Insecticidal proteins

Abstract

Compositions and methods for controlling corn plant pests are disclosed. In particular, novel insecticidal proteins that are toxic against coleopteran and / or lepidopteran insect pests are provided. Nucleic acid molecules encoding the novel insecticidal proteins are also provided. Also disclosed are methods of making the insecticidal proteins and methods of using the insecticidal proteins as well as nucleic acids encoding the insecticidal proteins of the invention, e.g., to confer protection from insect damage in transgenic maize plants.

Description

This divisional application is a divisional application based on the original Chinese patent application with application number 201980019159.9, application date March 7, 2019, and invention name “Insecticide Protein”. Sequence Listing

[0001] As an alternative to a paper copy, a sequence listing in ASCII text format is provided, which is submitted pursuant to 37 CFR §1.821, is named "81547_ST25.txt", is 305 kilobytes in size, was generated on March 14, 2018, and was submitted via EFS-Web. This sequence listing is hereby incorporated by reference into this specification for its disclosure. Technical Field

[0002] The present invention relates to the fields of protein engineering, plant molecular biology, and pest control. More specifically, the present invention relates to novel proteins and variants thereof having insecticidal activity, nucleic acids (expressed to produce insecticidal proteins), methods for making these insecticidal proteins and corresponding nucleic acids, and methods for using these insecticidal proteins and corresponding nucleic acids to control insects. Background Art

[0003] Insect pests are a major cause of crop losses. In the United States alone, billions of dollars are lost annually due to infestations by various insect species. In addition to losses to field crops, insect pests are a burden to vegetable and fruit growers, to producers of ornamental flowers, and a nuisance to gardeners and homeowners.

[0004] Multiple species of corn rootworm are considered to be the most destructive corn pests. In the U.S., three important species are the corn rootworm (Diabrotica virgifera virgifera, also known as the western corn rootworm), the longicornis barberi (D. longicornis barberi, also known as the northern corn rootworm), and the undecimpunctata howardi (D. undecimpunctata howardi, also known as the southern corn rootworm). In the U.S. Corn Belt, only the western and northern corn rootworms are considered to be major corn pests. Additionally, an important corn rootworm pest in the southern U.S. is the Mexican corn rootworm (Diabrotica virgifera zeae). Corn rootworm larvae cause the most substantial plant damage by almost exclusively feeding on corn roots. This damage has been shown to increase plant lodging, reduce grain yield, and the yield of nutrients, as well as change the nutrient content of grain. Larval feeding also has an indirect impact on corn by opening access to the roots for bacterial and fungal infections that cause root rot and stalk rot. Adult corn rootworms are active in corn fields in late summer where they feed on ears, silks, and pollen, thereby disrupting normal pollination.

[0005] Corn rootworms are primarily controlled through intensive applications of chemical pesticides, which are active by inhibiting insect growth, preventing insect feeding or reproduction, or causing mortality. This results in good corn rootworm control, but these chemicals can sometimes affect other beneficial organisms. Another problem resulting from the widespread use of chemical pesticides is the emergence of resistant insect varieties. Yet another problem is the fact that corn rootworm larvae feed underground, making rescue treatments with insecticides difficult. Consequently, most insecticide applications are carried out preventively at planting time. This practice results in a significant environmental burden. This situation has been partially improved through various farmland management practices, but there is an increasing need for alternative pest control mechanisms.

[0006] Biological pest control agents, such as Bacillus thuringiensis (Bt) strains expressing pesticidal toxins like delta endotoxins (delta-endotoxins; also known as crystalline toxins or Cry proteins), have also been applied to crop plants with satisfactory results against insect pests. These delta endotoxins are proteins contained within a crystalline matrix that are known to have insecticidal activity when ingested by certain insects. Several natural or engineered Cry proteins from Bacillus thuringiensis have been expressed in transgenic crop plants and commercially developed to control certain lepidopteran and coleopteran insect pests. For example, starting in 2003, transgenic corn hybrids that control corn rootworms by expressing Cry3Bb1, Cry34Ab1 / Cry35Ab1, or modified Cry3A (mCry3A) or Cry3Ab (eCry3.1Ab) proteins were commercially available in the United States.

[0007] Although transgenic plants expressing Cry proteins have been shown to be very effective, it is now known that there are insect pests that are resistant to the Cry proteins expressed in certain transgenic plants. Therefore, there is still a need to identify new and effective pest control agents that provide economic benefits to farmers and are environmentally acceptable. Particularly needed are proteins that are toxic to Diabrotica species (a major corn pest) that have different modes of action compared to existing insect control products to mitigate resistance development. In addition, it is desirable to deliver insect control agents through these products that minimize the environmental burden (such as through transgenic plants). Summary of the Invention

[0008] In view of these needs, the present invention provides novel insecticidal proteins (i.e., NitromobCRW) and proteins substantially identical to NitromobCRW and variants thereof. The proteins of the present invention are toxic to corn rootworms (Dryoptera species). The proteins of the present invention may also be toxic to other Coleoptera and / or Lepidoptera. The present invention further relates to nucleic acid molecules encoding NitromobCRW or variants thereof, complements thereof, or nucleic acid molecules substantially identical to NitromobCRW and variants thereof.

[0009] Also included in the present invention are vectors containing such recombinant (or complementary) nucleic acids; plants or microorganisms comprising such nucleic acids and capable of expressing such nucleic acids; various plants transformed with such nucleic acids, such as transgenic corn plants; progeny of such plants (which contain the nucleic acid stably incorporated and inherited in a Mendelian manner), and / or seeds of such plants and such progeny. The present invention also includes methods of breeding to introduce transgenes comprising the nucleic acid molecules of the present invention into progeny plants and various germplasms.

[0010] The present invention also includes compositions and formulations containing NitromobCRW or variants thereof that are capable of inhibiting the ability of insect pests to survive, grow and / or reproduce, or that are capable of limiting insect-associated damage or loss of crop plants, for example, by applying NitromobCRW or variants thereof as part of a composition or formulation to insect-infested areas or plants, or to preventively treat areas or plants susceptible to insect infestation to confer protection against insect pests.

[0011] The invention further relates to methods of making NitromobCRW or variants thereof, and to various methods of using these nucleic acids, for example, to control insects in microorganisms or to confer protection from insect damage in transgenic plants.

[0012] These novel proteins described herein are active against insects. For example, in embodiments, the proteins of the invention can be used to control economically important insect pests, including coleopteran insects such as western corn rootworm (WCR), northern corn rootworm (NCR), southern corn rootworm (SCR), and / or Mexican corn rootworm (Mexican corn rootworm). The insecticidal proteins of the invention can be used alone or in combination with other insect control strategies to impart enhanced pest control efficiency against the same insect pests and / or increase the spectrum of target insects with minimal environmental impact.

[0013] Other aspects and advantages of the present invention will become clear to those skilled in the art from a study of the invention described below and from the non-limiting examples. Brief description of the sequences in the sequence listing

[0014] The nucleic acid sequences listed in the attached sequence listing are represented using standard alphabetical abbreviations for nucleotide bases (as defined in 37 CFR § 1.822). The listed nucleic acid and amino acid sequences define molecules having nucleotide and amino acid monomers arranged in the manner described (i.e., polynucleotides and polypeptides, respectively). The listed nucleic acid and amino acid sequences also each define the genus of a polynucleotide or polypeptide (which includes nucleotide and amino acid monomers arranged in the manner described). Taking into account the redundancy of the genetic code, it will be understood that the nucleotide sequence comprising the coding sequence also describes the genus of a polynucleotide that encodes the same polypeptide as the polynucleotide consisting of the reference sequence. It will also be understood that the amino acid sequence describes the genus of the polynucleotide ORF that encodes the polypeptide.

[0015] Only one chain of each nucleic acid sequence is shown, but by any reference to the chain presented, it should be understood that the complementary chain is included. Since the complementary sequence and reverse complementary sequence of the primary nucleic acid sequence must be disclosed by the primary nucleic acid sequence, unless otherwise specified (or it is obvious from the context of the sequence in which it appears), the complementary sequence and reverse complementary sequence refer to the nucleic acid sequence. In addition, as understood in the art, the nucleotide sequence of the RNA chain is determined by the DNA sequence from which it is transcribed (but uracil (U) nucleoside base replaces thymine (T)), and the RNA sequence is included by any reference to the DNA sequence encoding it. In the accompanying sequence table: SEQ ID NO: 1 is the nucleotide sequence optimized for NitromobCRW E. coli. SEQ ID NO: 2 is the nucleotide sequence of Nitromob CRW variant I98L. SEQ ID NO: 3 is the nucleotide sequence of Nitromob CRW variant V99L. SEQ ID NO: 4 is the nucleotide sequence of Nitromob CRW variant I175L. SEQ ID NO: 5 is the nucleotide sequence of Nitromob CRW variant I208L. SEQ ID NO: 6 is the nucleotide sequence of Nitromob CRW variant I215L. SEQ ID NO: 7 is the nucleotide sequence of Nitromob CRW variant I215F. SEQ ID NO: 8 is the nucleotide sequence of Nitromob CRW variant I215Y. SEQ ID NO: 9 is the nucleotide sequence of Nitromob CRW variant Y213L / I215L. SEQ ID NO: 10 is the nucleotide sequence of Nitromob CRW variant I245L. SEQ ID NO: 11 is the nucleotide sequence of Nitromob CRW variant I255L. SEQ ID NO: 12 is the nucleotide sequence of Nitromob CRW variant I265L. SEQ ID NO: 13 is the nucleotide sequence of Nitromob CRW variant I257L. SEQ ID NO: 14 is the nucleotide sequence of Nitromob CRW variant G216A. SEQ ID NO: 15 is the nucleotide sequence of Nitromob CRW variant G216L. SEQ ID NO: 16 is the nucleotide sequence of Nitromob CRW variant V122L. SEQ ID NO: 17 is the nucleotide sequence of Nitromob CRW variant V167L. SEQ ID NO: 18 is the nucleotide sequence of Nitromob CRW variant V220L. SEQ ID NO: 19 is the nucleotide sequence of NitromobCRW insertion variant L214-Leu-I215. SEQ ID NO: 20 is the nucleotide sequence of NitromobCRW insertion variant I215-Leu-G216. SEQ ID NO: 21 is the nucleotide sequence of Nitromob CRW variant Y213F / I215L. SEQ ID NO: 22 is the nucleotide sequence of Nitromob CRW variant I175L / I215L. SEQ ID NO: 23 is the nucleotide sequence of Nitromob CRW variant I208L / I215L. SEQ ID NO: 24 is the nucleotide sequence of Nitromob CRW variant I215L / I255L. SEQ ID NO: 25 is the nucleotide sequence of Nitromob CRW variant I255L / I257L. SEQ ID NO: 26 is the nucleotide sequence of Nitromob CRW variant L214S / I215L. SEQ ID NO: 27 is the nucleotide sequence of Nitromob CRW variant V203S / M204L. SEQ ID NO: 28 is the nucleotide sequence of Nitromob CRW variant T218L. SEQ ID NO: 29 is the nucleotide sequence of Nitromob CRW variant T218F. SEQ ID NO: 30 is the nucleotide sequence of Nitromob CRW variant V185L. SEQ ID NO: 31 is the nucleotide sequence of Nitromob CRW variant V193L / I215L. SEQ ID NO: 32 is the nucleotide sequence of Nitromob CRW variant E196L / I215L. SEQ ID NO: 33 is the nucleotide sequence of Nitromob CRW variant E186L / I215L. SEQ ID NO: 34 is the nucleotide sequence of Nitromob CRW variant V177L / I215L. SEQ ID NO: 35 is the nucleotide sequence of Nitromob CRW variant Y213L. SEQ ID NO: 36 is the nucleotide sequence of Nitromob CRW variant V203S / M204L / I215L. SEQ ID NO: 37 is the native nucleotide sequence of NitromobCRW. SEQ ID NO: 38 is the codon-optimized nucleotide sequence of NitromobCRW variant Y213L / I215L maize. SEQ ID NO: 39 is the natural amino acid sequence of NitromobCRW. SEQ ID NO: 40 is the amino acid sequence of Nitromob CRW variant I98L. SEQ ID NO: 41 is the amino acid sequence of Nitromob CRW variant V99L. SEQ ID NO: 42 is the amino acid sequence of Nitromob CRW variant I175L. SEQ ID NO: 43 is the amino acid sequence of Nitromob CRW variant I208L. SEQ ID NO: 44 is the amino acid sequence of Nitromob CRW variant I215L. SEQ ID NO: 45 is the amino acid sequence of Nitromob CRW variant I215F. SEQ ID NO: 46 is the amino acid sequence of Nitromob CRW variant I215Y. SEQ ID NO: 47 is the amino acid sequence of Nitromob CRW variant Y213L / I215L. SEQ ID NO: 48 is the amino acid sequence of Nitromob CRW variant I245L. SEQ ID NO: 49 is the amino acid sequence of Nitromob CRW variant I255L. SEQ ID NO: 50 is the amino acid sequence of Nitromob CRW variant I265L. SEQ ID NO: 51 is the amino acid sequence of Nitromob CRW variant I257L. SEQ ID NO: 52 is the amino acid sequence of Nitromob CRW variant G216A. SEQ ID NO: 53 is the amino acid sequence of Nitromob CRW variant G216L. SEQ ID NO: 54 is the amino acid sequence of Nitromob CRW variant V122L. SEQ ID NO: 55 is the amino acid sequence of Nitromob CRW variant V167L. SEQ ID NO: 56 is the amino acid sequence of Nitromob CRW variant V220L. SEQ ID NO: 57 is the amino acid sequence of the NitromobCRW insertion variant L214-Leu-I215. SEQ ID NO: 58 is the amino acid sequence of NitromobCRW insertion variant I215-Leu-G216. SEQ ID NO: 59 is the amino acid sequence of Nitromob CRW variant Y213F / I215L. SEQ ID NO: 60 is the amino acid sequence of Nitromob CRW variant I175L / I215L. SEQ ID NO: 61 is the amino acid sequence of Nitromob CRW variant I208L / I215L. SEQ ID NO: 62 is the amino acid sequence of Nitromob CRW variant I215L / I255L. SEQ ID NO: 63 is the amino acid sequence of Nitromob CRW variant I255L / I257L. SEQ ID NO: 64 is the amino acid sequence of Nitromob CRW variant L214S / I215L. SEQ ID NO: 65 is the amino acid sequence of Nitromob CRW variant V203S / M204L. SEQ ID NO: 66 is the amino acid sequence of Nitromob CRW variant T218L. SEQ ID NO: 67 is the amino acid sequence of Nitromob CRW variant T218F. SEQ ID NO: 68 is the amino acid sequence of Nitromob CRW variant V185L. SEQ ID NO: 69 is the amino acid sequence of Nitromob CRW variant V193L / I215L. SEQ ID NO: 70 is the amino acid sequence of Nitromob CRW variant E196L / I215L. SEQ ID NO: 71 is the amino acid sequence of Nitromob CRW variant E186L / I215L. SEQ ID NO: 72 is the amino acid sequence of Nitromob CRW variant V177L / I215L. SEQ ID NO: 73 is the amino acid sequence of Nitromob CRW variant Y213L. SEQ ID NO: 74 is the amino acid sequence of Nitromob CRW variant V203S / M204L / I215L. SEQ ID NO: 75 is the NitromobCRW-Cterm-SUMO nucleotide sequence. SEQ ID NO: 76 is the amino acid sequence of the NitromobCRW-Cterm-SUMO extension peptide. SEQ ID NO: 77 is the amino acid sequence of NitromobCRW Y213L / I215L-Cterm-SUMO. definition

[0016] For the sake of clarity, certain terms used in this specification are defined and presented below:

[0017] "Activity" of the insecticidal proteins of the present invention means that the insecticidal proteins act as orally active insect control agents, have toxic effects, and / or are able to interfere with or prevent insect feeding, which may or may not cause the death of the insect. When the insecticidal proteins of the present invention are delivered to an insect, this result is typically the death of the insect, or the insect is unable to feed on a source that makes the insecticidal protein accessible to the insect. "Pesticide" is defined as a toxic biological activity that is able to control pests (such as insects, nematodes, fungi, bacteria or viruses), preferably by killing or destroying them. "Insecticidal" is defined as a toxic biological activity that is able to control insects, preferably by killing them. A "pesticide" is an agent having pesticidal activity. An "insecticide" is an agent having insecticide activity.

