Insecticide protein

Abstract

Compositions and methods for controlling insect pests are disclosed. In particular, novel insecticidal proteins that are toxic to at least coleopteran insect pests are provided. Also provided are nucleic acid molecules encoding the novel insecticidal proteins. Also disclosed are methods of making the insecticidal proteins and methods of using the insecticidal proteins and nucleic acids encoding the insecticidal proteins of the invention, for example, in transgenic plants to confer protection from insect damage.

Description

Technical Field

[0001] 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 controlling insects using these insecticidal proteins and corresponding nucleic acids. Background Art

[0002] Insect pests are a major cause of crop losses. In the United States alone, billions of dollars are lost each year due to infestations of various genera of insects. 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.

[0003] Multiple species of corn rootworm are considered the most destructive corn pests. In the United States alone, three species, Diabrotica virgifera virgifera (also known as the western corn rootworm), D. longicornis barberi (also known as the northern corn rootworm), and D. undecimpunctata howardi (also known as the southern corn rootworm), cause more than $1 billion in losses to corn each year in the U.S. Corn Belt. An important corn rootworm pest in the southern United States is the Mexican corn rootworm (Diabrotica virgifera zeae). In South America, the South American leafworm (Diabrotica Speciosa) is considered an important pest of corn. The western corn rootworm spread to Europe in 1992 and has been causing economic losses throughout major corn-growing regions since 2008. Corn rootworm larvae cause the most substantial plant damage by feeding almost exclusively on corn roots. This damage has been shown to increase plant lodging, reduce grain yield and vegetative yield as well as alter the nutrient content of the grain. Larval feeding also indirectly affects corn by opening access to the roots for bacterial and fungal infections that cause root and stem rot. Adult corn rootworms are active in corn fields in late summer where they feed on ears, silks, and pollen, thereby interfering with normal pollination.

[0004] Corn rootworms are primarily controlled by intensive applications of chemical pesticides, which are active by inhibiting insect growth, preventing insect feeding or reproduction, or causing mortality. Good corn rootworm control can be achieved, but these chemicals can sometimes affect other beneficial organisms as well. Another problem caused by the widespread use of chemical pesticides is the emergence of resistant insect populations. Yet another problem is due to the fact that corn rootworm larvae feed underground, making rescue treatments with insecticides difficult. Therefore, most insecticide applications are made preventively at the time of planting. This practice results in a huge environmental burden. This situation has been partially improved by various farmland management practices, but there is an increasing need for alternative mechanisms of pest control.

[0005] Biological pest control agents, such as Bacillus thuringiensis (Bt) strains expressing pesticidal toxins like delta-endotoxins (Δ-endotoxins; also known as crystalline toxins or Cry proteins), have also been applied to crop plants on a small scale, producing satisfactory results against certain insect pests. These delta-endotoxins are proteins contained within a crystalline matrix, which are known to possess insecticidal activity when ingested by certain insects. Such Cry proteins from Bacillus thuringiensis have been expressed in transgenic crop plants and commercially developed to control certain Lepidoptera and Coleopteran insect pests. For example, starting in 2003, transgenic corn hybrids that control corn rootworms by expressing Cry3Bb1, Cry34Ab1 / Cry35Ab1, modified Cry3A (mCry3A) or eCry3.1Ab proteins have been commercially available in the United States.

[0006] Although it has been shown that transgenic plants expressing Cry proteins are very effective, insect pests that are resistant to Cry proteins expressed in certain transgenic plants are now known. Therefore, there is still a need to identify new and effective pest control proteins 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 Cry proteins in existing insect control products to mitigate resistance development. In addition, it is desirable to deliver insect control proteins through these products that minimize the environmental burden (such as through transgenic plants). Summary of the invention

[0007] In view of these needs, the present invention provides insecticidal proteins, particularly derived from Streptomyces bacteria (but may also be derived from other types of bacteria). Examples of such insecticidal proteins are illustrated herein and such insecticidal proteins, whether derived from Streptomyces or other different bacteria, are collectively referred to as Streptomyces insecticidal proteins (SMIPs). The present invention also provides variants of the SMIPs of the present invention, as well as proteins substantially identical to the SMIPs of the present invention and their variants. Examples of amino acid sequences of the SMIPs of the present invention include, but are not limited to, any one of SEQ ID NOs: 1-17. The SMIPs of the present invention are toxic to insect pests. For example, the SMIPs of the present invention can be used to control economically important insect pests, including coleopteran insects, such as western corn rootworm (WCR; corn rootworm), northern corn rootworm (NCR; long-horned barbel rootworm), southern corn rootworm (SCR; cucumber eleven-spotted leafworm rootfeeding subspecies) and / or Mexican corn rootworm (MCR; Mexican corn rootworm).

[0008] The present invention also provides nucleic acid molecules comprising one or more nucleotide sequences encoding SMIP and / or SMIP variants, their complementary sequences, or nucleotide sequences substantially identical to SMIP and / or SMIP variants. Examples of nucleotide sequences encoding SMIP or variant SMIP of the present invention include, but are not limited to, any one of SEQ ID NO: 18-40.

