIC807 of US 2008 / 0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128 of PCT US 2006 / 033867; engineered Hemipteran toxic proteins of US Patent Application Publication Number US20160150795, AXMI-027, AXMI-036, and AXMI-038 of US Patent Number 8,236,757; AXMI-031, AXMI-039, AXMI-040, AXMI-049 of US Patent Number 7,923,602; AXMI-018, AXMI-020 and AXMI-021 of WO 2006 / 083891; AXMI-010 of WO 2005 / 038032; AXMI-003 of WO 2005 / 021585; AXMI-008 of US Patent Application Publication Number 2004 / 0250311; AXMI-006 of US Patent Application Publication Number 2004 / 0216186; AXMI-007 of US Patent Application Publication Number 2004 / 0210965; AXMI-009 of US Patent Application Number 2004 / 0210964; AXMI-014 of US Patent Application Publication Number 2004 / 0197917; AXMI- 004 of US Patent Application Publication Number 2004 / 0197916; AXMI-028 and AXMI-029 of WO 2006 / 119457; AXMI-007, AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI- 004 of WO 2004 / 074462; AXMI-150 of US Patent Number 8,084,416; AXMI-205 of US Patent Application Publication Number 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 US Patent Application Publication Number 2011 / 0263488; AXMI046, AXMI048, AXMI050, AXMI051, AXMI052, AXMI053, AXMI054, AXMI055, AXMI056, AXMI057, AXMI058, AXMI059, AXMI060, AXMI061, AXMI067, AXMI069, AXMI071, AXMI072, AXMI073, AXMI074, AXMI075, AXMI087, AXMI088, AXMI093, AXMI070, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, AXMI114, AXMI116,Attorney Docket: 212196-WO-SEC-1 AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI125, AXMI126, AXMI127, AXMI129, AXMI151, AXMI161, AXMI164, AXMI183, AXMI132, AXMI137, AXMI138 of US Patent US8461421 and US8,461,422; AXMI-R1 and related proteins of US Patent Application Publication Number 2010 / 0197592; AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011 / 103248; AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230 and AXMI231 of WO 2011 / 103247; AXMI-115, AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of US Patent Number 8,334,431; AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US Patent Application Publication Number 2010 / 0298211; AXMI-066 and AXMI-076 of US Patent Application Publication Number 2009 / 0144852; AXMI128, AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146, AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178, AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188, AXMI189 of US Patent Number 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101, AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, dsAXMI111, AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of US Patent US8461421; AXMI192 of US Patent US8,461,415; AXMI281 of US Patent Application Publication Number US20160177332; AXMI422 of US Patent Number US8,252,872; and Cry proteins such as Cry1A and Cry3A having modified proteolytic sites of US Patent Number 8,319,019; and a Cry1Ac, Cry2Aa and Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 of US Patent Application Publication Number 2011 / 0064710. The Cry proteins MP032, MP049, MP051, MP066, MP068, MP070, MP091S, MP109S, MP114, MP121, MP134S, MP183S, MP185S, MP186S, MP195S, MP197S, MP208S, MP209S, MP212S, MP214S, MP217S, MP222S, MP234S, MP235S, MP237S, MP242S, MP243, MP248, MP249S, MP251M, MP252S, MP253, MP259S, MP287S, MP288S, MP295S, MP296S, MP297S, MP300S, MP304S, MP306S, MP310S, MP312S, MP314S,Attorney Docket: 212196-WO-SEC-1 MP319S, MP325S, MP326S, MP327S, MP328S, MP334S, MP337S, MP342S, MP349S, MP356S, MP359S, MP360S, MP437S, MP451S, MP452S, MP466S, MP468S, MP476S, MP482S, MP522S, MP529S, MP548S, MP552S, MP562S, MP564S, MP566S, MP567S, MP569S, MP573S, MP574S, MP575S, MP581S, MP590, MP594S, MP596S, MP597, MP599S, MP600S, MP601S, MP602S, MP604S, MP626S, MP629S, MP630S, MP631S, MP632S, MP633S, MP634S, MP635S, MP639S, MP640S, MP644S, MP649S, MP651S, MP652S, MP653S, MP661S, MP666S, MP672S, MP696S, MP704S, MP724S, MP729S, MP739S, MP755S, MP773S, MP799S, MP800S, MP801S, MP802S, MP803S, MP805S, MP809S, MP815S, MP828S, MP831S, MP844S, MP852, MP865S, MP879S, MP887S, MP891S, MP896S, MP898S, MP935S, MP968, MP989, MP993, MP997, MP1049, MP1066, MP1067, MP1080, MP1081, MP1200, MP1206, MP1233, and MP1311 of US Patent 11,492,639. Other Cry proteins are well known to one skilled in the art (see, Crickmore, et al., "Bacillus thuringiensis toxin nomenclature" (2011), at lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / which can be accessed on the world-wide web using the "www" prefix). The insecticidal activity of Cry proteins is well known to one skilled in the art (for review, see, van Frannkenhuyzen, (2009) J. Invert. Path.101:1- 16). The use of Cry proteins as transgenic plant traits is well known to one skilled in the art and Cry-transgenic plants including but not limited to Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2, Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt have received regulatory approval (see, Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GM Crop Database Center for Environmental Risk Assessment (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 using the “www” prefix). More than one pesticidal proteins well known to one skilled in the art can also be expressed in plants such as Vip3Ab & Cry1Fa (US2012 / 0317682); Cry1BE & Cry1F (US2012 / 0311746); Cry1CA & Cry1AB (US2012 / 0311745); Cry1F & CryCa (US2012 / 0317681); Cry1DA & Cry1BE (US2012 / 0331590); Cry1DA & Cry1Fa (US2012 / 0331589); Cry1AB & Cry1BE (US2012 / 0324606); Cry1Fa & Cry2Aa and Cry1I & Cry1E (US2012 / 0324605); Cry34Ab / 35Ab & Cry6Aa (US20130167269); Cry34Ab / VCry35Ab & Cry3Aa (US20130167268); Cry1Da & Cry1Ca (US 9796982); Cry3Aa & Cry6Aa (US 9798963); and Cry3A & Cry1Ab or Vip3Aa (US9,045,766). Pesticidal proteins also includeAttorney Docket: 212196-WO-SEC-1 insecticidal lipases including lipid acyl hydrolases of US Patent Number 7,491,869, and cholesterol oxidases such as from Streptomyces (Purcell et al. (1993) Biochem Biophys Res Commun 15:1406-1413). Pesticidal proteins also include VIP (vegetative insecticidal proteins) toxins of US Patent Numbers 5,877,012, 6,107,279, 6,137,033, 7,244,820, 7,615,686, and 8,237,020, and the like. Other VIP proteins are well known to one skilled in the art (see, lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / vip.html which can be accessed on the world-wide web using the "www" prefix). Pesticidal proteins also include toxin complex (TC) proteins, obtainable from organisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, US Patent Numbers 7,491,698 and 8,084,418). Some TC proteins have “stand alone” insecticidal activity and other TC proteins enhance the activity of the stand-alone toxins produced by the same given organism. The toxicity of a “stand-alone” TC protein (from Photorhabdus, Xenorhabdus or Paenibacillus, for example) can be enhanced by one or more TC protein “potentiators” derived from a source organism of a different genus. There are three main types of TC proteins. As referred to herein, Class A proteins (“Protein A”) are stand-alone toxins. Class B proteins (“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity of Class A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 and XptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi. Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidal proteins also include spider, snake and scorpion venom proteins. Examples of spider venom peptides include but are not limited to lycotoxin-1 peptides and mutants thereof (US Patent Number 8,334,366). Gene silencing