[0018] "Associated with" refers to two nucleic acids that are physically or functionally associated. For example, a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence that encodes an RNA or protein if the two sequences are operably linked or configured so that the regulatory DNA sequence will affect the expression level of the coding or structural DNA sequence.

[0019] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA). Preferably, the RNA is then translated in an organism to produce a protein.

[0020] "Controlling" insects means inhibiting the ability of insect pests to survive, grow, feed, and / or reproduce through toxic effects, or limiting insect-associated damage or loss of crop plants. "Controlling" insects may or may not mean killing the insects, although it preferably means killing the insects.

[0021] "Delivery" of an insecticidal protein means that the insecticidal protein comes into contact with the insect, produces a toxic effect, and controls the insect. The insecticidal protein can be delivered in a number of recognized ways, for example, via transgenic plants expressing the insecticidal protein, one or more formulated protein compositions, one or more sprayable protein compositions, bait matrices, or any other art-recognized toxin delivery system.

[0022] "An insect control effective amount" means a concentration of an insecticidal protein that inhibits the ability of insects to survive, grow, feed, and / or reproduce through toxic effects, or limits insect-related damage or loss of crop plants. An "insect control effective amount" may or may not mean killing insects, although it preferably means killing insects.

[0023] As used herein, "expression cassette" means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to a target nucleotide sequence, which is operably linked to a termination signal. It also typically comprises the sequence required for the appropriate translation of the nucleotide sequence. The expression cassette comprising the target nucleotide sequence may have at least one of its components that is heterologous relative to at least one of its other components. The expression cassette can also be naturally occurring but has been an expression cassette obtained in a recombinant form useful for heterologous expression. However, typically, the expression cassette is heterologous relative to the host, i.e., the specific nucleic acid sequence of the expression cassette is not naturally present in the host cell and must have been introduced into the host cell or the ancestor of the host cell through a transformation event. The expression of the nucleotide sequence in the expression cassette can be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some specific external stimuli. In multicellular organisms (e.g., plants), the promoter can also be specific for a specific tissue, organ, or developmental stage.

[0024] The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. The expression cassette may also comprise a native promoter driving its native gene, but has been obtained in a recombinant form useful for heterologous expression. This use of the expression cassette renders it non-naturally occurring in the cell into which it is introduced.

[0025] The expression cassette can also optionally be included in a transcription and / or translation termination region (i.e., termination region) that plays a role in plants. A variety of transcription terminators are available for use in the expression cassette and are responsible for transcription termination and correct mRNA polyadenylation when exceeding the heterologous nucleotide sequence of interest. The termination region can be natural for the transcription initiation region, can be natural for the operably connected nucleotide sequence of interest, can be natural for plant hosts, or can be derived from another source (i.e., external or heterologous for promoter, nucleotide sequence of interest, plant hosts, or any combination thereof). Suitable transcription terminators include, but are not limited to, CAMV 35S terminator, tml terminator, nopaline synthase terminator, and / or pea rbcs E9 terminator. These terminators can be used in both monocotyledons and dicotyledons. In addition, the natural transcription terminator of the encoding sequence can be used. Any known terminator available for use in plants that plays a role can be used in the context of the present invention.

[0026] When used with reference to polynucleotides (such as genes, ORFs or parts thereof, or transgenics of plants), the term "expression" refers to the process by which the genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA or snRNA) by the "transcription" of a gene (i.e., by the enzymatic action of an RNA polymerase), and, where applicable (e.g., if the gene encodes a protein), into protein by the "translation" of the mRNA. Gene expression can be regulated at many stages of the process. For example, in the case of an antisense construct or a dsRNA construct, expression can refer only to the transcription of the antisense RNA or only to the transcription of the dsRNA. In an embodiment, "expression" refers to the transcription and stable accumulation of a sense (mRNA) or functional RNA. "Expression" can also refer to the production of a protein.

[0027] "Gene" is a defined region within the genome that comprises a coding nucleic acid sequence, and typically also comprises other primary regulatory nucleic acids responsible for controlling the expression (i.e., transcription and translation) of the coding portion. A gene may also comprise other 5' and 3' untranslated sequences and terminator sequences. Other elements that may exist are, for example, introns. As found in nature, the regulatory nucleic acid sequence of a gene may not be operably connected with the associated nucleic acid sequence under normal circumstances, and therefore can not be a mosaic gene.

[0028] "Gene of interest" refers to any nucleic acid molecule that, when transferred to a plant, confers a desired trait on the plant (e.g., antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other harmful organisms, herbicide tolerance, abiotic stress tolerance, male sterility, modified fatty acid metabolism, modified carbohydrate metabolism, improved nutritional value, improved performance in an industrial process, or altered reproductive capacity). A "gene of interest" can also be a gene transferred to a plant for the production of a commercially valuable enzyme or metabolite in the plant.

[0029] A "heterologous" nucleic acid sequence or nucleic acid molecule is one that is not naturally associated with the host cell into which the nucleic acid sequence is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence. A heterologous nucleic acid sequence or nucleic acid molecule can comprise a chimeric sequence, such as a chimeric expression cassette in which the promoter and coding region are derived from multiple organisms. The promoter sequence can be a constitutive promoter sequence, a tissue-specific promoter sequence, a chemically inducible promoter sequence, a wound-inducible promoter sequence, a stress-inducible promoter sequence, or a developmental stage-specific promoter sequence.

[0030] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

[0031] "Homologous recombination" is the reciprocal exchange of nucleic acid segments between homologous nucleic acid molecules.

[0032] "Identity" or "percentage identity" refers to the degree of similarity between two nucleic acid or protein sequences. For sequence comparison, typically, a sequence serves as a reference sequence for comparison with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer (if necessary, specifying subsequence coordinates), and the parameters of the sequence algorithm program are specified. Then, the sequence comparison algorithm calculates the sequence identity percentages of one or more test sequences relative to the reference sequence based on the specified program parameters. In the context of two nucleic acids or two amino acid sequences, the phrase "substantially identical" refers to two or more sequences or subsequences that have at least about 50% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In certain embodiments, substantially identical sequences have at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or even at least about 90% or 95% nucleotide or amino acid residue identity. In certain embodiments, substantial identity exists over a region of at least about 50 residues, or over a region of at least about 100 residues, or the sequences are substantially identical over at least about 150 residues. In further embodiments, the sequences are substantially identical when they are identical over the entire length of the coding region.

[0033] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search by similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Street, Wisconsin, WI). Dr., Madison, WI), or by visual inspection (see generally Ausubel et al., infra).

[0034] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. These word hits are then extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of word hits in each direction ceases when the cumulative alignment score falls by the amount X from its maximum achieved value; when the cumulative score approaches 0 or below due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0035] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0036] Another widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson et al. Nuc. Acids Res., 22:4673-4680, 1994). The number of matched bases or amino acids is divided by the total number of bases or amino acids and multiplied by 100 to obtain the percent identity. For example, if two 580 base pair sequences have 145 matched bases, they would be 25% identical. If the two sequences being compared are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there are 100 matched amino acids between a 200 amino acid protein and a 400 amino acid protein, the two proteins are 50% identical relative to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches is divided by 150 (for nucleic acid bases) or 50 (for amino acids) and multiplied by 100 to obtain the percent identity.

[0037] Another indication that two nucleic acids are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specifically hybridizes" means that a molecule binds, double-strands, or hybridizes only to a specific nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Substantially binds" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and encompasses minor mismatches that can be accommodated by reducing the stringency of the hybridization medium to achieve desired detection of the target nucleic acid sequence.

[0038] In the context of nucleic acid hybridization experiments (such as DNA hybridization and RNA hybridization), "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Chapter 2, Part I "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York. Typically, highly stringent hybridization and wash conditions are selected to be higher than the thermal melting point (T for the specific sequence) at a defined ionic strength and pH. m Typically, under "stringent conditions," a probe will hybridize to its target sequence, but to no other sequences.

[0039] T m It is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T for a specific probe. m. An example of stringent hybridization conditions for hybridization of complementary nucleic acids (which have more than 100 complementary residues on the filter in a Southern or Northern blot) is 50% formamide with 1 mg of heparin at 42°C, and the hybridization is carried out overnight. An example of a high stringency wash condition is 0.15 M NaCl at 72°C for about 15 minutes. An example of a stringent wash condition is a 0.2x SSC wash at 65°C for 15 minutes (see Sambrook, below for a description of SSC buffer). Typically, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex of, for example, more than 100 nucleotides is 1x SSC at 45°C for 15 minutes. An example of a low stringency wash for a duplex of, for example, more than 100 nucleotides is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10-50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0-8.3, and the temperature is typically at least about 30° C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2x (or higher) observed compared to an unrelated probe in a particular hybridization assay indicates that specific hybridization has been detected. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if their encoded proteins are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0040] The following are examples of sets of hybridization / wash conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence under the following conditions: in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 2X SSC, 0.1% SDS at 50°C; more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 1X SSC, 0.1% SDS at 50°C; still more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 0.5X SSC, 0.1% SDS at 50°C; preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and washing in 0.1X SSC, 0.1% SDS at 50°C. Washing is in SDS at 50°C; more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C, and in 0.1X SSC, 0.1% SDS at 65°C.

[0041] Another indication that two nucleic acids or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross-reactive or specifically binds to the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, e.g., where the two proteins differ only in conservative substitutions.

[0042] A nucleotide sequence is "homologous to" a reference nucleic acid sequence when the sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.

[0043] An "isolated" nucleic acid molecule or isolated toxin is one that has been artificially separated from its natural environment and is therefore not a product of nature. An isolated nucleic acid molecule or toxin may be present in a purified form or may be present in a non-natural environment, such as, for example, without limitation, in a recombinant microbial cell, plant cell, plant tissue, or plant.

[0044] A "nucleic acid molecule" or "nucleic acid sequence" is a segment of single-stranded or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is typically a segment of DNA. In some embodiments, the nucleic acid molecule of the present invention is an isolated nucleic acid molecule.

[0045] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein.

[0046] As used herein, " codon optimized " sequence means the nucleotide sequence of recombinant, transgenic or synthetic polynucleotide, wherein these codons are selected to reflect the specific codon preference that host cell can have.This is to complete in such a way, and this mode is in order to keep the amino acid sequence of the polypeptide encoded by codon optimized polynucleotide.In certain embodiments, the nucleotide sequence of recombinant DNA construct comprises the sequence that has been codon optimized for the cell (for example, animal, plant or fungal cell) that has been waited for expression therein for this construct.For example, construct to be expressed in plant cell can make its whole or part of sequence (for example, first gene suppression element or gene expression element) be carried out codon optimized for expression in plant.See for example, U.S. Patent number 6,121,014, incorporated herein by reference.

[0047] A "plant" is any plant at any stage of development, particularly a seed plant.

[0048] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell can be in the form of an isolated single cell or a cultured cell, or as part of a higher-order organizational unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0049] "Plant cell culture" means a culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0050] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0051] A "plant organ" is a distinct and clearly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0052] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. This includes any plant tissue in plants or in culture. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term in conjunction with or alone with any specific type of plant tissue, as listed above or otherwise encompassed by this definition, is not intended to exclude any other type of plant tissue.

[0053] "Promoter" is an untranslated DNA sequence upstream of a coding region that contains an RNA polymerase binding site and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.

[0054] "Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements include promoters operably linked to a nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.

[0055] "Transformation" is a process for introducing heterologous nucleic acid into a host cell or organism. In specific embodiments, "transformation" means that a DNA molecule is stably integrated into the genome (nucleus or plastid) of the organism of interest.

[0056] "Transformed / transgenic / recombinant" refers to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host genome, or the nucleic acid molecule can exist as an extrachromosomal molecule. Such extrachromosomal molecules are capable of autonomous replication. Transformed cells, tissues, or plants should be understood to encompass not only the end product of the transformation process, but also its transgenic progeny. A "non-transformed," "non-transgenic," or "non-recombinant" host refers to a wild-type organism, such as a bacterium or plant, that does not contain the heterologous nucleic acid molecule.

[0057] Nucleotides are represented by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are also represented by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). DETAILED DESCRIPTION

[0058] The present invention relates to novel insecticidal proteins that are active against Coleoptera, such as Diabrotica zeae (Western Corn Rootworm; WCR), Diabrotica barbatae (Northern Corn Rootworm; NCR), and / or Diabrotica elevenstigmata (Southern Corn Rootworm; SCR), and / or other Diabrotica species, including Diabrotica dasyphylla (Mexican Corn Rootworm), and / or other Coleopteran insect pests, such as the Colorado Potato Beetle. In embodiments, the novel insecticidal proteins of the invention are active against Lepidoptera species. The invention also relates to nucleic acids, the expression of which produces the insecticidal proteins of the invention, and to methods of making and using these insecticidal proteins to control insect pests. In embodiments, expression of these nucleic acids produces insecticidal proteins that are useful for controlling Coleopteran insects, such as Western, Northern, and / or Southern Corn Rootworm, particularly when expressed in transgenic plants, such as transgenic corn plants.

[0059] The present invention further encompasses nucleic acid molecules comprising a nucleotide sequence encoding an insecticidal protein of the present invention. The nucleotide sequence can be optimized for expression in bacteria (e.g., E. coli) or for expression in plants (e.g., Zea mays). Nucleotide sequences optimized for expression in heterologous organisms (e.g., bacteria of a different origin) or plants do not occur naturally. In one aspect of this embodiment, the nucleic acid molecule comprises a nucleotide sequence of any one of SEQ ID NOs: 1 to 38, or a complementary sequence thereof. Specific exemplary teachings of methods for making nucleic acid molecules encoding insecticidal proteins of the present invention can be found in the Examples herein. One skilled in the art will recognize that modifications may be made to the exemplary methods for making insecticidal proteins encompassed by the present invention.

[0060] The skilled artisan will recognize that a transgene for commercial use, such as a nucleic acid molecule comprising any one of SEQ ID NOs: 1 to 38 or a complementary sequence thereof, may require relatively minor modifications to the nucleic acid sequence to comply with government regulatory standards. Such modifications will not affect the function of the resulting molecule, which will be substantially identical to SEQ ID NOs: 1 to 38. The skilled artisan will recognize that the modified nucleic acid molecule will be substantially identical to the starting molecule and is encompassed by the present invention.

[0061] The present invention also encompasses nucleic acid molecules comprising (a) a nucleotide sequence of any one of SEQ ID NOs: 1 to 38; (b) a nucleotide sequence that is at least 45% identical, at least 50% identical, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the nucleotide sequences of SEQ ID NOs: 1 to 38; (c) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide comprises SEQ ID NOs: 39 to 74 and has insect control activity; (d) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide comprises SEQ ID NOs: 39 to 74 and has insect control activity; any one of the amino acid sequences of NOs: 39 to 74 having at least 45% identity, at least 50% identity, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; or (e) a nucleotide sequence complementary to the nucleotide sequence of any one of (a) to (d) above.