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

[0010] The invention also provides compositions and formulations comprising a SMIP or variant SMIP of the invention, which are capable of inhibiting the ability of pests to survive, grow and / or reproduce, or to limit insect-associated damage or loss to crops, e.g., applying the SMIP or variant thereof as part of a composition or formulation to an area or plant infested by insects, or preventatively treating an area or plant susceptible to insect infestation to confer protection against insect pests.

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

[0012] The SMIPs and / or variant SMIPs of the invention can be used alone or in combination with other insect control agents and strategies to impart enhanced pest control efficacy against the same insect pests and / or to increase the spectrum of target insects with minimal environmental impact.

[0013] Other aspects and advantages of the present invention will become apparent to those skilled in the art upon study of the following description and non-limiting examples of the invention.

[0014] Brief description of the sequences in the sequence listing

[0015] SEQ ID NO: 1 is the amino acid sequence of StrepF5053CRW (SMIP1Aa) of Streptomyces.

[0016] SEQ ID NO: 2 is the amino acid sequence of SMIP1Aa-A71L.

[0017] SEQ ID NO: 3 is the amino acid sequence of SMIP1Aa-A90L.

[0018] SEQ ID NO:4 is the amino acid sequence of SMIP1Aa-C92L.

[0019] SEQ ID NO:5 is the SMIP1Aa-C92S amino acid sequence.

[0020] SEQ ID NO:6 is the SMIP1Aa-G84L amino acid sequence.

[0021] SEQ ID NO:7 is the SMIP1Aa-G85L amino acid sequence.

[0022] SEQ ID NO: 8 is the SMIP1Aa-V76L amino acid sequence.

[0023] SEQ ID NO:9 is the SMIP1Aa-W78F amino acid sequence.

[0024] SEQ ID NO: 10 is the SMIP1Aa-A71L / A90L amino acid sequence.

[0025] SEQ ID NO: 11 is the SMIP1Aa-A71L / W78F amino acid sequence.

[0026] SEQ ID NO: 12 is the SMIP1Aa-W78F / C92L amino acid sequence.

[0027] SEQ ID NO: 13 is the SMIP1Aa-Y82F / I83L amino acid sequence.

[0028] SEQ ID NO: 14 is the SMIP1Aa-A2G / A3G amino acid sequence.

[0029] SEQ ID NO: 15 is the SMIP1Aa-A2V / A3V amino acid sequence.

[0030] SEQ ID NO: 16 is the amino acid sequence of SUMO-SMIP1Aa.

[0031] SEQ IS NO: 17 is the amino acid sequence of Strep8K308CRW (SMIP2Aa) of Streptomyces.

[0032] SEQ ID NO: 18 is the nucleotide sequence of StrepF5053CRW (SMIP1Aa).

[0033] SEQ ID NO: 19 is the nucleotide sequence of Strep8K308CRW (SMIP2Aa).

[0034] SEQ ID NO: 20 is the E. coli optimized nucleotide sequence of SMIP1Aa.

[0035] SEQ ID NO: 21 is the nucleotide sequence of SMIP1Aa-A71L optimized for E. coli.

[0036] SEQ ID NO: 22 is the E. coli optimized nucleotide sequence of SMIP1Aa-A90L.

[0037] SEQ ID NO: 23 is the nucleotide sequence of SMIP1Aa-C92L optimized for E. coli.

[0038] SEQ ID NO: 24 is the nucleotide sequence optimized for SMIP1Aa-C92S E. coli.

[0039] SEQ ID NO: 25 is the E. coli optimized nucleotide sequence of SMIP1Aa-G84L.

[0040] SEQ ID NO: 26 is the nucleotide sequence optimized for SMIP1Aa-G85L in E. coli.

[0041] SEQ ID NO: 27 is the E. coli optimized nucleotide sequence of SMIP1Aa-V76L.

[0042] SEQ ID NO: 28 is the nucleotide sequence optimized for SMIP1Aa-W78F Escherichia coli.

[0043] SEQ ID NO: 29 is the nucleotide sequence optimized for SMIP1Aa-A71L / A90L in E. coli.

[0044] SEQ ID NO: 30 is the nucleotide sequence optimized for SMIP1Aa-A71L / W78F Escherichia coli.

[0045] SEQ ID NO: 31 is the nucleotide sequence optimized for SMIP1Aa-W78F / C92L Escherichia coli.

[0046] SEQ ID NO: 32 is the nucleotide sequence of SMIP1Aa-Y82F / I83L optimized for E. coli.

[0047] SEQ ID NO: 33 is the E. coli optimized nucleotide sequence of SMIP1Aa-A2G / A3G.

[0048] SEQ ID NO: 34 is the nucleotide sequence of SMIP1Aa-A2V / A3V optimized for E. coli.

[0049] SEQ ID NO: 35 is the E. coli optimized nucleotide sequence of SUMO-SMIP1Aa.