[0230] In some embodiments, the stacked trait may be in the form of silencing of one or more polynucleotides of interest resulting in suppression of one or more target pest polypeptides. In some embodiments, the silencing is achieved using a suppression DNA construct.

[0231] In some embodiments, one or more polynucleotide encoding the polypeptides of the IPD115, IPD119, IPD125, OR IPD130 polypeptide or fragments or variants thereof may be stacked with one or more polynucleotides encoding one or more polypeptides having insecticidal activity or agronomic traits as set forth supra and optionally may further include one or more polynucleotides providing for gene silencing of one or more target polynucleotides as discussedAttorney Docket: 212196-WO-SEC-1 infra.

[0232] “Suppression DNA construct” is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in “silencing” of a target gene in the plant. The target gene may be endogenous or transgenic to the plant. “Silencing,” as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein / enzyme expressed by the target gene, and / or the level of the enzyme activity or protein functionality. The term “suppression” includes lower, reduce, decline, decrease, inhibit, eliminate and prevent. “Silencing” or “gene silencing” does not specify mechanism and is inclusive, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi- based approaches and small RNA-based approaches.

[0233] A suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest. Depending upon the approach to be utilized, the region may be 100% identical or less than 100% identical (e.g., at least 50% or any integer between 51% and 100% identical) to all or part of the sense strand (or antisense strand) of the gene of interest.

[0234] Suppression DNA constructs can be constructed once the target gene of interest is selected, and include, without limitation, cosuppression constructs, antisense constructs, viral-suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, double-stranded RNA-producing constructs, and more generally, RNAi (RNA interference) constructs and small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.

[0235] “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.

[0236] “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns or the coding sequence.