[0062] The present invention further encompasses an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence comprising: (a) a nucleotide sequence of any one of SEQ ID NOs: 1 to 38; (b) a nucleotide sequence having at least 45% identity, at least 50% identity, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the nucleotide sequence of any one of SEQ ID NOs: 1 to 38; (c) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide comprises SEQ ID NOs: 39 to 74 and has insect control activity; (d) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide comprises SEQ ID NOs: 39 to 74 and has insect control activity; NO:39 to 74 has at least 45% identity, at least 50% identity, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of NO:39 to 74; or (e) a nucleotide sequence complementary to the nucleotide sequence of any one of (a) to (d) above. In some embodiments, the present invention encompasses an expression cassette comprising a heterologous nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide has at least 93% identity to the amino acid sequence of SEQ ID NO:39 to 74. The expression cassette comprises a promoter operably linked to the heterologous nucleotide sequence, and the expression cassette is not naturally occurring.

[0063] The present invention also encompasses recombinant vectors or constructs, which may also be referred to as vectors or constructs, comprising expression cassettes and / or nucleic acid molecules of the present invention. In such vectors, the nucleic acids are preferably in expression cassettes comprising regulatory elements for expressing the nucleotide molecules in host cells capable of expressing the nucleotide molecules. Such regulatory elements typically comprise a promoter and a termination signal and preferably also comprise elements that allow for efficient translation of the polypeptide encoded by the nucleic acid of the present invention. The vector comprising the nucleic acid can replicate (preferably as an extrachromosomal molecule) in a specific host cell and can therefore be used to amplify the nucleic acid of the present invention in these host cells. The present invention also encompasses host cells containing expression cassettes or nucleic acid molecules of the present invention. In one embodiment, the host cell for such vectors is a microorganism (such as a bacterium, particularly Bacillus thuringiensis or Escherichia coli), or a fungus (such as yeast). In another embodiment, the host cell for such recombinant vectors is an endophyte or epiphyte. In yet another embodiment, such vectors are viral vectors and are used to replicate nucleotide sequences in a specific host cell (such as an insect cell or a plant cell). Recombinant vectors are also used to transform the nucleotide molecules of the present invention into host cells, whereby these nucleotide molecules are stably integrated into the DNA of the transgenic host. In one embodiment, the transgenic host is a plant, e.g., a monocot, such as a corn plant or a wheat plant. In an embodiment, the transgenic host plant is a dicot, such as a soybean plant or a cotton plant.

[0064] In another embodiment, at least one of the nucleic acids of the present invention is inserted into an appropriate expression cassette (comprising a promoter and a termination signal). Expression of the nucleic acid can be constitutive, or an inducible promoter that initiates transcription in response to various types of stimuli can be used. In another embodiment, the cell in which the insecticidal protein of the present invention is expressed is a microorganism, such as a virus, bacteria, or fungus. In yet another embodiment, a virus (such as a baculovirus) contains a nucleic acid of the present invention in its genome and, after infecting an appropriate eukaryotic cell suitable for viral replication and expression of the nucleic acid, expresses a large amount of the corresponding insecticidal protein. The insecticidal protein thus produced is used as an insecticide. Alternatively, a baculovirus engineered to contain the nucleic acid is used to infect insects in vivo and kill them through expression of the insecticidal toxin, or through a combination of viral infection and expression of the insecticidal toxin. In a further embodiment, the present invention also encompasses a method for producing a polypeptide having insecticidal activity, comprising culturing a host cell under conditions in which a nucleic acid molecule encoding the polypeptide is expressed.

[0065] Bacterial cells are also hosts for expressing nucleic acid of the present invention. In one embodiment, non-pathogenic symbiotic bacteria (so-called endophytes) that can live and replicate in plant tissues have been used, or non-pathogenic symbiotic bacteria (so-called epiphytes) that can be settled in phyllosphere or rhizosphere have been used. This type of bacterium includes the bacterium of the following genus: Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Corynebacterium, Enterobacter, Erwinia, Flavobacterium, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas. Symbiotic fungi (such as Trichoderma and Glycobacterium) are also possible hosts for expressing nucleic acid of the present invention for the same purpose.

[0066] These gene manipulation techniques are specific for different available hosts and are known in the art. For example, expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes after tac or trc promoters in Escherichia coli (in transcription or translation fusion). In order to express the operon encoding multiple ORFs, the simplest method is to insert the operon into a vector (such as pKK223-3) in transcriptional fusion, allowing the homologous ribosome binding site of the heterologous gene to be utilized. Overexpression techniques in gram-positive species (such as Bacillus) are also known in the art, and can be used in the context of the present invention (Quax et al., in: Industrial Microorganisms: Basic and Applied Molecular Genetics [industrial microorganisms: basic and applied molecular genetics], editor Baltz et al., American Society for Microbiology [American Society for Microbiology], Washington (1993)). Alternative systems for overexpression rely on, for example, yeast vectors and include the use of Pichia, Saccharomyces, and Kluyveromyces (Sreekrishna, In: Industrial microorganisms: basic and applied molecular genetics, Baltz, Hegeman, and Skatrud, eds., American Society for Microbiology, Washington (1993); Dequin and Barre, Biotechnology L2: 173-177 (1994); van den Berg et al., Biotechnology 8: 135-139 (1990)).

[0067] Certain insecticidal proteins have been expressed in plants, and seeds from such plants are sold to farmers each year for use in controlling various insect pests. Such self-protective insecticidal products are subject to review and registration by various regulatory agencies, including, for example, the U.S. Environmental Protection Agency (EPA).

[0068] Dietary exposure is the primary route by which humans can be exposed to insecticidal proteins expressed in transgenic plants. Mammalian acute oral toxicity and protein digestibility are endpoints in the EPA's human health risk assessments. Further scientific evidence for the safety of insecticidal proteins lies in their rapid in vitro degradation using simulated gastric fluid. For example, results from seven in vitro assays using representative Cry1, Cry2, and Cry3 proteins established that these proteins typically degrade rapidly within 30 seconds. These results support the broader conclusion that members of these groups of Cry proteins (which share significant amino acid sequence identity) are likely to degrade rapidly after ingestion in humans. Similar testing is being performed on each transgenic protein expressed in the plant. Another consideration is whether the insecticidal protein might elicit an allergic reaction. The demonstrated rapid in vitro degradation of transgenic insecticidal proteins should minimize the likelihood of this occurring. In contrast, food allergens are typically retained in in vitro gastrointestinal models, while common food proteins without a history of allergies are rapidly degraded in simulated gastric fluid (Metcalfe et al., 1996).

[0069] Simulated gastric fluid (SGF) is determined under the strict controlled conditions representing the upper digestive tract of mammals to measure the in vitro digestibility of test protein. For example, at 37 ℃, through the time period of one hour, the test Cry protein (at a concentration of 0.5-5mg / ml) produced by bacteria is exposed to pepsin (from porcine gastric mucosa, dissolved in 2mg / mlNaCl, pH 1.2) at a ratio of 10 units of pepsin activity / μg test protein. Sample is taken out at the time point of 1, 2, 5, 10, 30 and 60 minutes, and quenched immediately by adding preheated (95 ℃-2 minutes) stop buffer (65% 0.5M sodium bicarbonate (pH 11), 35% Tricine loading buffer) so that pepsin is inactivated immediately, and return to heat and continue for 5 minutes. Once the assay is complete, time point samples and controls (test protein only, pepsin only) are examined by SDS-PAGE on 10%-20% Tris-Tricine gels (peptides visible down to 1 kDa) to track the kinetics and level of digestion by pepsin. If significant polypeptide fragments of the test protein or proteins herein are visible at, for example, the 5 and / or 10 minute time points, then it is not digestible or not fully digestible by the SGF assay and can be qualitatively scored as "no" or "undigestible." If the test protein or any significant polypeptide fragment is visible at, for example, the 5 minute time point, then it can be digested by the SGF assay and can be qualitatively scored as "yes" or "digestible."

[0070] The present invention also encompasses polypeptides comprising an amino acid sequence that is at least 45% identical, at least 50% identical, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% identical, or 100% identical to any one of SEQ ID NOs: 39 to 74, and further comprising an introduced protease cleavage site. The introduced protease cleavage site is not naturally occurring and is introduced into the polypeptide sequence as a substitution mutation or as an insertion or deletion mutation. The introduced protease cleavage site can be introduced by inserting at least one leucine residue into the polypeptide sequence comprising any one of SEQ ID NOs: 39 to 74. The introduced mutation may destabilize the polypeptide, thereby allowing proteases to access cleavage sites that were previously inaccessible to proteases due to the compact and / or stable folding of the protein or due to steric hindrance. The protease cleavage site introduced can be a sudden change introduced in the polypeptide sequence, and this sudden change is recognized by protease (for example, chymotrypsin, trypsin or pepsin) as the site of proteolytic cleavage. In certain embodiments, the protease cleavage site introduced can change existing protease cleavage site so that it is recognized by different proteases. Protease cleavage sites for chymotrypsin, trypsin and pepsin are well known in the art. Chymotrypsin preferentially cuts peptide amide bonds, and wherein the carboxyl side (P1 position) of the amide bond is a large hydrophobic amino acid (tyrosine, tryptophan and phenylalanine). Trypsin mainly cuts the peptide chain at the carboxyl side of amino acid lysine or arginine, unless any one of them is proline subsequently. Pepsin is the most effective aspect the peptide bond between cutting hydrophobic amino acids and preferably aromatic amino acids (such as phenylalanine, tryptophan, tyrosine and leucine). These cleavage sites are preferential cleavage sites, do not include all cleavage sites identified by chymotrypsin, trypsin or pepsin, and do not also include all cleavage sites of all proteases.

[0071] An example of a polypeptide engineered to include an introduced protease cleavage site is the NitromobCRW variant Y213L / I215L (SEQ ID NO: 47). This substitution mutation changes the YNA Y L I G" to "YNA L L L" motif. The introduced protease cleavage site can be recognized by pepsin and / or chymotrypsin and is not present in the wild-type NitromobCRW protein sequence. In some embodiments, the introduced protease cleavage site may be at or near the site of the mutation, for example, residues 190-230 of the polypeptide. The NitromobCRW variant Y213L / I215L may have an altered or less stable tertiary structure compared to wild-type NitromobCRW. In some embodiments, the introduced protease cleavage site may be located distal to the introduced mutation. For example, the introduced Y213 and / or I215 mutations may "loosen" the three-dimensional folding of the NitromobCRW polypeptide, thereby making a previously inaccessible (and therefore not cleaved) protease cleavage site accessible to the protease. This results in the introduced mutations introducing a protease cleavage site that is not present in the unaltered polypeptide. In some embodiments, the introduced mutations and / or the introduced protease cleavage site are located between amino acid residues 1 to 300 of any one of SEQ ID NOs: 39 to 74. In some embodiments, the introduced mutations and / or the introduced protease cleavage site are located between amino acid residues 1 to 300 of any one of SEQ ID NOs: 39 to 74. In another embodiment, the introduced mutations and / or the introduced protease cleavage site are located between amino acid residues 97 to 300 of any one of SEQ ID NOs: 39 to 74. In another embodiment, the introduced mutations and / or the introduced protease cleavage site are located between amino acid residues 97 to 266 of any one of SEQ ID NOs: 39 to 74. In another embodiment, the introduced mutations and / or the introduced protease cleavage site are located between amino acid residues 175 to 266 of any one of SEQ ID NOs: 39 to 74. In another embodiment, the introduced mutations and / or the introduced protease cleavage site are located between amino acid residues 185 to 250 of any one of SEQ ID NOs: 40 to 74. In another embodiment, the introduced mutations and / or the introduced protease cleavage site are located between amino acid residues 200 to 230 of any one of SEQ ID NOs: 40 to 74.

[0072] The present invention also encompasses polypeptides comprising an amino acid sequence that is at least 45% identical, at least 50% identical, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, at least 99% identical, or 100% identical to any one of SEQ ID NOs: 39 to 74, and further comprising an introduced mutation that improves digestibility in an SGF assay compared to a polypeptide comprising the amino acid sequence of SEQ ID NO: 39. The mutation can be a substitution mutation, an insertion, or a deletion. The mutation can be an insertion of at least one leucine residue.

[0073] The present invention also includes methods for improving digestibility of a polypeptide having at least 45% identity, at least 50% identity, at least 55% identity, at least 60% identity, at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 92% identity, at least 95% identity, at least 98% identity, at least 99% identity, or 100% identity to any of SEQ ID NOs: 39 to 74, comprising introducing at least one mutation into the amino acid sequence of the polypeptide. In embodiments, the introduced mutation improves digestibility of the polypeptide in an SGF assay. The mutation may improve digestibility by introducing a protease cleavage site. In other embodiments, the mutation may improve digestibility by altering the specificity of the protease at that site. For example, this may result in a mutation that is currently a chymotrypsin or trypsin site being mutated into a pepsin site. In other embodiments, the mutation may destabilize the protein, making the site accessible to a protease for cleavage. The protease-accessible site may be distal to the introduced mutation. In preferred embodiments, the mutation does not alter, or does not significantly alter, the activity or insecticidal activity of the polypeptide. In some embodiments, the polypeptide having the introduced mutation has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the insecticidal activity of NitromobCRW. This method is exemplified in the Examples of this specification, where, for example, the NitromobCRW variant Y213L / I215L was found to have improved digestibility in the SGF assay. It also retained very high insecticidal activity.

[0074] In some embodiments of the above methods, the one or more introduced mutations may be located between amino acid residues 1 to 300 of any one of SEQ ID NOs: 39 to 74. In further embodiments, the one or more introduced mutations may be located between amino acid residues 97 to 300 of any one of SEQ ID NOs: 39 to 74. In further embodiments, the one or more introduced mutations may be located between amino acid residues 97 to 266 of any one of SEQ ID NOs: 39 to 74. In further embodiments, the one or more introduced mutations may be located between amino acid residues 175 to 266 of any one of SEQ ID NOs: 39 to 74. In further embodiments, the one or more introduced mutations may be located between amino acid residues 185 to 250 of any one of SEQ ID NOs: 39 to 74. In further embodiments, the one or more introduced mutations may be located between amino acid residues 200 to 230 of any one of SEQ ID NOs: 39 to 74.

[0075] In other embodiments, mutations can be introduced into Y213 and / or I215 of SEQ ID NO:39 or their proximal ends. In further embodiments, mutations can be Y213L / I215L. In other embodiments, mutations can be insertions or deletions of amino acid residues, such as, for example, insertions of at least one leucine residue. The residue or residues can be adjacent to Y213 and / or I215 of SEQ ID NO:39 or adjacent thereto, such as, for example, NitromobCRW variants L214-Leu-I215 (SEQ ID NO:57) or I215-Leu-G216 (SEQ ID NO:58). Leucine residues can also be inserted into the proximal ends of Y213 and / or I215, wherein "proximal ends" can be at least 1, at least 2, at least 4, at least 6, at least 8, at least 10, or at least 20 amino acids away from Y213 and / or I215.

[0076] The insecticidal proteins of the present invention have insect control activity when tested against insect pests in bioassays. In one embodiment, the insecticidal proteins of the present invention are active against coleopteran insects and / or lepidopteran insects. It will be understood by those skilled in the art that the proteins of the present invention may have a different range of insecticidal activity compared to other proteins of the present invention. In some embodiments, NitromobCRW mutant variants may have insecticidal activity against a wider range of insect pests (e.g., more coleopteran or lepidopteran species) than other variants of NitromobCRW. In other embodiments, variants of NitromobCRW may have insecticidal activity against Lepidopteran species but not against Coleopteran species. In some embodiments, variants of NitromobCRW may have insecticidal activity against a wider range of Coleopteran or Lepidopteran species compared to unmodified NitromobCRW (SEQ ID NO: 39).