[0050] SEQ ID NO:36 is the nucleotide sequence of SMIP2Aa optimized for E. coli

[0051] SEQ ID NO:37 is the maize-optimized nucleotide sequence of SMIP1Aa.

[0052] SEQ ID NO:38 is the maize optimized nucleotide sequence of SMIP1Aa-A71L / A90L.

[0053] SEQ ID NO:39 is the maize optimized nucleotide sequence of SMIP1Aa-W78F / C92L.

[0054] SEQ ID NO:40 is the nucleotide sequence optimized for SMIP2Aa maize DETAILED DESCRIPTION

[0055] This description is not intended to be an exhaustive list of all the different ways in which the present invention may be implemented, or of all the features that may be added to the present invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. Therefore, the present invention contemplates that, in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted. Furthermore, in view of this disclosure, numerous variations of the various embodiments suggested herein and additions will be apparent to those skilled in the art without departing from the present invention. Therefore, the following description is intended to set forth some specific embodiments of the present invention, and does not exhaustively describe all permutations, combinations, and variations thereof.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention.

[0057] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings of the pertinent sentence and / or paragraph in which the reference is cited.

[0058] The nucleotide sequences provided herein are presented from left to right in the 5' to 3' direction and are represented using the standard code representing nucleotide bases as described in 37 CFR §§1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25, for example: adenine (A), cytosine (C), thymine (T), and guanine (G).

[0059] Amino acids are also indicated using WIPO Standard ST.25, for example: 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; 1), 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).

[0060] Unless the context indicates otherwise, it is expressly contemplated that the various features of the invention described herein can be used in any combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features stated herein can be excluded or omitted. For example, if the specification states that a composition comprises components A, B, and C, it is expressly contemplated that any one or combination of A, B, or C can be omitted and abandoned, either singly or in any combination.

[0061] definition

[0062] As used in accordance with this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0063] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" is a reference to one or more plants and includes equivalents thereof known to those skilled in the art, and so forth.

[0064] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0065] The term "about" is used herein to mean approximately, roughly, about, or around. When the term "about" is used in conjunction with a numerical range, it defines the range by extending the boundaries above and below the stated values. In general, the term "about" is used herein to define a numerical value to a variation of 20%, preferably 10% above and below (higher or lower) above and below the stated value. With respect to temperature, the term "about" means ±1°C, preferably ±0.5°C. When the term "about" is used in the context of the present invention (e.g., in combination with a temperature or molecular weight value), an exact value (i.e., no "about") is preferred.

[0066] As used herein, phrases such as “between about X and Y,” “between about X and about Y,” “from X to Y,” and “from about X to about Y” (and similar phrases) should be interpreted to include X and Y unless the context indicates otherwise.

[0067] As used herein, the term "amplified" means using at least one nucleic acid molecule as a template to construct multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule. Amplification systems include polymerase chain reaction (PCR) systems, ligase chain reaction (LCR) systems, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), Q-β replicase systems, transcription-based amplification systems (TAS) and strand displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, PERSING et al., ed., American Society for Microbiology, Washington, DC, (1993). The amplified product is referred to as an "amplicon".

[0068] The "activity" of the insecticidal protein of the present invention means that the insecticidal protein acts as an orally active insect control agent, has a toxic effect, and / or is able to interfere with or prevent insect feeding, which may or may not cause the death of the insect. When the insecticidal protein of the present invention is delivered to an insect, this result is typically the death of the insect, or the insect does not 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 harmful organisms (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 with pesticidal activity. An "insecticide" is an agent with insecticide activity.

[0069] As used herein, the term "chimeric construct" or "chimeric gene" or "chimeric polynucleotide" or "chimeric nucleic acid" (or similar terms) refers to a construct or molecule comprising two or more polynucleotides from different sources assembled into a single nucleic acid molecule. The term "chimeric construct", "chimeric gene", "chimeric polynucleotide" or "chimeric nucleic acid" refers to any construct or molecule that contains, but is not limited to (1) polynucleotides (e.g., DNA) including regulatory polynucleotides and coding polynucleotides that are not found together in nature (i.e., at least one polynucleotide in the construct is heterologous relative to at least one of its other polynucleotides), or (2) polynucleotides encoding portions of a protein that are not naturally contiguous, or (3) portions of a promoter that are not naturally contiguous. In addition, a chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid may comprise regulatory polynucleotides and coding polynucleotides derived from different sources, or comprise regulatory polynucleotides and coding polynucleotides derived from the same source but arranged in a manner different from that found in nature. In some embodiments of the invention, a chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid comprises an expression cassette comprising a polynucleotide of the invention under the control of a regulatory polynucleotide, particularly a regulatory polynucleotide functional in plants or bacteria.

[0070] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (eg, mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA). Preferably, the RNA is then translated in an organism to produce a protein.