[0237] “Cosuppression” refers to the production of sense RNA transcripts capable of suppressing the expression of the target protein. “Sense” RNA refers to RNA transcript that includes theAttorney Docket: 212196-WO-SEC-1 mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see, Vaucheret, et al., (1998) Plant J. 16:651-659 and Gura, (2000) Nature 404:804-808).

[0238] Another variation describes the use of plant viral sequences to direct the suppression of proximal mRNA encoding sequences (PCT Publication WO 1998 / 36083).

[0239] Recent work has described the use of “hairpin” structures that incorporate all or part, of an mRNA encoding sequence in a complementary orientation that results in a potential “stem-loop” structure for the expressed RNA (PCT Publication WO 1999 / 53050). In this case the stem is formed by polynucleotides corresponding to the gene of interest inserted in either sense or anti- sense orientation with respect to the promoter and the loop is formed by some polynucleotides of the gene of interest, which do not have a complement in the construct. This increases the frequency of cosuppression or silencing in the recovered transgenic plants. For review of hairpin suppression, see, Wesley, et al., (2003) Methods in Molecular Biology, Plant Functional Genomics: Methods and Protocols 236:273-286.

[0240] A construct where the stem is formed by at least 30 nucleotides from a gene to be suppressed and the loop is formed by a random nucleotide sequence has also effectively been used for suppression (PCT Publication WO 1999 / 61632).

[0241] The use of poly-T and poly-A sequences to generate the stem in the stem-loop structure has also been described (PCT Publication WO 2002 / 00894).

[0242] Yet another variation includes using synthetic repeats to promote formation of a stem in the stem-loop structure. Transgenic organisms prepared with such recombinant DNA fragments have been shown to have reduced levels of the protein encoded by the nucleotide fragment forming the loop as described in PCT Publication WO 2002 / 00904.

[0243] RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing (PTGS) or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defenseAttorney Docket: 212196-WO-SEC-1 mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire, et al., (1999) Trends Genet. 15:358). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA of viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized.

[0244] The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein, et al., (2001) Nature 409:363). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir, et al., (2001) Genes Dev. 15:188). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hu...

Claims

Attorney Docket: 212196-WO-SEC-1 THAT WHICH IS CLAIMED:

1. A recombinant polynucleotide encoding an insecticidal polypeptide selected from the group consisting of: a) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 1-33; b) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 34-73; c) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 74-86; and d) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 87-104; wherein said recombinant polynucleotide further comprises a heterologous sequence operably linked to the recombinant polynucleotide.

2. A DNA construct comprising a polynucleotide, wherein the polynucleotide encodes an insecticidal polypeptide selected from the group consisting of: a) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 1-33; b) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 34-73; c) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 74-86; and d) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 87-104; wherein said recombinant polynucleotide further comprises a heterologous sequence operably linked to the recombinant polynucleotide.

3. A transgenic plant, plant cell, or progeny thereof comprising the DNA construct of claim 2.

4. The transgenic plant, plant cell, or progeny thereof of claim 3, wherein the plant, plant cell, or progeny is soy or corn.

5. A host cell transformed with the DNA construct of claim 2.Attorney Docket: 212196-WO-SEC-1 6. The recombinant polynucleotide of claim 1, wherein the polynucleotide has codons optimized for expression in an agriculturally important crop.

7. An insecticidal polypeptide comprising an amino acid sequence selected from the group consisting of: a) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 1-33; b) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 34-73; c) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 74-86; and d) a polypeptide having greater than 80% sequence identity compared to the sequence of any one of SEQ ID NOs: 87-104; wherein the insecticidal polypeptide is operably linked to a heterologous signal peptide or transit peptide.

8. A composition comprising the recombinant insecticidal polypeptide of claim 7.

9. A method of controlling Lepidoptera and / or Coleoptera insect infestation in the transgenic plant or plant cell comprising the DNA construct of claim 2.

10. The method of claim 9, wherein the Lepidoptera and / or Coleoptera insect population is resistant to a Bt toxin.

11. A method of inhibiting growth or killing an agricultural insect pest population, comprising contacting the insect pest population with an insecticidally-effective amount of the polypeptide of claim 7.

12. A method of controlling Lepidoptera and / or Coleoptera insect infestation in a transgenic plant and providing insect resistance management, comprising expressing in the plant the polynucleotide of claim 1.

13. The method of claim 12, wherein the insect pest or insect pest population is resistant to a Bt toxin.

14. A transgenic plant comprising the recombinant polynucleotide of claim 1.Attorney Docket: 212196-WO-SEC-1 15. A method of inhibiting growth or killing an insect pest or pest population, comprising contacting the insect pest or pest population with a transgenic plant or plant part of claim 15.

16. A transgenic seed derived from the transgenic plant of claim 14.

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