[0077] Insects of the order Lepidoptera include, but are not limited to, any insect now known or later identified that is classified as such, including those species within the suborders Hypogyroptera, Proboscis, and Heterotrichum, and any combination thereof. Exemplary Lepidoptera include, but are not limited to, Ostrinia spp., such as O. nubilalis (European corn borer); Plutella spp., such as P. xylostella (diamondback moth); Spodoptera spp., such as S. frugiperda (fall armyworm), S. ornithogalli (yellowstriped armyworm), S. praefica (western yellowstriped armyworm), S. eridania (southern armyworm), and S. exigua (beet armyworm); Agrotis spp., such as A. ipsilon (black cutworm), A. segetum (common cutworm), and A. cutworm), A. gladiaria (claybacked cutworm), and A. orthogonia (palewestern cutworm); Striacosta spp., such as S. albicosta (western bean cutworm); Helicoverpa spp., such as H. zea (corn earworm), H. punctigera (native budworm), S. littoralis (Egyptian cotton leafworm), and H. armigera (cotton bollworm); Heliothis spp., such as H. virescens (tobacco budworm); budworm); Diatraea spp., such as D. grandiosella (southwestern corn borer) and D.saccharalis (sugarcane borer); Trichoplusia spp., such as T. ni (cabbage looper); Sesamia spp., such as S. nonagroides (Mediterranean cornborer); Pectinophora spp., such as P. gossypiella (pink bollworm); Cochylis spp., such as C. hospes (banded sunflower moth); Manduca spp., such as M. sexta (tobacco hornworm) and M. quinquemaculata (tomato hornworm). hornworm; Elasmopalpus spp., such as E. lignosellus (lesser cornstalk borer); Pseudoplusia spp., such as P. includens (soybean looper); Anticarsia spp., such as A. gemmatalis (velvet bean caterpillar); Plathypena spp., such as P. scabra (green cloverworm); Pieris spp., such as P. brassicae (cabbage butterfly); Papaipema spp. spp., such as P. nebris (stalk borer); Pseudaletia spp., such as P. unipuncta (common armyworm); Peridroma spp., such as P. saucia (variegated cutworm); Keiferia spp., such as K. lycopersicella (tomato pinworm); Artogeia spp., such as A.rapae) (imported cabbageworm); Phthorimaea spp., such as P. operculella (potato tuberworm); Crymodes spp., such as C. devastator (glassy cutworm); Feltia spp., such as F. ducens (dingy cutworm); and any combination of the foregoing. In one aspect of this embodiment, the insecticidal protein of the invention is active against black cutworm, sugarcane borer, and / or southwestern corn borer.

[0078] Insects of the order Coleoptera include, but are not limited to, any Coleoptera now known or later identified, including those of the suborders Protocoleoptera, Myxophaga, Carnivora, and Polyphaga, and any combination thereof.

[0079] In one aspect of this embodiment, the insecticidal proteins of the invention are active against species of the genus Diabrotica. Diabrotica is a genus of beetles in the order Coleoptera, commonly known as "corn rootworms" or "cucumber beetles." Exemplary Diabrotica species include, but are not limited to: D. barteata (northern corn rootworm), D. zeae (western corn rootworm), D. undecimpunctata (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. beetle), South American leaf beetle (D. speciosa) (chrysanthemum beetle), Mexican corn rootworm (Mexican corn rootworm), Benni rootworm (D. beiensis), Christa rootworm (D. cristata), Kowei rootworm (D. curvipustulata), two-spotted rootworm (D. dissimilis), elegant rootworm (D. elegantula), Imer rootworm (D. emorsitans), grass rootworm (D. graminea), Hispani rootworm (D. graminea), D. hispanolae, D. lemniscata, D. linsleyi, D. milleri, D. nummularis, D. occlusa, D. porracea, D. scutellata, D. tibialis, D. trifasciata, and D. viridula; and any combination thereof.

[0080] Other non-limiting examples of coleopteran insect pests according to the present invention include Leptinotarsa spp., such as L. decemlineata (Colorado potato beetle); Chrysomela spp., such as C. scripta (cottonwood leaf beetle); Hypothenemus spp., such as H. hampei (coffee berry borer); Sitophilus spp., such as S. zeamais (maize weevil); Epitrix spp., such as E. hirtipennis (tobacco flea beetle); (tobacco flea beetle) and E. cucumeris (potato flea beetle): Phyllotreta spp., such as P. cruciferae (crucifer flea beetle) and P. pusilla (western black flea beetle); Anthonomus spp., such as A. eugenii (pepper weevil); Hemicrepidus spp., such as H. memnonius (wireworms); Melanotus spp., such as M. communis (wireworms); Ceutorhychus spp. spp., such as C. assimilis (cabbage seedpod weevil); Aeolus spp., such as A. mellillus (wireworm); Aeolus spp., such as A. mancus (wheat wireworm); Horistonotus spp., such as H. uhlerii (sand wireworm); Sphenophorus spp., such as S. maidis (maize billbug), S. zeae (timothy billbug), S. parvulus (bluegrass billworm). billbug) and S. callosus (southern cornbillbug); Phyllophaga spp. (white grub); Chaetocnema spp., such as C. pulicaria (corn flea beetle); Popillia spp., such as P.japonica (Japanese beetle); Epilaxna spp., such as E. varivestis (Mexican bean beetle); Cerotoma spp., such as C. trifurcate (Bean leaf beetle); Epicauta spp., such as E. pestifera and E. lemniscata (Blister beetles); and any combination of the foregoing.

[0081] The insecticidal proteins of the present invention may also be active against Hemiptera, Diptera, Lygus spp., and / or other piercing-sucking insects (e.g., Orthoptera or Thysanoptera). Insects of the order Diptera include, but are not limited to, any Diptera insect now known or hereafter identified, including, but not limited to, Liriomyza spp., such as L. trifolii (leaf miner) and L. sativae (vegetable leaf miner); Scrobipalpula spp., such as S. absoluta (tomato leaf miner); Delia spp., such as D. platura (seedcorn maggot), D. brassicae (cabbage maggot), and D. radicum (cabbage root fly); Psilia spp., such as P. rosae (carrot rust fly); Tetanops spp., such as P. rosae (carrot rust fly); spp.), such as T. myopaeformis (sugarbeet root maggot); and any combination of the foregoing.

[0082] Insects of the order Orthoptera include, but are not limited to, any Orthoptera insect now known or later identified, including, but not limited to, Melanoplus spp., such as M. differentialis (Differential grasshopper), M. femurrubrum (Redlegged grasshopper), M. bivittatus (Twostriped grasshopper); and any combination thereof.

[0083] Insects of the order Thysanoptera include, but are not limited to, any Thysanoptera insect now known or later identified, including, but not limited to, Frankliniella spp., such as F. occidentalis (western flower thrips) and F. fusca (tobacco thrips); and Thrips spp., such as T. tabaci (onion thrips), T. palmi (melon thrips); and any combinations of the foregoing.

[0084] The insecticidal proteins of the present invention may also be active against nematodes. As used herein, the term "nematode" encompasses any currently known or later identified organism classified as a member of the phylum Nematoda of the animal kingdom, including but not limited to nematodes in the class Adenophora (including, for example, the order Rhizophora, Isopharyngea, Monodonta, Lanceteria, Trichodesmata, Trachelona, Muspas, Lachnophora, Chromophora, Taenelena, Chain Rings, and Monogyria) and / or the class Tubulonema (including, for example, the order Rhabditis, Strongylida, Ascaris, Spirulina, Camelida, Digastricales, Sphaeroides, and Aphelenchus).

[0085] Nematodes include, but are not limited to, parasitic nematodes such as root-knot nematodes, cyst nematodes, and / or decay nematodes. Exemplary genera of nematodes according to the present invention include, but are not limited to, root-knot nematodes, Heterodera (cyst nematodes), Globodera (cyst nematodes), Penelope (piercing nematodes), Renilla (kidney-shaped kidney-shaped nematodes), Pratylenchus (rot nematodes), Aphelenchus (leaf nematodes), Heliconema (helical nematodes), Nematode (lance nematodes), Pseudocercosus (short and thick root nematodes), Long-nematode, Pearl Nematode (pseudo-root-knot nematodes), Subanguina, Stinging Nematode (thorn nematodes), Microcircle Nematode (microcircle nematodes), Ring Nematodes (ring nematodes), Stem Nematodes (stem nematodes), Trypanoidea (tie nematodes), Semirothrix (semirothrix nematodes), Sheath Nematodes (sheath nematodes), Subroot Nematodes (subroot nematodes), Root-knot Nematodes (Hypsoperine), Ditylenchus (large stem nematodes), Melinius spp., Punctodera, Quinisulcius, Shield Nematodes (shield nematodes), Xiphinema (dagger nematodes), Dwarf Nematodes (dwarf nematodes), Penelope nematodes (puncture nematodes), Bursaphelenchus (roundworms), and any combination thereof.

[0086] Exemplary plant parasitic nematodes according to the present invention include, but are not limited to, Belonolaimus gracilis, Belonolaimus longicaudatus, Bursaphelenchus xylophilus (pine wood nematode), Criconemoides ornata, Ditylenchus destructor (potato rot nematode), Ditylenchus dipsaci (stem and bulb nematode), Globodera pallida (potato cyst nematode), Globodera rostochiensis (golden nematode), Heterodera glycines (soybean cyst nematode), Heteroderas schachtii (sugar beet cyst nematode), and Ditylenchus schachtii (sweet beet cyst nematode). nematode); Heterodera zeae, corn cystnematode; Heterodera avenae,cereal cyst nematode), Heterodera carotae, Heterodera trifolii, Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimus magnistylus, Longidorus breviannulatus, Meloidogynearenaria, Meloidogyne chitwoodi, Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica, Mesocriconema xenoplax, Nacobbus aberrans, Naccobus dorsalis, Paratrichodorus christiei, Paratrichodorus minor), Pratylenchus brachyurus, Pratylenchus crenatus, Pratylenchus hexincisus, Pratylenchus negletus, Pratylenchus penetrans, Pratylenchus projectus, Pratylenchus scribneri, Pratylenchus tenuicaudatus, Pratylenchus thornei, Pratylenchus zeae, Punctodera chaccoensis, Quinisulcius acutus, Radopholus similis, Rotylenchulus reniformis, Tylenchorhynchus dubius), Tylenchulus semipenetrans, Siphinema americanum, X. Mediterraneum, and any combination of the foregoing.

[0087] In another embodiment, the present invention encompasses a method for producing an insecticidal protein active against insects, the method comprising: (a) obtaining a host cell comprising a gene, which gene itself comprises an expression cassette and / or nucleic acid molecule of the present invention; and (b) growing the transgenic host cell in a manner to express an insecticidal protein active against insects.

[0088] In yet further embodiments, the present invention encompasses methods of controlling insects comprising delivering to the insects an insect-control-effective amount of an insecticidal protein of the present invention.

[0089] In one embodiment, at least one of the insecticidal proteins of the present invention is expressed in a higher organism, such as a plant. In this case, a transgenic plant expressing an insect-controlling amount of the insecticidal protein protects itself from insect pests. When an insect begins to feed on the transgenic plant, it also ingests the expressed insecticidal protein. This prevents the insect from further feeding on plant tissue and / or may even injure or kill the insect. A nucleic acid of the present invention is inserted into an expression cassette, which can then be stably integrated into the plant's genome. In another embodiment, the nucleic acid is contained in a non-pathogenic, self-replicating virus. Plants transformed according to the present invention can be monocots or dicots and include, but are not limited to, corn, wheat, oats, turf grass, pasture grass, flax, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis species, and woody plants such as coniferous and deciduous trees.

[0090] In another embodiment, the present invention encompasses a method for producing a plant or plant part having enhanced insect resistance compared to a control plant or plant part, the method comprising: (a) introducing a nucleic acid molecule comprising an expression cassette of the present invention; and (b) growing the plant part into a plant that expresses the heterologous nucleic acid molecule of the expression cassette and that has enhanced insect resistance compared to a control plant or plant part not transformed with the nucleic acid molecule comprising the expression cassette. In a preferred embodiment, the expression cassette can encode a polypeptide comprising an amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or similar to SEQ ID NOs: 39 to 74. In a preferred embodiment, the expression cassette can encode a polypeptide comprising an amino acid sequence that is at least 60% identical to SEQ ID NO: 47. "Enhanced" insect resistance can be measured as increased insecticidal activity. Enhanced insect resistance can be 0%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% greater insecticidal activity compared to control plants. Plants or plant parts having enhanced insect resistance compared to control plants or plant parts can be produced by plant transformation, plant tissue culture, or breeding methods. Plants or plant parts can be produced by sexual or asexual propagation methods. Any suitable control plant or plant part can be used, such as plants grown under the same environment and having the same or similar genetic background. In embodiments, the control plant or plant part has the same genetic background and is grown under the same environment as the described plant, but does not contain a molecule of the invention, while the described plant contains a molecule of the invention.

[0091] In another embodiment, the present invention encompasses a method for increasing insect resistance in a plant or plant part compared to a control plant or plant part, the method comprising expressing in the plant or plant part a nucleic acid molecule or expression cassette of the present invention, wherein expression of the heterologous nucleic acid of the expression cassette results in a plant or plant part having increased insect resistance compared to a control plant or plant part. In embodiments, the expression cassette or nucleic acid molecule comprises a promoter operably linked to a heterologous nucleic acid molecule comprising a nucleotide sequence comprising: (a) the nucleotide sequence of any one of SEQ ID NOs: 1 to 38; (b) a nucleotide sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1 to 38; (c) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide comprises SEQ ID NOs: 39 to 74 and has insect control activity; (d) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide comprises SEQ ID NOs: 39 to 74 and has insect control activity. NO:39 to 74 amino acid sequence has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; or (e) a nucleotide sequence complementary to the nucleotide sequence of any one of the above-mentioned (a) to (d). Nucleic acid molecules or expression cassettes can be introduced into plants. In certain embodiments, nucleic acid molecules or expression cassettes can be introduced into plant parts, and plants comprising nucleic acid molecules or expression cassettes can be produced from these plant parts.

[0092] In another embodiment, the present invention encompasses a method for producing a plant having enhanced insect resistance compared to a control plant, the method comprising detecting a heterologous nucleic acid comprising a nucleic acid molecule or expression cassette of the present invention in a plant part, and producing a plant from the plant part, thereby producing a plant having enhanced insect resistance compared to a control plant. In a further embodiment, the present invention encompasses a method for identifying a plant or plant part having enhanced insect resistance compared to a control plant or plant part, the method comprising detecting a nucleic acid molecule or expression cassette of the present invention in the plant or plant part, thereby identifying a plant or plant part having enhanced insect resistance. In a further embodiment, the expression cassette or its diagnostic fragment is detected in an amplified product from a nucleic acid sample of the plant or plant part. The diagnostic fragment can be a nucleic acid molecule of at least 10 consecutive nucleotides long that is unique to the expression cassette of the present invention.

[0093] In yet another embodiment, the present invention encompasses a method of producing a plant having enhanced insect resistance compared to a control plant or plant part, the method comprising crossing a first parent plant with a second parent plant, wherein at least the first parent plant comprises in its genome a heterologous nucleic acid comprising a nucleic acid molecule or expression cassette of the present invention; and producing progeny generations, wherein the progeny generations comprise at least one plant having in its genome the heterologous nucleic acids and exhibiting enhanced insect resistance compared to a control plant.

[0094] In preferred embodiments, the methods of the present invention confer enhanced insect resistance to plants or plant parts against coleopteran and / or lepidopteran insect pests. Insect control of coleopteran insect pests is demonstrated in the examples. In further embodiments, the methods of the present invention confer enhanced insect resistance to Diabrotica species (including Diabrotica spp., Diabrotica barbatus, Diabrotica elevenstigmata, Diabrotica peciosa, and / or Diabrotica sp.) and / or related species on plants or plant parts.