[0071] "Controlling" insects means inhibiting the ability of insect pests to survive, grow, feed, and / or reproduce through toxic effects, or limiting insect-related damage or losses in crop plants. "Controlling" insects may or may not mean killing the insects, although it preferably means killing the insects.

[0072] As used herein, "codon optimized" sequence means the following nucleotide sequence, wherein these codons are selected to reflect the specific codon preference that host cells or organisms may have. This is typically accomplished in such a manner that the amino acid sequence of the polypeptide encoded by the nucleotide sequence to be optimized is maintained. In certain embodiments, the DNA sequence of the recombinant DNA construct includes a sequence that has been codon optimized for the cell (e.g., animal, plant, or fungal cell) to be expressed therein for the construct. For example, the construct to be expressed in plant cells can have its entire or partial sequence (e.g., the first gene suppression element or gene expression element) codon optimized for expression in plants. See, e.g., U.S. Patent number 6,121,014, incorporated herein by reference.

[0073] The terms “comprises” or “comprising” when used in the present specification indicate the presence of stated features, integers, steps, operations, elements, or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0074] As used herein, the transitional phrase "consisting essentially of (and grammatical variations) means that the scope of the claims is to be interpreted as encompassing the specified materials or steps recited in the claims and those that do not materially alter one or more of the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of" when used in the claims of the present invention is not intended to be interpreted as being equivalent to "comprising".

[0075] In the context of the present invention, "corresponding to or corresponds to" means that when the amino acid sequences of variant or homolog proteins are aligned with each other, the amino acids "corresponding to" certain enumerated positions in the variant or homolog protein are those aligned with these positions in the reference protein, but not necessarily in these exact numerical positions relative to the specific reference amino acid sequence of the present invention. For example, if SEQ ID NO: 1 is the reference sequence and is aligned with SEQ ID NO: 17, then the amino acid Arg (R) at position 52 (R52) of SEQ ID NO: 17 "corresponds to" the Arg (R) at position 20 (R20) of SEQ ID NO: 1, or, for example, Tyr (Y) at position 96 (Y96) of SEQ ID NO: 17 "corresponds to" the His (H) at position 64 (H64) of SEQ ID NO: 1.

[0076] "Delivery" of a composition or toxic protein means that the composition or toxic protein contacts the insect, which promotes oral ingestion of the composition or toxic protein, resulting in toxic effects and control of the insect. The composition or toxic protein can be delivered in many recognized ways, including but not limited to transgenic plant expression, one or more formulated protein compositions, one or more sprayable protein compositions, bait matrix, or any other art-recognized protein delivery system.

[0077] The term "domain" refers to a group of amino acids that are conserved at specific positions along the alignment of the sequences of evolutionarily related proteins. Although amino acids at other positions may differ between homologs, highly conserved amino acids at specific positions indicate amino acids that are likely to be essential in the structure, stability or function of the protein. They are identified by their high conservation in the aligned sequences of a family of protein homologs and can be used as identifiers to determine whether any polypeptide in question belongs to a previously identified polypeptide group.

[0078] "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 crop plant losses. "Insect control effective amount" may or may not mean killing insects, although it preferably means killing insects.

[0079] 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 connected to a target nucleotide sequence, which is operably connected to a termination signal. It also typically comprises a 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, which is heterologous relative to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette that has been 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 may 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 the case of a multicellular organism (e.g., a plant), the promoter may also be specific for a specific tissue, or an organ, or a developmental stage.

[0080] The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. The expression cassette may also include 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 makes it so that it does not naturally occur in the cell into which it is introduced.

[0081] The expression cassette can also optionally include 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 nucleotide sequence of interest that is operably connected, can be natural for the plant host, or can be derived from another source (i.e., foreign or heterologous for promoter, nucleotide sequence of interest, plant host, 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 terminator known to be available for use in plants that plays a role can be used in the context of the present invention.

[0082] When used with reference to polynucleotides (such as genes, ORFs or parts thereof, or transgenics of plants), the term "expression" refers to the process of converting the genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA or snRNA) by the "transcription" of a gene (i.e., via the enzymatic action of an RNA polymerase), and into a protein by the "translation" of mRNA in applicable cases (e.g., if the gene encodes a protein). 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 may refer to only the antisense RNA or only the transcription of the dsRNA, respectively. In an embodiment, "expression" refers to the transcription and stable accumulation of a sense (mRNA) or functional RNA. "Expression" may also refer to the production of a protein.

[0083] "Gene" is a restricted region that is located in the genome and comprises a coding nucleic acid sequence, and typically also comprises other major regulatory nucleic acids that are responsible for controlling the expression (i.e., transcription and translation) of the coding portion. A gene can also comprise other 5' and 3' untranslated sequences and termination sequences. Other possible elements 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.

[0084] "Target gene" refers to any nucleic acid molecule that, when transferred to an organism, such as a bacterium or a plant, confers a desired trait (such as 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) on the bacterium or plant. "Target gene" can also be a gene transferred to a bacterium or a plant for producing a commercially valuable enzyme or metabolite in the plant.

[0085] "Heterologous" nucleic acid sequence or nucleic acid molecule is a nucleic acid sequence or nucleic acid molecule that is not naturally associated with the host cell into which the nucleic acid sequence is introduced, including multiple copies of non-naturally occurring nucleic acid sequences. Heterologous nucleic acid sequences or nucleic acid molecules can comprise chimeric sequences, such as chimeric expression cassettes, in which promoters and coding regions are derived from multi-source organisms. Promoter sequences can be constitutive promoter sequences, tissue-specific promoter sequences, chemically inducible promoter sequences, wound-inducible promoter sequences, stress-inducible promoter sequences, or developmental stage-specific promoter sequences.

[0086] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

[0087] "Homologous recombination" is the reciprocal exchange of nucleic acid fragments between homologous nucleic acid molecules.

[0088] As used herein, "hypothetical protein" refers to a protein whose existence has been predicted but lacks experimental evidence of its expression in vivo. Sequencing a genome or organism (such as bacteria or plants) usually produces a large number of predicted open reading frames, and the functions of these reading frames are not easy to specify. These proteins, whether isolated or conservative hypothetical proteins, account for about 20% to about 40% of the proteins encoded in each newly sequenced genome. Even if there is enough evidence to show that the product of the gene is expressed, it is difficult to specify its function through technologies such as microarrays and mass spectrometry because it lacks identity with the protein sequence with annotated biochemical functions. Generally, most protein sequences are inferred from computational analysis of genomic DNA sequences. Hypothetical proteins are usually created by gene prediction software during genome analysis. When bioinformatics tools for gene identification find large open reading frames without characteristic homologs in protein databases, such tools usually return the name "hypothetical protein" as an annotation.

[0089] The term "motif" or "consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. A motif is often a highly conserved portion of a domain, but may also include only a portion of a domain, or be located outside a conserved domain (if all of the amino acids of the motif are located outside the defined domain).

[0090] In the context of two nucleic acid or amino acid sequences, the term "identity" or "identical" or "substantially identical" refers to two or more sequences or subsequences that have at least 60%, preferably at least 80%, more preferably 90%, even more preferably 95%, and most preferably at least 99% 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. Preferably, this basic identity is present throughout a region having a sequence of at least about 50 residues or bases in length, more preferably throughout a region of at least about 100 residues or bases, and most preferably these sequences are substantially identical over at least about 150 residues or bases. In particularly preferred embodiments, the sequences throughout the length of the coding region are substantially identical. In addition, substantially identical nucleic acid or amino acid sequences substantially perform the same function.

[0091] 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, subsequence coordinates are specified), and the parameters of the sequence algorithm program are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.

[0092] 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 Analysis Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or by visual inspection (see generally Ausubel et al., below).

[0093] An example of an algorithm suitable for determining percentages of sequence identity and sequence similarity is the BLAST algorithm, which is described in the following literature: Altschul et al., J. Mol. Biol. [Molecular Biology] 215: 403-410 (1990). The software performing BLAST analysis is available to the public through the National Center for Biotechnology Information (the National Center for Biotechnology Information, US National Library of Medicine, 8600 Rockville Pike, Bethesda, Maryland 20894 USA). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words with length W in the query sequence, which match or meet some positive threshold score T when compared with words with the same length in the database sequence. T is referred to as the neighboring word score threshold (Altschul et al., 1990). These initial neighboring word hits serve as seeds for initiating searches to find longer HSPs containing them. These word hits are then extended in both directions along each sequence for as far as 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 mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score decreases 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 a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains as defaults. 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)).

[0094] 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.

[0095] Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specific hybridization" refers to a molecule that binds, doubles or hybridizes only to a specific nucleotide sequence under stringent conditions when the 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 a small amount of mismatches that can be accommodated by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0096] 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 the following literature: Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes [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 [Overview of hybridization principles and nucleic acid probe assay strategies]" Elsevier [Elsevier Group], New York. Generally, high stringency hybridization and washing conditions are selected to be higher than the thermal melting point (T) of the specific sequence at a defined ionic strength and pH. m Typically, under "stringent conditions," a probe will hybridize to its target subsequence, but not to other sequences.

[0097] Tm 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 at 42°C with 1 mg of heparin, and the hybridization is carried out overnight. An example of high stringency washing conditions is 0.15M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is a 0.2×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 signals. An example of a medium stringency wash for, for example, a duplex of more than 100 nucleotides is 1×SSC at 45°C for 15 minutes. An example of a low stringency wash for, for example, a duplex of more than 100 nucleotides is 4-6×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 2-fold (or higher) observed in a particular hybridization assay compared to an unrelated probe indicates that specific hybridization has been detected. If the proteins encoded by nucleic acids that do not hybridize to each other under stringent conditions are substantially identical, they are still substantially identical. For example, this occurs when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code.