[0095] In preferred embodiments, the methods of the present invention confer enhanced insect resistance to monocotyledonous plants.

[0096] The present invention further encompasses transgenic plants comprising a heterologous nucleic acid molecule or expression cassette of the present invention that confers enhanced insect resistance when transcribed or translated. In preferred embodiments, the heterologous nucleic acid molecule comprises a sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1 to 38, or the complement thereof. In another embodiment, the transgenic plant comprises a heterologous nucleic acid molecule comprising a sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 1 to 38, or a complementary sequence thereof. In embodiments, the transgenic plant is a dicot. In preferred embodiments, the transgenic plant is a monocot. In other embodiments, the transgenic plant is alfalfa, fennel, apple, apricot, artichoke, arugula, asparagus, avocado, banana, bean, beet, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery, cherry, cilantro, citrus, clementine, coffee bean, corn, cotton, cucumber, Douglas fir, eggplant, endive, kohlrabi, eucalyptus, fennel, fig, gourd, grape, grapefruit, honeydew melon, jicama, kiwi, lettuce, leek, In a preferred embodiment, the transgenic plant is millet, switchgrass, maize, sorghum, wheat, oats, oats, turf grass, pasture grass, flax, rice, sugarcane, rapeseed or barley.

[0097] In another embodiment, the transgenic plant of the present invention comprises a heterologous nucleic acid molecule comprising a promoter sequence. In another embodiment, the transgenic plant of the present invention may comprise a heterologous nucleic acid molecule encoding at least one other desired proterties. Additional proterties may be encoded on a heterologous nucleic acid molecule identical to the molecule of the present invention, or may be encoded on a second heterologous nucleic acid molecule. Additional desired proterties may confer insect resistance to a second insect pest, insect resistance to the same insect pest, abiotic stress tolerance, male sterility, herbicide resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, nematode resistance, modified fatty acid metabolism, modified carbohydrate metabolism, improved nutritional value, improved performance in industrial processes, or altered reproductive capacity. Additional desired proterties may also induce commercially valuable enzymes or metabolites to be produced in the plant.

[0098] In embodiments, the desired additional trait is a second pesticide.The second pesticide may be active against any plant pest, including insects, nematodes, fungi, viruses, or bacteria. Examples of insect plant pests include, but are not limited to, Nilaparvata spp. (e.g., N. lugens (brown planthopper)); Laodelphax spp. (e.g., L. striatellus (small brown planthopper)); Nephotettix spp. (e.g., N. virescens or N. cincticeps (green leafhopper), or N. nigropictus (rice leafhopper)); Sogatella spp. (e.g., S. furcifera (white-backed planthopper)); Blissus spp. (e.g., B. leucopterus (wheat leafhopper)); leucopterus (chinchbug); Scotinophora spp. (e.g., S. vermidulate, rice blackbug); Acrosternum spp. (e.g., A. hilare (green stink bug); Parnara spp. (e.g., P. guttata, riceskipper); Chilo spp. (e.g., C. suppressalis (rice striped stem borer), C. auricilius (gold-fringed stemborer), or C. polychrysus (dark-headed stem borer). borer); Chilotraea spp. (e.g., C. polychrysa (rice stalk borer); Sesamia spp. (e.g., S.inferens (pink rice borer); Tryporyza spp. (e.g., T. innotata (white rice borer), or T. incertulas (yellow rice borer); Cnaphalocrocis spp. (e.g., C. medinalis (rice leafroller); Agromyza spp. (e.g., A. oryzae (leafminer), or A. parvicornis (corn blot leafminer); C. spp. (e.g., D. grandiosella (southwestern corn borer)); borer; Narnaga species (e.g., N. aenescens, green rice caterpillar); Xanthodes species (e.g., X. transversa, green caterpillar); Spodoptera species (e.g., Spodoptera frugiperda (fall armyworm), S. exigua (beet armyworm), S. littoralis (climbing cutworm), or S. praefica (western yellowstriped armyworm); Mythimna species (e.g., Mythmna seperata, Pseudaletia seperata); Heliothis species (e.g., corn earworm (corn moth)); Colaspis species (e.g., C. brunnea, grapecolaspis); Lissorhoptrus species (e.g., Lissorhoptrus spp.). spp.) (e.g., L. oryzophilus (rice water weevil); Echinocnemus spp. (e.g., E. squamos (rice plant weevil)); Diclodispa spp. (e.g., D. armigera, rice hispa); Oulema spp. (e.g., O.oryzae (leaf beetle); Sitophilus spp. (e.g., S. oryzae, rice weevil); Pachydiplosis spp. (e.g., P. oryzae, rice gall midge); Hydrellia spp. (e.g., H. griseola (small rice leaf miner), or H. sasakii, rice stem maggot); Chlorops spp. (e.g., C. oryzae, stem maggot); Diabrotica species (e.g., O. nubilalis (Western corn rootworm), O. barnettii (Northern corn rootworm), O. eleven-spotted (Southern corn rootworm), O. mexicana (Mexican corn rootworm); Diabrotica spp. (Banded cucumber beetle); Ostrinia species (e.g., O. nubilalis (European corn borer); Spodoptera species (e.g., Agrotissima spp. (Black cutworm); Ostrinia species (e.g., O. serrata (Small corn stalk borer)); Cyclocephala species (e.g., C. borealis (Northern masked chafer) or C. immaculata (Southern masked chafer)); Cyclocephala species (e.g., C. borealis (Northern masked chafer) or C. immaculata (Southern masked chafer)); chafer); Acrochafer species (e.g., Acrochafer japonica (Japanese beetle)); Acrochafer species (e.g., Copper flea beetle (corn flea beetle)); Cryptorhynchus species (e.g., Corn weevil (maize weevil)); Rhopalosiphum spp. (e.g., R. maidis (corn leaf aphid); Anuraphis spp. (e.g., A. maididiradicis (corn root aphid)); Locust species (e.g., Locust rubrum (red-legged locust), Locust aberrantum (long-fronted locust)), or M. sanguinipes (migratory grasshopper); Hylemya spp. (e.g., H. platura (seedcorn maggot)); Anaphothrips species. spp.) (e.g., Thrips (A.obscrurus (grass thrips); Solenopsis spp. (e.g., S. milesta (thief ant)); Tetranychus spp. (e.g., T. urticae (twospotted spidermite), T. cinnabarinus (carmine spider mite); Spodoptera spp. (e.g., corn earworm (cotton bollworm) or cotton bollworm (American bollworm)); Pink bollworm spp. (e.g., P. gossypiella (pink bollworm)); Earias spp. (e.g., E. vittella (spotted (e.g., B. abutiloneus (banded-winged whitefly), T. vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g., B. argentifolii (silverleaf whitefly); Aphis spp. (e.g., A. gossypii (cotton aphid)). aphid); Lygus species (e.g., L. lineolaris (tarnished plant bug) or L. hesperus (western tarnished plant bug)); Euschistus species (e.g., E. conspersus (consperse stink bug)); Chlorochroa species (e.g., C. sayi (Say stink bug)); Nezara species (e.g., Nezara viridis (Nezara viridis)).viridula (southern green stinkbug); Thrips species (e.g., Thrips tabaci (onion thrips); Thrips species (e.g., Tobacco thrips or Western flower thrips); Chrysomelidae species (e.g., Colorado potato beetle, L. juncta (false potato beetle), or L. texana (Texan false potato beetle); Lema species (e.g., L. trilineata (three-lined potato beetle); Flea beetle species (e.g., Cucumber flea beetle (potato flea beetle), Tobacco flea beetle (flea beetle), or Tuber flea beetle (E. tuberis, tuber flea beetle). beetle); Echinops spp. (e.g., E. vittata (striped blister beetle); Phaedon spp. (e.g., P. cochleariae (mustard leaf beetle); Coccinella spp. (e.g., Mexican bean beetle); Acheta spp. (e.g., A. domesticus (house cricket); Empoasca spp. (e.g., E. fabae (potato leafhopper); Myzus spp. (e.g., M. persicae (green peach aphid); Paratrioza spp. spp.) (e.g., P. cockerelli (psyllid)); Conoderus spp. (e.g., C. falli, southern potato wireworm or C. vespertinus, tobacco wireworm); Pseudomonas spp. (e.g., P. operculella, potato tuberworm); Macrosiphum spp. (e.g., M.euphorbiae (potato aphid); Thyanta species (e.g., T. Pallidovirens (redshouldered stinkbug); Lymantria species (e.g., P. operculella (potato tuberworm); Spodoptera species (e.g., P. operculella (tomato fruitworm); Lymantria species (e.g., Trichoderma species (tomato pinworm); Limonius spp. (wireworms); Sphingomyelia species, such as M. sexta (tobacco hornworm) and M. quinquemaculata (tomato hornworm); Liriomyza species (e.g., Liriomyza sativae, Liriomyza trifoliata, or L. huidobrensis (leaf miner)); Drosophila species (e.g., Liriomyza sativae, Liriomyza trifoliata, or L. huidobrensis (leaf miner)); spp.) (e.g., Drosophila melanogaster, D. yakuba, D. pseudoobscura, or D. simulans); Carabus spp. (e.g., C. granulatus); Chironomus spp. (e.g., C. tentanus); Ctenocephalides spp. (e.g., C. felis, cat flea); Diaprepes spp. (e.g., D. abbreviation, root weevil); Ips spp. (e.g., I. pini (pine engraver)); Tribolium spp. (e.g., T. innotata, red floor beetle); Glossina spp. spp.) (e.g., G. Morsitans (tsetse fly)); Anopheles spp. (e.g., A. gambiae (malaria mosquito)); Heliothis spp. (e.g., Helicoverpa armigera (African Bollworm)); Acyrthosiphon spp. (e.g., A. pisum (peaaphid)); Apis spp. (e.g., A. mellifera (Isella mellifera)).melifera (honeybee); Homalodisca spp. (e.g., H. coagulate (glassy-winged sharpshooter); Aedes spp. (e.g., Ae. aegypti (yellow fever mosquito); Bombyx spp. (e.g., B. mori (silkworm); Locusta spp. (e.g., L. migratoria (migratory locust); Boophilus spp. (e.g., B. microplus (cattle tick); Acanthoscurria spp. (e.g., A. Gomesiana (red-haired chololate bird-eating spider); eater); Diploptera spp. (e.g., D. punctata, pacific beetle cockroach); Heliconius spp. (e.g., H. erato (red passion flower butterfly) or H. melpomene (postman butterfly); Curculio spp. (e.g., C. glandium, acorn weevil); Plutella xylostella (e.g., P. xylostella, diamondback moth); Amblyomma spp. (e.g., A. variegatum, cattle tick); Anteraea spp. (e.g., A. yamamai (silkmoth); and Armigeres spp. spp.) (e.g. A. subalbatus).

[0099] The insecticidal proteins of the present invention can be used in combination with other pesticides (e.g., Bt Cry proteins) to increase the target range of pests. In addition, the insecticidal proteins of the present invention can be used in combination with insecticides (which have different modes of action or target different receptors in the insect gut) to have specific utility for preventing and / or managing insect resistance.

[0100] The second pesticide may be an insecticidal protein derived from Bacillus thuringiensis. The Bacillus thuringiensis insecticidal protein can be any of a number of insecticidal proteins, including, but not limited to, Cry1 protein, Cry3 protein, Cry6 protein, Cry7 protein, Cry8 protein, Cry9 protein, Cry11 protein, Cry22 protein, Cry23 protein, Cry36 protein, Cry37 protein, Cry34 protein together with Cry35 protein, binary insecticidal proteins CryET33 and CryET34, binary insecticidal proteins TIC100 and TIC101, binary insecticidal protein PS149B1, VIP (vegetative insecticidal protein, disclosed in U.S. Patents 5,849,870 and 5,877,012, incorporated herein by reference), TIC900 or related proteins, TIC901, TIC1201, TIC407, TIC417, modified Cry3A protein, or a hybrid protein or chimera made from any of the foregoing insecticidal proteins. In other embodiments, the Bacillus thuringiensis insecticidal protein is selected from the group consisting of Cry3Bb1, Cry34Ab1 together with Cry35Ab1, mCry3A (U.S. Patent No. 7,276,583, incorporated herein by reference), eCry3.1Ab (U.S. Patent No. 8,309,516, incorporated herein by reference), and Vip3A proteins (including Vip3Aa (U.S. Patent No. 6,137,033, incorporated herein by reference)).

[0101] In other embodiments, the transgenic plants of the present invention may comprise a second insecticide (which may be derived from a source other than Bacillus thuringiensis). The second insecticide may be an agent selected from the group consisting of an alpha-amylase, a peroxidase, a cholesterol oxidase, a potato glycoprotein, a protease, a protease inhibitor, a urease, an alpha-amylase inhibitor, a pore-forming protein, a chitinase, a lectin, an engineered antibody or antibody fragment, a Bacillus cereus insecticidal protein, a Xenorhabdus species (such as X. nematophila or X. bovienii) insecticidal protein, a Photorhabdus species (such as P. luminescens or P. asymobiotica) insecticidal protein, a Brevibacillus species (such as B. laterosporous) insecticidal protein, a Lysinibacillus species (such as B. spp.) (such as spherical lysine Bacillus (L. Sphearicus)) insecticidal proteins, Chromobacterium species (such as C. subtsugae or C. piscinae) insecticidal proteins, Yersinia species (such as Yersinia entomophage (Y. entomophaga)) insecticidal proteins, Paenibacillus species (such as type G Paenibacillus species (P. propylaea)) insecticidal proteins, Clostridium species (such as double enzyme Clostridium (C. bifermentans)) insecticidal proteins, Pseudomonas species (Pseudomonas spp.) (such as Pseudomonas fluorescens) and lignin. In other embodiments, the second agent can be at least one insecticidal protein derived from an insecticidal toxin complex (Tc) (the complex is from Photorhabdus, Xenorhabus, Serratia, or Yersinia). In other embodiments, the insecticidal protein can be an ADP-ribosyltransferase derived from an insecticidal bacterium (such as a Photorhabdus species). In still other embodiments, the insecticidal protein can be Axmi205 or derived from Axmi205 (U.S. Patent Nos. 8,575,425 and 9,394,345, each of which is incorporated herein by reference). In other embodiments, the insecticidal protein can be a VIP protein (such as VIP1 and / or VIP2 from Bacillus cereus). In still other embodiments, the insecticidal protein can be a binary toxin derived from an insecticidal bacterium (such as ISP1A and ISP2A from Brevibacillus laterosporus or BinA and BinB from Bacillus sphaericus).In other embodiments, the insecticidal protein can be LachbCRW (PCT Application No. PCT / US2017 / 045,256), HmassCRW (PCT Application No. PCT / US2017 / 058,179), or WoodsCRW (PCT Application No. PCT / US2018 / 012,730) protein or protein variant. In still other embodiments, the insecticidal protein can be engineered or can be a hybrid or chimera of any of the foregoing insecticidal proteins.