[0098] The following are examples of settings of hybridization / washing conditions that can be used to clone homologous nucleotide sequences (which 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 2×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 1×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 0.5×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 0.5×SSC, 0.1% SDS at 50°C. EDTA at 50°C, and washing in 0.1X SSC, 0.1% SDS at 50°C; more preferably in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, 1 mM EDTA at 50°C, and washing in 0.1X SSC, 0.1% SDS at 65°C.

[0099] Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid reacts immunologically with 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.

[0100] The term "isolated" nucleic acid molecule, polynucleotide or protein is a nucleic acid molecule, polynucleotide or protein that is no longer present in its natural environment. The isolated nucleic acid molecule, polynucleotide or protein of the present invention can exist in a purified form, or can be present in a recombinant host, such as a transgenic bacterium or a transgenic plant. Therefore, the requirements for "isolated" nucleic acid molecules as listed herein encompass nucleic acid molecules when the nucleic acid molecule is included in the transgenic plant genome.

[0101] 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, a nucleic acid molecule is typically a segment of DNA. In some embodiments, a nucleic acid molecule of the present invention is an isolated nucleic acid molecule.

[0102] "Operably linked" refers to the association of polynucleotides on a single nucleic acid fragment such that the function of one affects the function of the other. For example, when a promoter is able to affect the expression of a coding polynucleotide or a functional RNA (i.e., the coding polynucleotide or the functional RNA is under the transcriptional control of the promoter), the promoter is operably linked to the coding polynucleotide or the functional RNA. A coding polynucleotide in either the sense or antisense orientation can be operably linked to a regulatory polynucleotide.

[0103] As used herein, "pesticide," "insecticide," and the like refer to the ability of the SMIP toxins of the invention to control pests or the amount of SMIP toxins that can control pests as defined herein. Thus, the pesticidal SMIP toxins can kill or inhibit the ability of pests (e.g., insect pests) to survive, grow, feed, or reproduce.

[0104] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein.

[0105] "Plant" is any plant at any stage of development, particularly a seed plant. Exemplary plants include, but are not limited to, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice (Oryza sativa, including but not limited to indica and / or japonica), rapeseed (Brassica napus), rye (rye, Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (sunflower, Helianthus annus), wheat (Triticum aestivum), soybean (soybean, Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium hirsutum), hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musas spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), apple (Malus pumila), blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), grape (Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach (Prunus persica), persica), plum (Prunus domestica), pear (Pyrus communis), watermelon (Citrullus vulgaris), duckweed (Lemna spp.), oats (Avena sativa), barley (Hordium vulgare), vegetables, ornamentals, conifers, and turf grasses (e.g., ornamental, recreational, or forage), and biomass grasses (e.g., switchgrass and miscanthus).

[0106] Vegetables include, but are not limited to, Solanum species (e.g., tomatoes, Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), carrots (Caucus carota), cauliflower (Brassica oleracea), celery (Apium graveolens), eggplant (eggplant, Solanum melongena), asparagus (Asparagus officinalis), okra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), members of the genus Cucurbita such as C. hubbard, winter squash (C. moschata), zucchini (C. pepo), C. crookneck, C. argyrosperma, C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis, C. foetidissima, C. lundelliana, and C. martinezii, and members of the genus Cucumis such as cucumber (Cucumber, Cucumis sativus), cantaloupe (C. cantalupensis), and muskmelon (C. melo).

[0107] Ornamental plants include, but are not limited to, azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherima), and chrysanthemums.

[0108] Conifers that can be used to practice the present invention include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), jack pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western arborvitae (Thuja plicata) and Alaska yellow fir (Alaska yellow-cedar (Chamaecyparis nootkatensis).

[0109] Turf grasses include, but are not limited to, zoysia grass, bentgrass, fescue, bluegrass, Augustine grass, Bermuda grass, buffalo grass, ryegrass, and orchardgrass.

[0110] Also included are plants that serve primarily as laboratory models, such as Arabidopsis thaliana.

[0111] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as part of a higher organization unit such as a plant tissue, a plant organ, or a whole plant.

[0112] "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.

[0113] "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.

[0114] A "plant organ" is a distinct and distinct structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0115] As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. Any plant tissue in a plant or in culture is included. 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 covered by this definition is not intended to exclude any other type of plant tissue.

[0116] "Polynucleotide" refers to a polymer consisting of many nucleotide monomers covalently bonded in a chain. Such "polynucleotides" include DNA, RNA, modified oligonucleotides (e.g., oligonucleotides containing bases that are not typical for biological RNA or DNA, such as 2'-O-methylated oligonucleotides), etc. In some embodiments, nucleic acids or polynucleotides can be single-stranded, double-stranded, multi-stranded, or a combination thereof. Unless otherwise indicated, specific nucleic acids or polynucleotides of the present invention optionally include or encode complementary polynucleotides in addition to any polynucleotides explicitly indicated.

[0117] "Polynucleotide of interest" refers to any polynucleotide that, when transferred into an organism (e.g., a plant), confers a desired trait to that organism, such as insect resistance, disease resistance, herbicide tolerance, antibiotic resistance, improved nutritional value, improved performance in an industrial process, production of a commercially valuable enzyme or metabolite, or altered reproductive capacity.