[0102] In some embodiments, the transgenic plants of the present invention may contain and / or express at least one non-proteinaceous second pesticide. In some embodiments, the second pesticide may be present on the plant surface, for example, as a topical application. In a preferred embodiment, the second pesticide is an interfering RNA molecule. An interfering RNA molecule typically comprises at least one RNA fragment directed against a target gene, a spacer sequence, and a second RNA fragment complementary to the first RNA fragment, thereby forming a double-stranded RNA structure. When an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them, RNA interference (RNAi) occurs. The resulting hydrolyzate is a small RNA fragment of approximately 19-24 nucleotides in length, which is referred to as small interfering RNA (siRNA). These siRNAs then diffuse or are carried throughout the organism, including across the cell membrane, where they hybridize with mRNA (or other RNA) and cause RNA hydrolysis. Interfering RNA is recognized by the RNA interference silencing complex (RISC), in which the effector strand (or "guide strand") of the RNA is located. This guide strand serves as a template for the recognition and destruction of the duplex sequence. Each time siRNA hybridizes with its complementary RNA target, this process is repeated, effectively preventing those mRNAs from being translated, and thus "silencing" the expression of the specific gene from which the mRNA is transcribed. Interfering RNA is known in the art to be useful for insect control (see, for example, publication WO 2013 / 192256, which is incorporated herein by reference). Interfering RNA designed for insect control produces non-natural double-stranded RNA that utilizes the natural RNAi pathway in insects to trigger the downregulation of target genes, which may result in cessation of feeding and / or growth and may cause the death of insect pests. Interfering RNA molecules can confer insect resistance to the same target pests as the protein of the present invention, or different pests can be targeted. Target insect plant pests can be fed by chewing, sucking, or piercing. Interfering RNA is known in the art to be useful for insect control. In an embodiment, dsRNA useful for insect control is described in WO Publication Nos. WO 2018 / 026770, WO 2018 / 026773, and WO 2018 / 026774 (incorporated herein by reference). In embodiments, dsRNA useful for insect control are described in U.S. Patent Nos. 9,238,8223, 9,340,797, or 8,946,510 (incorporated herein by reference). In embodiments, dsRNA useful for insect control are described in U.S. Patent Application Nos. 12 / 868,994, 13 / 831,230, 14 / 207,313, or 14 / 207318 (incorporated herein by reference). In other embodiments, the interfering RNA can confer resistance to non-insect plant pests, such as nematode pests or viral pests.

[0103] Co-expression of more than one pesticide in the same transgenic plant can be achieved by creating a single recombinant vector (containing the coding sequences for more than one pesticide in a so-called molecular stack) and genetically engineering the plant so that all of the pesticides are contained and expressed in the transgenic plant. Such molecular stacks can also be created using mini-chromosomes, as described, for example, in U.S. Patent 7,235,716. Alternatively, a transgenic plant containing a nucleic acid encoding a first pesticide can be retransformed with a different nucleic acid encoding a second pesticide, etc. Alternatively, a plant (Parent 1) can be genetically engineered for expression of the genes of the present invention. A second plant (Parent 2) can be genetically engineered for expression of a second pesticide. By crossing Parent 1 with Parent 2, progeny plants expressing all of the genes introduced into Parent 1 and Parent 2 are obtained.

[0104] Transgenic plants or seeds comprising and / or expressing an insecticidal protein of the invention may also be treated with an insecticide or insecticidal seed coating, as described in U.S. Patent Nos. 5,849,320 and 5,876,739 (incorporated herein by reference). In embodiments, where the insecticide or insecticidal seed coating of the invention and the transgenic plant or seed are active against the same target insect (e.g., a coleopteran pest or a rootworm target pest), the combination is useful (i) in methods for further enhancing the activity of the composition of the invention against the target insect and / or (ii) in methods for preventing the development of resistance to the composition of the invention by providing yet another mechanism of action against the target insect. Thus, in embodiments, the invention provides methods for enhancing control of rootworm insect populations, comprising providing a transgenic plant or seed of the invention and applying an insecticide or insecticidal seed coating of the invention to the plant or seed.

[0105] Even where an insecticide or insecticidal seed coating is active against different insects, the insecticide or insecticidal seed coating is useful for expanding the spectrum of insect control, for example, by adding an insecticide or insecticidal seed coating that is active against Lepidoptera insects to transgenic seeds of the invention (which in some embodiments are active against Coleoptera and some Lepidoptera), the resulting coated transgenic seeds control both Lepidoptera and Coleoptera pests.

[0106] Examples of such insecticides and / or insecticide seed coatings include, but are not limited to, carbamates, pyrethroids, organophosphates, friprole, neonicotinoids, organochlorides, nereistoxins, or combinations thereof. In another embodiment, the insecticide or insecticide seed coating is selected from the group consisting of carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyphos, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, tebupirimiphos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamic acid, and combinations thereof. Commercial products comprising such insecticides and insecticide seed coatings include, but are not limited to, the following: (Carbofuran), (Methomyl, Methomyl, Nanaid), (carbaryl), (Bifenthrin), (tefluthrin), (Cypermethrin), (Cypermethrin), Delta (Deltamethrin), (λ-cyhalothrin), (Permethrin), (Permethrin), (Bifenthrin), (Bifenthrin), (tefluthrin)), (lambda cyhalothrin), (chlorpyrifos), (Chlorine Oxyphosphorus), (Methoate), (Phorate), (phorate, flucythinate), (Phorate), (terbufos), (Dimethoate), isochlorophos, (fipronil)), (Thiamethoxam), (Imidacloprid), (Imidacloprid), (thiamethoxam) and (Cyfluthrin, Pyrifos).

[0107] The present invention also encompasses compositions comprising an insecticide protein according to the present invention in an insect-control-effective amount. In further embodiments, the composition comprises a suitable agricultural carrier and a polypeptide of the present invention having insecticidal activity. The agricultural carrier may include adjuvants, mixtures, enhancers, and the like that facilitate the application of the active ingredient (e.g., a polypeptide of the present invention, including a polypeptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with any one of SEQ ID NOs: 39 to 74). Suitable carriers should not be phytotoxic to valuable crops (particularly at the concentrations used when the composition is applied in the presence of crops) and should not chemically react with the compounds of the active ingredients herein (i.e., the polypeptide of the present invention or other composition components). Such mixtures may be designed for direct application to crops, or may be concentrates or formulations that are typically diluted with additional carriers and adjuvants prior to application. They may include inert or active ingredients and may be solid (such as, for example, dusts, powders, granules, water-dispersible granules or wettable powders) or liquid (such as, for example, emulsifiable concentrates, solutions, emulsions or suspensions). Suitable agricultural carriers may include liquid carriers such as water, toluene, xylene, naphtha, crop oils, acetone, methyl ethyl ketone, cyclohexanone, trichloroethylene, perchloroethylene, ethyl acetate, amyl acetate, butyl acetate, propylene glycol monomethyl ether and diethylene glycol methyl ether, methanol, ethanol, isopropyl alcohol, amyl alcohol, ethylene glycol, propylene glycol, glycerol, and the like. Water is generally the carrier of choice for diluting concentrates. Suitable solid carriers may include talc, pyrophyllite clay, silica, attapulgite clay, kieselguhr, chalk, diatomaxeous earth, lime, calcium carbonate, bentonite, Fuller's earth, cottonseed hulls, wheat flour, soy flour, pumice, wood flour, walnut shell flour, lignin, and the like. In another embodiment, the polypeptides of the invention can be encapsulated in a synthetic matrix (such as a polymer) and applied to the surface of a host (such as a plant). Insect uptake of host cells allows the insect control agent to be delivered to the insect and results in a toxic effect on the insect pest.

[0108] In other embodiments, the compositions of the present invention can be powders, dusts, pills, granules, sprays, emulsions, colloids or solutions. The compositions of the present invention can be prepared by dehydrating, freeze-drying, homogenizing, extracting, filtering, centrifuging, sedimenting or concentrating a culture of bacterial cells. The compositions of the present invention can contain at least 1%, at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% of the polypeptide of the present invention by weight.

[0109] In an embodiment, the composition of the present invention may include at least a second pesticide (e.g., which may be transgenically expressed from a plant and / or incorporated into the composition), which may be insecticidal, nematocidal, fungicidal, or bactericidal. At least the second pesticide may have an insecticidal effect against the same insect as the polypeptide of the present invention or a different insect. The second pesticide may be a polypeptide. The pesticide may be an interfering RNA (e.g., dsRNA). The second pesticide may be a microorganism (e.g., a bacterium) comprising a nucleic acid molecule encoding the pesticide and / or comprising a pesticide (e.g., a polypeptide or interfering RNA). The microorganism may be attenuated, heat-inactivated, or freeze-dried. The microorganism may be dead or unable to reproduce. The second pesticide can be an insecticide, such as carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, clothianidin, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, terbufos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, thiosulfuron, and combinations thereof, or commercial products containing insecticides and insecticidal seed coatings as described above.

[0110] The composition of the present invention (for example, a composition comprising a polypeptide of the present invention and an agriculturally acceptable carrier) can be used in traditional agricultural methods. An agriculturally acceptable carrier is a formulation that can be used to apply the composition comprising the polypeptide of the present invention to plants or seeds. For example, the composition of the present invention can be mixed with water and / or fertilizer, and can be applied to a desired place before and / or after emergence with any device, such as an airplane spray barrel, irrigation equipment, a direct injection spray device, a backpack spray barrel, a livestock dip tank, the farm equipment used in ground spraying (for example, a nozzle sprayer, a hand sprayer), etc. The desired place can be soil, plant, etc.

[0111] The compositions of the present invention can be applied to seeds or plant propagules in any physiological state at the following time: any time between seed harvest and sowing; or during sowing or after sowing; and / or after germination. Preferably, the seeds or plant propagules are in a sufficiently durable state so that no damage or minimal damage, including physical damage or biological damage, is caused during the treatment process. The formulation can be applied to the seeds or plant propagules using conventional coating techniques and machines (such as fluidized bed technology, drum milling method, static rotation (rotostatic) seed processor and drum coater).

[0112] The present invention also includes a method for controlling a population of lepidopteran and / or coleopteran pests, comprising contacting the population with an insecticidal amount of a polypeptide of the present invention, wherein the polypeptide is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 39 to 74. Contacting comprises ingestion or uptake of the polypeptide by members of the pest population. The polypeptide may be incorporated into the insect's dietary food, or may be expressed or present in plant tissue that the insect then ingests. In further embodiments, controlling a population of lepidopteran and / or coleopteran pests comprises killing the insects by contacting them with an insecticidal amount of a polypeptide of the present invention.

[0113] The present invention also includes a method for protecting a plant from an insect pest, the method comprising expressing in a plant or plant cell a nucleotide sequence or expression cassette encoding an insecticidal polypeptide of the present invention. In embodiments, the nucleotide sequence is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequence of SEQ ID NOs: 2 to 36, or encodes a polypeptide comprising an amino acid sequence identical to SEQ ID NOs: NOs:39 to 74 have at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity. In further embodiments, the plant or plant cell produces an insecticidal polypeptide that has insecticidal activity against lepidopteran and / or coleopteran pests.

[0114] The present invention further comprises a method for increasing plant yield, which method comprises growing a plant or its seeds having stably incorporated into its genome a nucleic acid molecule of the expression cassette of the present invention in a field, and wherein the field is infested with a pest against which the polypeptide has insecticidal activity.

[0115] Once a desired nucleic acid has been transformed into a particular plant species, it can be propagated within that species or moved into other varieties of the same species (particularly including commercial varieties) using conventional breeding techniques.

[0116] In embodiments, the nucleic acids of the present invention are expressed in transgenic plants, thereby causing biosynthesis of the corresponding insecticidal proteins in the transgenic plants. In this manner, transgenic plants with enhanced resistance to insects, particularly corn rootworms, are produced. The nucleic acids of the present invention can optionally be modified and optimized for expression in transgenic plants. While genes from microbial organisms can in many cases be expressed at high levels in plants without modification, low expression in transgenic plants can be caused by microbial nucleic acids using codons that are not preferred in plants. It is known in the art that all organisms have specific preferences for codon usage, and the codons of the nucleic acids described herein can be altered to conform to plant preferences while maintaining the amino acids encoded thereby. Furthermore, high expression in plants is best achieved with coding sequences having a GC content of at least about 35%, preferably greater than about 45%, more preferably greater than about 50%, and most preferably greater than about 60%. Microbial nucleic acids with low GC content may not express well in plants due to the presence of the ATTTA motif, which can destabilize the message, and the AATAAA motif, which can cause inappropriate polyadenylation. In an embodiment, the sequence can be modified to accommodate the specific codon preferences and GC content preferences of monocots or dicots, as these preferences have been shown to be different (Murray et al. Nucl. Acids Res. [Nucleic Acids Research] 17: 477-498 (1989)). In addition, these nucleic acids are screened for the presence of irrational splice sites that may cause message shortening. Site-directed mutagenesis, PCR, and synthetic gene construction, as known, can be used, for example, using the methods described in disclosed patent applications EP 0 385 962, EP 0 359 472, and WO 93 / 07278, to modify all changes (such as those described above) that need to be made within these nucleic acids.

[0117] In one embodiment of the invention, the coding sequence of the insecticidal protein of the present invention is made according to the procedure disclosed in U.S. Patent No. 5,625,136 (incorporated herein by reference). In this procedure, maize preferred codons are used, i.e., single codons that most frequently encode amino acids in maize. Maize preferred codons for specific amino acids can be derived from, for example, known gene sequences from maize. Maize codon usage for 28 genes from maize plants is found in Murray et al., Nucleic Acids Research [nucleic acid sequence] 17: 477-498 (1989), the disclosure of which is incorporated herein by reference.

[0118] In this way, these nucleotide sequences can be optimized for expression in any plant. It will be appreciated that all or any portion of the gene sequence can be optimized or synthetic. That is, synthetic or partially optimized sequences can also be used.

[0119] For more effective translation initiation, the sequence adjacent to the start methionine can be modified. For example, they can be modified by comprising a known sequence effective in plants. Joshi has proposed a suitable consensus sequence for plants (NAR 15:6643-6653 (1987)), and Clontech has proposed another consensus translation initiator (1993 / 1994 catalogue, page 210). These consensus sequences are suitable for use with nucleic acid of the present invention. In an embodiment, these sequences are incorporated into the construct comprising nucleic acid, reaching and including ATG (and not modifying the second amino acid), or alternatively reaching and including the GTC after the ATG (with the possibility of modifying the second amino acid of this transgenic).

[0120] In transgenic plants, the expression of these nucleic acids is driven by promoters that play a role in plants. The selection of promoters will vary according to the time and space required for expression, and also according to the target species. Therefore, the expression of nucleic acids of the present invention in leaves, stalks or stems, spikes, inflorescences (such as spikes, panicles, cobs, etc.), roots, and / or seedlings is preferred. However, in many cases, protection is sought for more than one type of insect pest, and therefore expression in multiple tissues is desirable. Although it has been shown that many promoters from dicots are operable in monocots and vice versa, it is desirable to select dicot promoters for expression in dicots, and to select monocot promoters for expression in monocots. However, there is no restriction on the origin of the selected promoter; as long as they can effectively drive nucleic acid expression in the desired cell, it is enough.

[0121] In one embodiment, a constitutively expressed promoter is used, including actin or ubiquitin or CMP promoters or CaMV 35S and 19S promoters. Nucleic acid of the present invention can also be expressed under the regulation of a promoter that is regulated by a chemical method. Preferred techniques for chemical induction of gene expression are detailed in open application EP 0 332 104 (Ciba-Geigy) and U.S. Patent No. 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a promoter.

[0122] In another embodiment, a class of wound-inducible promoters can be used. Numerous promoters have been described that express at the site of wounding and also at sites of infection by plant pathogens. Ideally, such promoters would be locally active only at the site of infection, and in this way, the insecticidal proteins of the invention would accumulate only in the cells where they are synthesized to kill the invading insect pests. Such preferred promoters include those described by Stanford et al. Mol. Gen. Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier and Lehle, Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), and Warner et al. Plant J. 3:191-201 (1993).

[0123] Tissue-specific or tissue-preferred promoters for expressing genes encoding the insecticidal proteins of the present invention in plants, particularly corn, are those that express directly in roots, pith, leaves, or pollen, particularly roots. Such promoters, such as those isolated from PEPC or trpA, are disclosed in U.S. Pat. No. 5,625,136, or those isolated from MTL, are disclosed in U.S. Pat. No. 5,466,785. Both U.S. patents are incorporated herein by reference in their entirety.