[0118] "Promoter" is a non-translated DNA sequence upstream of the 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.

[0119] As used herein, the term "recombination" refers to a nucleic acid molecule (e.g., DNA or RNA) or a protein or an organism in a form that is not usually found in nature and that is produced by human intervention. As used herein, a "recombinant nucleic acid molecule" is a nucleic acid molecule comprising a combination of polynucleotides that do not naturally exist together and are the result of human intervention, for example, a nucleic acid molecule composed of a combination of at least two polynucleotides that are heterologous to each other, or an artificially synthesized (e.g., a polynucleotide synthesized using an assembled nucleotide sequence) nucleic acid molecule comprising a polynucleotide that deviates from a polynucleotide that is usually present in nature, or a nucleic acid molecule comprising a transgenic artificially incorporated into the genomic DNA of a host cell and the host cell genome-related flanking DNA. Another example of a recombinant nucleic acid molecule is a DNA molecule produced by inserting a transgenic into the genomic DNA of a plant, which can ultimately result in the expression of a recombinant RNA / or protein molecule in the organism. As used herein, a "recombinant plant" is a plant that is not usually present in nature, is the result of human intervention, and contains transgenic and / or heterologous nucleic acid molecules incorporated into its genome. Due to such genome changes, the recombinant plant is significantly different from the associated wild-type plant.

[0120] "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.

[0121] Streptomyces insecticidal protein (SMIP) is a protein encoded by a gene found in at least the genome of Actinomycetaceae bacteria (particularly in Streptomyces), which contains an aegerolysin-like pore-forming domain and is active against insect pests in at least the genus Radix Gnaphalidae. A "variant SMIP" is an engineered SMIP by replacing and / or deleting at least one amino acid found in a naturally occurring SMIP and / or inserting an amino acid into the SMIP sequence. As used herein, unless otherwise indicated, when the term "Streptomyces insecticidal protein" or "SMIP" is used alone, it is meant to include non-engineered proteins as well as engineered variant proteins.

[0122] The terms "substitution", "insertion" or "addition" and "deletion" are used herein with reference to amino acid or nucleotide sequences. "Substitution" refers to the replacement of one or more nucleotides or amino acids with different nucleotides or amino acids, respectively. "Insertion" or "addition" is a change in a nucleotide or amino acid sequence that results in the addition of one or more nucleotides or amino acid residues, respectively, compared to a naturally occurring sequence. "Deletion" is defined as a change in a nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent. Amino acid substitutions are typically single residue substitutions; insertions will generally be on the order of about 1 to 20 amino acids, although significantly larger insertions can be tolerated. The range of deletions is about 1 to about 20 residues, although in some cases, deletions can be much larger. Substitution, deletion, insertion or any combination thereof can be used to obtain the final variant polypeptide. Typically, some amino acids are changed to minimize changes in the molecule. However, in some cases, greater changes can be tolerated. In certain embodiments, an amino acid substitution can be the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, such as replacing isoleucine with valine, i.e., a conservative amino acid substitution. Insertion or deletion can optionally be in the range of 1 to 5 amino acids. In an embodiment, substitutions can be made according to known "conservative substitutions". A "conservative substitution" refers to the substitution of an amino acid in one class with an amino acid in the same class, where the class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature. In contrast, in certain embodiments, the substitution is non-conservative. A "non-conservative substitution" refers to the substitution of an amino acid in one class with an amino acid from another class.

[0123] "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.

[0124] "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 also exist as an extrachromosomal molecule. Such extrachromosomal molecules can replicate autonomously. 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.

[0125] The present invention provides compositions and methods for controlling harmful insect pests. In particular, the present invention relates to insecticidal proteins, particularly Streptomyces insecticidal proteins (SMIPs), which are active against at least coleopteran insects, such as corn rootworms (Western corn rootworm; WCR), Diabrotica barbatus (Diabrotica barber) (northern corn rootworm; NCR) and / or cucumber rootworm subspecies undecemlineatus (southern corn rootworm; SCR) and / or other rootworm species (including Mexican corn rootworm (Mexican corn rootworm). Natural SMIPs are produced by Gram-negative bacteria in the genus Streptomyces and related bacteria. Examples of such SMIPs described herein include those produced by Streptomyces strain F5053, which produces Strept5053CRW (SMIP1Aa) insecticidal protein, and Streptomyces strain 8K308, which produces Strept8K308CRW (SMIP2Aa) insecticidal protein. In some embodiments, the novel insecticidal proteins of the present invention may be active against lepidopteran insect pests, including but not limited to black cutworm (black cutworm), sugarcane borer (sugarcane borer; SCB) and / or southwestern corn borer (southwestern corn borer; SWCB)). The present invention also relates to nucleic acids, the expression of which produces the SMIPs of the present invention, and to methods of making and using these SMIPs to control insect pests. In certain embodiments, expression of these nucleic acids produces insecticidal proteins that can be used to control at least coleopteran insects (such as western corn rootworm, northern corn rootworm and / or southern corn rootworm), particularly when expressed in transgenic plants (such as transgenic corn plants).