[0124] In addition, promoters that function in plastids can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5'UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters suitable for use in the present invention include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0125] In another aspect, the nucleotide sequences of the present invention can be operably associated with promoters that are inducible by wounding or infection by pests or pathogens (e.g., insect or nematode plant pests). Numerous promoters have been described that express at sites of wounding and / or at sites of pest attack (e.g., insect / nematode feeding) or infection by plant pathogens. Ideally, such promoters should be locally active only at or near the site of attack, and in this way, expression of the nucleotide sequences of the present invention will be concentrated in the cells that have been invaded or fed. Such promoters include, but are not limited to, those described by Stanford et al., Mol. Gen. Genet. 215:200-208 (1989); Xu et al., Plant Molec. Biol. 22:573-588 (1993); Logemann et al., Plant Cell 1:151-158 (1989); Rohrmeier and Lehle, Plant Molec. Biol. 22:783-792 (1993); Firek et al., Plant Molec. Biol. 22: 129-142 (1993); Warner et al., Plant J. 3: 191-201 (1993); U.S. Patent No. 5,750,386; U.S. Patent No. 5,955,646; U.S. Patent No. 6,262,344; U.S. Patent No. 6,395,963; U.S. Patent No. 6,703,541; U.S. Patent No. 7,078,589; U.S. Patent No. 7,196,247; U.S. Patent No. 7,223,901; and U.S. Patent Application Publication No. 2010043102.

[0126] In some embodiments of the present invention, a "minimal promoter" or "basic promoter" is used. The minimal promoter can recruit and bind to RNA polymerase II complex and its auxiliary proteins to allow transcription initiation and extension. In certain embodiments, the minimal promoter is constructed to comprise only the nucleotides / nucleotide sequences of the selected promoter necessary for the binding of transcription factors and the transcription of the target nucleotide sequence, and this target nucleotide sequence is operably associated with a minimal promoter including but not limited to a TATA box sequence. In other embodiments, the minimal promoter lacks the cis sequence for recruiting and binding transcription factors, and these transcription factors regulate (e.g., enhance, repress, confer tissue specificity, confer induction or repressibility) transcription. The minimal promoter is typically placed upstream (i.e., 5') of the nucleotide sequence to be expressed. Therefore, the nucleotides / nucleotide sequences from any promoter available with the present invention can be selected to be used as the minimal promoter.

[0127] Numerous other sequences can be incorporated into the expression cassettes described herein. These sequences include sequences that have been shown to enhance expression, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0128] Nucleic acid of the present invention may be more preferred for the targeted expression of different cellular locations in plants. In some cases, the location in the cytosol may be desirable, and in other cases, the location in a certain subcellular organelle may be preferred. Use technology well known in the art to carry out the subcellular localization of the transgenic encoding enzyme. Typically, the DNA encoding the target peptide from the gene product of known organelle targeting is operated and fused to the upstream of the nucleic acid. Many such target sequences for chloroplasts are known and have demonstrated their functions in heterologous constructs. The expression of nucleic acid of the present invention is also targeted to the endoplasmic reticulum or the vacuole of the host cell. The technology for realizing it is well known in the art.

[0129] The carrier that is suitable for plant transformation is well known in the art.For agrobacterium-mediated transformation, binary vectors or the carrier carrying at least one T-DNA border sequence are suitable, and for direct gene transfer, any vector is all suitable, and the linear DNA containing only the purpose construct may be preferred.In the case of direct gene transfer, it is possible to use conversion or co-transformation (Schocher et al., Biotechnology [biological technology] 4:1093-1096 (1986)) with a single DNA species. For direct gene transfer and agrobacterium-mediated transformation, conversion is usually (but not necessarily) carried out with a selective marker, and this selective marker can provide resistance to antibiotics (kanamycin, hygromycin or methotrexate) or herbicides (basta). The plant transformation vector comprising nucleic acid molecules of the present invention can also include following genes (for example phosphomannose isomerase; PMI), these genes provide the positive selection of transgenic plants, as disclosed in United States Patents 5,767,378 and 5,994,629 (incorporated herein by reference). However, the selection of selective markers is not crucial for the present invention.

[0130] In an embodiment, nucleic acid can be transformed into nuclear genome. In another embodiment, nucleic acid of the present invention is directly transformed into plastid genome. The major advantage of plastid transformation is that plastids can usually express bacterial genes without substantial codon optimization, and plastids can express multiple open reading frames under the control of a single promoter. In U.S. Patent Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT application No. WO 95 / 16783, and in McBride et al., (1994), Proc. Nati. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 91,7301-7305, plastid transformation technology is widely described. Basic chloroplast transformation technology relates to, for example, using biolistic or protoplast transformation (for example, calcium chloride or PEG-mediated transformation), the cloned plastid DNA region positioned at the selective marker flank is introduced into a suitable target tissue together with the gene of interest. These 1 to 1.5 kb flanking regions (designated targeting sequences) promote homologous recombination with the plastid genome and thus allow replacement or modification of specific regions of the plastid. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes (conferring resistance to spectinomycin and / or streptomycin) were used as selective markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Nati. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell [Plant Cell] 4, 39-45). This produces stable homoplasmic transformants at a frequency of approximately 1 per 100 target leaf bombardments. The presence of a cloning site between these markers allows the creation of plastid targeting vectors for the introduction of foreign genes (Staub, JM and Maliga, P. (1993) EMBO J. 12, 601-606). A substantial increase in transformation frequency was achieved by replacing a recessive rRNA or r-protein antibiotic resistance gene with a dominant selectable marker (the bacterial aadA gene, which encodes the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenylyltransferase) (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this marker had been successfully used for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089).Other selective markers useful for plastid transformation are known in the art and are included within the scope of the present invention. Typically, approximately 15-20 cell division cycles are required after transformation to reach a homoplasmic state. Plastid expression (wherein the gene is inserted into the copies of all thousands of circular plastid genomes present in each plant cell by homologous recombination) takes advantage of the large copy number of genes that exceed nuclear expression to allow expression levels that can easily exceed 10% of total soluble plant protein. In a preferred embodiment, the nucleic acid of the present invention is inserted into a plastid-targeted vector and transformed into the plastid genome of the desired plant host. Plants that are homologous to the plastid genome comprising the nucleic acid of the present invention are obtained, and these plants are able to preferentially highly express the nucleic acid. Examples

[0131] The present invention can be further described by reference to the following specific examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001); T. J. Silhavy, M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984); and Ausubel, F. M. et al., Current Protocols in Molecular Biology, New York, John Wiley and Sons Inc., (1988); Reiter et al., Methods in Arabidopsis Research, World Scientific Publishing House. Press [World Scientific Press] (1992), and Schultz et al., Plant Molecular Biology Manual [Plant Molecular Biology Manual], Kluwer Academic Publishers [Kluwer Academic Publishers] (1998). Example 1: Identification of proteins with insecticidal activity against western corn rootworm

[0132] An insecticidal protein (SEQ ID NO: 39) was identified from Nitrococcus mobilis. An E. coli-optimized version of the gene (SEQ ID NO: 1) was synthesized and cloned into the pET29a vector to generate the construct p(Nitromob). The p(Nitromob) construct was transformed into E. coli BL21*(DE3) and incubated overnight at 18°C in Luria-Bertani medium with IPTG for protein expression. A soluble fraction of lysate was prepared from these cultures using a French pressure cell followed by centrifugation of the entire lysate at 20,000 x g for thirty minutes. The supernatant (soluble fraction) was then tested for bioactivity against western corn rootworm (WCR; corn rootworm).

[0133] Use the dietary incorporation method to carry out biological activity assay. In brief, E. coli BL21*(DE3) lysate is mixed with an equal volume of heated artificial insect diet (Bioserv, Frenchtown, New Jersey (NJ)) in a 1.5mL centrifuge tube and then applied to a small culture dish. After the diet-sample mixture is cooled and solidified, 12 WCR larvae are added to each plate. The plate is sealed and maintained under ambient laboratory conditions with respect to temperature, light and relative humidity. Lysate from E. coli BL21*(DE3) culture carrying an empty pET29a vector is used as a negative control. Mortality is assessed on the 4th and 7th days, or optionally on the 3rd and 6th days. For this table and all subsequent tables showing insecticidal activity against CRW, the abbreviations in the "Remarks" column are as follows: s=small larvae, sm=small / medium larvae, m=medium larvae, mb=medium / large larvae, b=large larvae, vb=very large larvae. For this and all subsequent tables showing insecticidal activity of NitromobCRW or its variants, "SEQ ID NO." refers to the amino acid sequence of the protein. As shown in Table 1, lysates from cultures expressing p(Nitromob) exhibited potent bioactivity against WCR. The active Nitromob protein was renamed NitromobCRW. Table 1: Insecticidal activity of Nitromob against western corn rootworm Example 2: NitromobCRW variant has insecticidal activity against WCR

[0134] Mutations were introduced into NitromobCRW, and bacterial lysates containing NitromobCRW mutants were assayed for protein stability and insecticidal activity. Mutations included amino acid changes at multiple residues and insertion of leucine residues. These mutations were introduced to determine whether NitromobCRW mutants could be designed to retain insecticidal activity but be digestible in a simulated gastric fluid (SGF) assay. Such NitromobCRW variants could have commercial value, for example, by conferring insecticidal properties to plants through transgenic expression in plants.

[0135] Insecticidal activity was determined using a dietary incorporation assay performed essentially as described in Example 1, using 12 WCR larvae per experimental assay. The results are shown in Tables 2-6. The SEQ ID NOs correspond to the amino acid sequences of the variants. The treatments shown in Tables 5 and 6 also indicate the dilution of bacterial lysate used. All NitromobCRW mutant variants exhibited insecticidal activity on day 6 or day 7. Table 2: Insecticidal activity of NitromobCRW mutant variants against WCR Table 3: Insecticidal activity of NitromobCRW mutant variants against WCR Table 4: Insecticidal activity of NitromobCRW mutant variants against WCR Table 5: Insecticidal activity of NitromobCRW mutant variants against WCR Table 6: Insecticidal activity of NitromobCRWI215L against WCR Example 3: Simulated gastric fluid test on E. coli lysate preparation

[0136] This example describes an assay performed to determine SGF digestibility. Each NitromobCRW protein variant was produced in E. coli strain BL21*(DE3). The expression levels of the variants in the bacterial strains and the solubility of the variants are shown in Table 7. Bacterial lysates in 50 mM potassium phosphate (pH 7.0), 50 mM sodium chloride were diluted to 3 mg / mL (total protein concentration) for digestibility analysis. The digestion reaction was initiated at 37°C by adding 15 μL of lysate to 285 μL of simulated gastric fluid [10 units of pepsin / μg protein, or approximately 1579 units of pepsin / mL in G-Con solution (2 mg / mL sodium chloride, pH 1.2)]. At 5 minutes, 100 μ L of lysate-SGF reaction solution was removed and the reaction was stopped by adding it to 100 μ L of preheated (95° C.) stop solution, which consisted of 65% Tricine loading buffer (Bio-rad 2x Tricine loading buffer w / 10% β-mercaptoethanol) and 35% 500 mM sodium bicarbonate (pH 11.0). Zero time point (T0) was generated by adding 5 μ L of test lysate to 100 μ L of preheated (95° C.) stop solution and 95 μ L of simulated gastric fluid. All samples were heated at 95° C. for 5 minutes and then stored on ice until SDS-PAGE analysis. Before standard protein gel electrophoresis, 30 microlitres of each reaction solution were added to a 10%-20% Tris-tricine peptide gel. Immediately after electrophoresis, the Tris-tricine gel was fixed for 20 minutes with a 40% methanol: 10% acetic acid mixture. The gel was then stained with GelCode blue protein stain at room temperature for 1 hour. After 1 hour, the polyacrylamide gel was decolorized with distilled water for at least 12 hours. The results are qualitatively shown in Table 7. A "fail" of the T5 test means that complete or partially digested NitromobCRW protein variants can be detected by GelCode blue protein staining after gel electrophoresis, which indicates that the protein cannot be completely digested in the SGF assay. A "pass" of the T5 test means that no complete NitromobCRW protein variant can be detected, which indicates that the NitromobCRW protein variant is digestible in the SGF assay. As shown in the previous example, the insecticidal activity of the NitromobCRW protein variant is also indicated ("active"), where "yes" indicates insecticidal activity. Table 7: Digestion of mutant variants of NitromobCRW in the SGF assay

[0137] Surprisingly, of all the NitromobCRW variants produced, only NitromobCRW Y213L / I215L (SEQ ID NO: 47) ultimately passed the SGF assay T5 test. NitromobCRW I215L (SEQ ID NO: 44) showed better digestibility than the wild-type protein, but the variant did not pass the T5 test. Interestingly, NitromobCRW variants 214-Leu-215, 215-Leu-216, G216A, G216L, and L214S / I215L were insoluble, while the NitrobCRW1265L variant had low solubility. These data suggest that the domains, motifs, or folds in this region of the protein are crucial for protein function and / or protein stability. Example 4: Purified NitromobCRW variant Y213L / I215L has insecticidal effect against WCR

[0138] The variant was further characterized for its insecticidal properties. Two liters of E. coli BL21*(DE3) cells carrying pET-NitromobCRW Y213L / I215L were grown in LB medium at 37°C. IPTG (1 mM) was added to the culture when the OD reached 0.8-1.0, and the culture was then moved to 18°C and cultured for 18 hours. The cell pellet was harvested and resuspended in 20 mM Tris (pH 8.5) containing 10% glycerol. The cells were lysed using a French press; the lysate was then spun at 100k xg in an ultracentrifuge. The supernatant was collected, filtered, and then loaded onto a HiPrepQ anion exchange column, which was pre-equilibrated in 20 mM Tris (pH 8.5) containing 10% glycerol. The HiPrepQ column can effectively bind NitromobCRW Y213L / I215L; the protein is eluted from the column using a linear NaCl gradient. The high salt buffer consists of 20mMTris (pH 8.5), 0.5M NaCl containing 10% glycerol. The purest fractions are combined and then concentrated to approximately 2mL. The protein is loaded onto a Sephadex 200 gel filtration column that has been pre-equilibrated in 1X PBS. The purity of the fractions from the Sephadex 200 column is analyzed by SDS-PAGE (NitromobCRW Y213L / I215L (SEQ ID NO: 47) has a predicted molecular weight of 32.1kDa). The purest fractions are combined and then concentrated to 7.2mg / mL and then stored at -80°C. A series of concentrations of the pure protein are then tested on 12 WCR larvae using a dietary incorporation method essentially as described in Example 1. As shown in Table 8, NitromobCRW Y213L / I215L was effective against WCR; NitromobCRW Y213L / I215L at 50 pg / mL produced at least 75% mortality on day 6. Table 8: Insecticidal activity of purified NitromobCRW Y213L / I215L against WCR Example 5: Nitromob CRWCRWY213L / I215L has insecticidal properties against Cry-resistant western corn rootworm strains active

[0139] To determine whether NitromobCRW Y213L / I215L (SEQ ID NO: 47) toxicity is achieved through a mode of action separate from Cry3-related proteins, NitromobCRW Y213L / I215L lysate was purified as in Example 4 and tested for efficacy against a WCR strain resistant to eCry3.1Ab toxin (eCry3.1Ab-R; see Table 9), a WCR strain resistant to modified Cry3A (mCry3A) toxin (mCry3A-R; see Table 10), and a WCR strain resistant to Cry3Bb toxin (Cry3Bb-R; see Table 11). Dietary incorporation assays were performed on a range of NitromobCRW Y213L / I215L proteins, and mortality was assessed on days 3 and 6 (Table 9) or days 2 and 7 (Table 10) as described in Example 4. As shown in Tables 9, 10, and 11, NitromobCRW Y213L / I215L was tested twice at a variety of concentrations (μg / mL). The negative control consisted of 1X PBS alone. Each assay was performed with 12 WCR larvae. As shown in Tables 9, 10, and 11, NitromobCRW Y213L / I215L exhibited insecticidal activity against Cry-resistant WCR strains. Table 9: Insecticidal activity of purified NitromobCRW Y213L / I215L against eCry3.1Ab-resistant WCR Table 10: Insecticidal activity of purified NitromobCRW Y213L / I215L against mCry3A-resistant WCR Table 11: Insecticidal activity of purified NitromobCRW Y213L / I215L against Cry3Bb-resistant WCR Example 6: Nitromob CRWY213L / I215L has no insecticidal activity against Lepidoptera

[0140] Lysates from bacterial cultures expressing NitromobCRW Y213L / I215L (SEQ ID NO: 47) were tested for biological activity against a panel of lepidopteran insect pests using a dietary overlay bioassay. NitromobCRW insecticidal activity was tested against European corn borer (ECB), black cutworm (BCW), corn borer (CEW), and fall armyworm (FAW) using a dietary incorporation assay similar to that described in Example 1. Lysates from Bl21*(DE3) bacterial cultures carrying the gene encoding NitromobCRW Y213L / I215L (SEQ ID NO: 9) were used to test 12 L1 larvae in each experiment. Positive controls for BCW, CEW, and FAW consisted of larvae exposed to lysates from E. coli BL21*(DE3) expressing the Vip3 protein. 1X PBS alone and lysates from BL21*(DE3) bacterial cultures carrying an empty pET29 vector were used as negative controls. Mortality was assessed on day 7. Larvae that reached the L3 stage were not significantly affected by the treatment. If the larvae only reached the L2 stage, the treatment may result in growth inhibition. Growth inhibition occurs if the larvae remain in the L1 stage throughout the treatment. This can also be considered "effective mortality" because even if they still survive, the larvae will not develop beyond the L1 stage. For Tables 11-14, L1 = first instar, L2 = second instar, L3 = third instar. Under these experimental conditions, NitromobCRW had no activity against the lepidopteran pests tested (Tables 12-15). Table 12: Insecticidal activity of NitromobCRW Y213L / I215L against CEW Table 13: Insecticidal activity of NitromobCRW Y213L / I215L against FAW Table 14: Insecticidal activity of NitromobCRW Y213L / I215L against BCW Table 15: Insecticidal activity of NitromobCRW Y213L / I215L against ECB Example 7: Nitromob CRWY213L / I215L has insecticidal activity against northern corn rootworm

[0141] NitromobCRW Y213L / I215L was purified as in Example 1 and tested for efficacy against 12 northern corn rootworm (NCR) larvae at each concentration in a dietary incorporation assay performed essentially as described in Example 1. The negative control was IX PBS only. Table 16: Insecticidal activity of NitromobCRW Y213L / I215L against NCR Example 8: Nitromob CRWY213L / I215L has insecticidal activity against southern corn rootworm

[0142] NitromobCRW Y213L / I215L was purified as in Example 1 and tested for efficacy against 12 southern corn rootworm (SCR) larvae in a dietary incorporation assay performed essentially as described in Example 1. NitromobCRW Y213L / I215L (SEQ ID NO: 47) was tested at concentrations ranging from 100 μg / mL to 400 μg / mL. The negative control was 1X PBS only. As shown in Table 16, NitromobCRW Y213L / I215L exhibited insecticidal activity against SCR. Table 16: Insecticidal activity of NitromobCRW Y213L / I215L against SCR Example 9: Transformation of maize with NitromobCRWY213L / I215L

[0143] A binary vector construct suitable for Agrobacterium-mediated NitromobCRW Y213L / I215L transformation has been produced. The binary vector comprises the NitromobCRW Y213L / I215L coding sequence (SEQ ID NO:38) optimized for maize, which is operably connected to a promoter suitable for driving expression in plants at the 5' end and operably connected to a terminator sequence at the 3' end. For example, maize codon optimization is performed using the method described in U.S. Patent number 6,320,100 (incorporated herein by reference). Standard molecular biology techniques known to those skilled in the art are used to transform the construct into Agrobacterium tumefaciens. In order to prepare the Agrobacterium for transformation, the cells are cultured overnight in liquid YPC culture medium at 28°C and 220rpm. Agrobacterium transformation of immature maize embryos is performed essentially as described in Negrotto et al., 2000 (Plant Cell Reports [plant cell communication] 19:798-803). For this example, all media components were essentially as described by Negrotto et al. (supra). However, various media components known in the art may be substituted.

[0144] After transformation, selection and regeneration, use The analysis was performed to determine whether the gene encoding the selectable marker and the NitromobCRW Y213L / I215L maize codon-optimized coding sequence were present in the plants. The presence of the vector backbone was also tested. Plants that were negative for the vector backbone and comprised a copy of the transgenic gene were transferred to a greenhouse and assayed for resistance to WCR damage. Example 10: Maize plants expressing NitromobCRWY213L / I215L have insecticidal activity against WCR NitromobCRW Y213L / I215L was detected by ELISA using ng / mg total soluble protein (TSP) in leaves or root tissues from each event. Insecticidal activity was determined using the Root Segment Bioassay. In brief, when maize events expressing NitromobCRW variants reached the V3-V4 stage, samples of maize root tissue from each event were removed. Maize root tissue was placed in a culture dish and then infested with 12 WCR larvae. Feeding holes (FH) and scar damage were assessed for two root tissue samples (Rep1 and Rep2) on the 3rd day. Root tissue from untransformed (invalid) maize was used as a negative control. Insect damage was scored using the following: ND = Not Detected; FH = Feeding Holes; L = Mild Scarring; M = Moderate Scarring; H = Severe Scarring; ++ = Excellent Performer; + = Good Performer; - = Poor Performer Table 17: Insecticidal Activity of Transgenic NitromobCRW Y213L / I215L Maize Against WCR Example 11: NitromobCRW Y213L / I215L combined with interfering RNA has insecticidal activity against WCR

[0145] NitromobCRW and / or NitromobCRW variants were purified from bacterial lysates as in Example 1 or purified as proteins similar to Example 4. dsRNAs were prepared against essential targets known to have insecticidal activity. In non-limiting examples, dsRNAs can target genes encoding vacuolar ATP synthase, β-tubulin, 26S proteosome subunit p28 protein, EF1α48D, troponin I, tetraspanin, γ-coat body, β-coat body, and / or juvenile hormone epoxide hydrolase (WO Publication Nos. WO 2018 / 026770, WO 2018 / 026773, and WO 2018 / 026774; U.S. Patent No. 7,812,219; each of which is incorporated herein by reference). dsRNA and purified NitromobCRW proteins were tested for insecticidal efficacy against WCR in a dietary incorporation assay performed essentially as described in Example 1, but with the dsRNA added to artificial feed.

[0146] Asdadasdas Example 12. NiromobCRW with a C-terminal extension is active against CRW.

[0147] This example describes the effect of attaching a C-terminal peptide to the NitromobCRW protein. The pET-NitromobCRW-Y213L / I215L:C-terminal extension construct encoding SEQ ID NO:77 was cloned into E. coli. The C-terminal extension peptide comprises SEQ ID NO:76 and is a combination of a linker sequence (amino acids 1-35 of SEQ ID NO:76) and a SUMO tag (amino acids 36-133 of SEQ ID NO:76). SUMO is a small ubiquitin-like modified protein that, when fused to the protein of interest, can enhance the production of functional proteins in prokaryotic and eukaryotic expression systems based on significantly improved protein stability and solubility. Following expression and purification of the fusion protein, the SUMO tag is typically cleaved by a specific (SUMO) protease via its in vitro endopeptidase activity to produce the desired released protein chaperone. For this example, the C-terminal extension peptide (SEQ ID NO: 76) was not cleaved from the NitromobCRW protein, and the intact extension protein (NitromobCRW-Cterm-SUMO; SEQ ID NO: 77) was tested for SGF digestibility and insecticidal activity against WCR.

[0148] The SGF analysis of NitromobCRW-Y213L-I215L-Cterm-SUMO was the same as described above. The digestibility of NitromobCRW-Y213L-I215L-Cterm-SUMO was compared with NitromobCRW-Y213L-I215L without C-terminal extension (as a control). As described above, the activity of NitromobCRW-Y213L-I215L-Cterm-SUMO protein against WCR was also tested.

[0149] The results of the SGF digestibility assay showed that NitromobCRW-Y213L-I215L-Cterm-SUMO was digested before the 5-minute time point. Therefore, the addition of the Cterm SUMO tag had no effect on the digestibility of the NitromobCRW protein. The SGF gels of the tagged and untagged proteins were almost identical (data not shown). The results of the bioassay shown in Table 18 showed that the NitromobCRW protein with the C-terminal extension peptide had the same activity as the NitromobCRW protein without the C-terminal extension peptide. Table 18. Biological activity of NitromobCRW-Cterm-SUMO protein against WCR.

[0150] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or variations from the descriptions thereof will be suggested to those skilled in the art and will be included within the spirit and scope of this application and the appended claims.

[0151] All publications and patent applications mentioned in this specification are indicative of the levels of skill of those skilled in the art to which this invention pertains. All such publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An expression cassette comprising a promoter operably linked to a heterologous nucleic acid molecule comprising: (a) the nucleotide sequence of SEQ ID NO: 6; (b) a nucleotide sequence that is at least 99% identical to the nucleotide sequence of SEQ ID NO: 6; (c) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 44; or (d) a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide is at least 99% identical to the amino acid sequence of SEQ ID NO:

44.

2. A nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:

6.

3. A polypeptide comprising the amino acid sequence of SEQ ID NO:

44.

4. A vector or construct comprising the expression cassette of claim 1.

5. A bacterial host cell comprising the expression cassette of claim 1.

6. A method for producing a polypeptide having insecticidal activity, the method comprising culturing the bacterial host cell of claim 5 under conditions whereby a nucleic acid molecule encoding the polypeptide is expressed.

7. A method of producing a corn plant or corn plant part having enhanced insect resistance compared to a control corn plant or control corn plant part, the method comprising: (a) introducing a nucleic acid molecule comprising the expression cassette of claim 1 into a corn plant part; as well as (b) growing the corn plant part into a corn plant that expresses the nucleic acid molecule and has enhanced insect resistance compared to a control corn plant or a control corn plant part that does not contain the nucleic acid molecule comprising the expression cassette of claim 1.

8. The method of claim 7, wherein the expression cassette encodes a polypeptide comprising an amino acid sequence that is at least 99% identical to SEQ ID NO:

44.

9. A method of increasing insect resistance in a plant or plant part compared to a control plant or plant part, the method comprising expressing the expression cassette of claim 1 in the plant or plant part, wherein expression of the expression cassette results in increased insect resistance in the plant or plant part compared to a control plant or plant part.

10. The method of claim 9, further comprising introducing the expression cassette into the corn plant.

11. The method of claim 9, further comprising introducing the expression cassette into a corn plant part and producing a corn plant from the corn plant part.

12. A method for producing a corn plant having enhanced insect resistance compared to a control corn plant, the method comprising detecting a nucleic acid comprising the expression cassette of claim 1 in a corn plant part, and producing a corn plant from the corn plant part, thereby producing a corn plant having enhanced insect resistance compared to the control corn plant.

13. A method for identifying a corn plant or corn plant part having enhanced insect resistance compared to a control corn plant or control corn plant part, the method comprising detecting the nucleic acid of claim 2 in the corn plant or corn plant part, thereby identifying a corn plant or corn plant part having enhanced insect resistance.

14. The method of claim 13, wherein the expression cassette or diagnostic fragment thereof is detected in an amplification product of a nucleic acid sample from the corn plant or corn plant part.

15. A method for producing a corn plant having enhanced insect resistance compared to a control corn plant or a control corn plant part, the method comprising crossing a first parent corn plant with a second parent corn plant, wherein at least the first parent corn plant comprises in its genome a nucleic acid molecule comprising the expression cassette of claim 1; and producing offspring, wherein the offspring includes at least one corn plant having the nucleic acid in its genome and exhibiting enhanced insect resistance compared to the control corn plant.

16. The method of any one of claims 7 to 15, wherein the enhanced insect resistance is against Diabrotica virgifera virgifera, Diabrotica barberi, and / or Diabrotica undecimpunctata howardi.

17. The method of claim 7 , wherein the nucleic acid molecule further comprises a promoter sequence selected from a constitutive promoter sequence, a tissue-specific promoter sequence, a chemically inducible promoter sequence, a wound-inducible promoter sequence, a stress-inducible promoter sequence, and a developmental stage-specific promoter sequence.

18. A composition comprising a suitable agricultural carrier and a polypeptide having insecticidal activity, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

47.

19. The composition of claim 18, wherein the composition is selected from the group consisting of: Powders, dusts, pills, granules, sprays, emulsions, colloids and solutions.

20. The composition of claim 18, wherein the composition is prepared by dehydrating, freeze-drying, homogenizing, extracting, filtering, centrifuging, sedimenting or concentrating a culture of bacterial cells.

21. The composition of claim 18, comprising from about 1% to about 99% by weight of the polypeptide.

22. The composition of any one of claims 18 to 21, wherein the composition comprises at least a second pesticide.

23. The composition of claim 22, wherein the composition comprises an interfering RNA molecule.

24. A method for controlling a population of coleopteran pests, the method comprising contacting the population with an insect-control-effective amount of an insecticidal polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

44.

25. A method for killing a coleopteran pest, the method comprising contacting the pest with an insect control effective amount of a polypeptide having insecticidal activity, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

44.

26. A method for increasing yield in a corn plant, the method comprising growing corn plants or seeds thereof in a field, the corn plants or seeds thereof having stably incorporated into their genome the DNA construct of claim 4, and wherein the field is infested with a pest against which the polypeptide has insecticidal activity.

27. The expression cassette of claim 1, wherein the heterologous nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide, wherein the amino acid sequence of the polypeptide is at least 99% identical to the amino acid sequence of SEQ ID NO:

44.

28. A polypeptide comprising an amino acid sequence at least 99% identical to SEQ ID NO: 44, and further comprising an introduced protease cleavage site.

29. The polypeptide of claim 28, wherein the introduced protease cleavage site is located between amino acid residues 97 to 266 of SEQ ID NO:

44.

30. A polypeptide comprising an amino acid sequence at least 99% identical to SEQ ID NO: 44, further comprising a mutation introducing a protease cleavage site.

31. The polypeptide of claim 28, wherein the introduced protease cleavage site is introduced by insertion, deletion, or substitution of at least one amino acid residue.

32. The polypeptide of claim 31 , wherein the introduced protease cleavage site is introduced by insertion of a leucine residue.

33. A polypeptide comprising an amino acid sequence at least 99% identical to SEQ ID NO: 44, comprising a mutation that improves digestibility in an SGF assay compared to a polypeptide comprising the amino acid sequence of SEQ ID NO:

39.

34. A method for improving the digestibility of a polypeptide having at least 99% identity to SEQ ID NO: 39 in an SGF assay, the method comprising introducing at least one mutation into the amino acid sequence of a NitromobCRW polypeptide comprising SEQ ID NO:

39.

35. The method of claim 34, wherein the at least one mutation is an insertion, deletion, or mutation of at least one amino acid residue.

36. The method of claim 35, wherein at least one mutation is an insertion of a leucine residue.

37. The method of claim 34, wherein at least one mutation is introduced between amino acid residues 97 to 266 corresponding to SEQ ID NO:

39.

38. The method of claim 35, wherein at least one mutation is introduced at or proximal to amino acid residues corresponding to 213 and / or 215 of SEQ ID NO:

39.

39. The method of claim 38, wherein the mutation is I215L.

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