[0126] In some non-limiting embodiments, the present invention encompasses nucleic acid molecules, and optionally isolated nucleic acid molecules, comprising, consisting essentially of, or consisting of a nucleotide sequence encoding a protein that is toxic to insect pests (i.e., an insecticidal protein), wherein the nucleotide sequence (a) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-17, or a toxin fragment thereof; or (b) having 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-17, or a toxin fragment thereof; NO: 18-40 or a toxin encoding fragment thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity; or (c) is a synthetic sequence of (a) or (b) that has been codon-optimized for expression in a transgenic organism. In other embodiments, the insecticidal protein comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NO: 1-17 or a toxic fragment thereof. In other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of any one of SEQ ID NO: 18-40 or a toxin encoding fragment thereof.

[0127] In some non-limiting embodiments, the present invention encompasses a chimeric gene comprising a heterologous promoter operably linked to a nucleic acid molecule comprising, consisting essentially of, or consisting of a nucleotide sequence encoding a protein toxic to an insect pest, wherein the nucleotide sequence (a) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-17, or a toxin fragment thereof; or (b) encodes a protein comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-17, or a toxin fragment thereof; NO: 18-40 or its toxin encoding fragment has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity; or (c) is a synthetic sequence of (a) or (b) that has been codon optimized for expression in a transgenic organism. In other embodiments, the insecticidal protein comprises the amino acid sequence of any one of SEQ ID NO: 1-17 or its toxic fragment. In other embodiments, the nucleotide sequence comprises any one of SEQ ID NO: 18-40 or its toxin encoding fragment. In some aspects of these embodiments, the chimeric gene is an expression cassette.

[0128] In other non-limiting embodiments, the promoter contained in the chimeric gene or expression cassette of the present invention is a plant expressible promoter. In aspects of these embodiments, the plant expressible promoter is selected from the group consisting of: ubiquitin, tuberose yellow virus, corn TrpA, OsMADS 6, maize H3 histone, bacteriophage T3 gene 9 5'UTR, maize sucrose synthase 1, maize alcohol dehydrogenase 1, maize light harvesting complex, maize heat shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, legume glycine-rich protein 1, potato glycoprotein, lectin, CaMV 35S and S-E9 small subunit RuBP carboxylase promoter.

[0129] In some non-limiting embodiments, the insecticidal protein encoded by the nucleic acid molecule of the invention or the chimeric gene of the invention or the expression cassette of the invention is active against coleopteran insect pests. In some aspects of these embodiments, the coleopteran insect pest is in the genus Diabrotica. In other aspects, the genus Diabrotica insect pest is a corn rootworm (Western corn rootworm; WCR), a barker's rootworm (Northern corn rootworm; NCR) and / or a cucumber rootworm subspecies eleven-spotted (Southern corn rootworm; SCR) and / or other species of the genus Diabrotica (including the Mexican corn rootworm (Mexican corn rootworm; MCR).

[0130] In some non-limiting embodiments, the chimeric gene or expression cassette of the present invention comprises a nucleotide sequence encoding a SMIP of the present invention, wherein the nucleotide sequence is codon optimized for expression in a transgenic organism. In some embodiments, the transgenic organism is a bacterium or a plant.

[0131] In other non-limiting embodiments, the transgenic bacteria comprising the codon-optimized sequences of the present invention belong to Bacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteurella, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Sinorhizobium, Sword, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, Sphingolipids, Burkholderia, Candidatus Glomeribacter, Dai Shi, Grass Spirillum, Bradyrhizobium, Staphylococcus, Methylophilus, Greedy Phage, Streptococcus, Chitinophaga or Alcaligenes. In other embodiments, the transgenic bacteria are Escherichia coli. In other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of any one of SEQ ID NOs: 20-36.

[0132] In other non-limiting embodiments, the transgenic plant comprising the codon optimized sequence of the present invention is a monocot or a dicot. In other embodiments, the dicot is selected from the group consisting of soybean, sunflower, tomato, Brassica crops, cotton, sugar beet and tobacco. In other embodiments, the monocot is selected from the group consisting of barley, maize, oats, rice, sorghum, sugar cane and wheat. In other embodiments, the transgenic plant is a maize plant. In other embodiments, the nucleotide sequence comprises codons optimized for expression in maize. In still other embodiments, the nucleotide sequence comprises, consists essentially of, or consists of: any one of SEQ ID NO:37-40.

[0133] In some non-limiting embodiments, the present invention encompasses proteins that are toxic to insect pests, and optionally isolated proteins, i.e., insecticidal proteins, wherein the protein or isolated protein comprises, consists essentially of, or consists of: (a) an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-17, or a toxin fragment thereof; or (b) an amino acid sequence comprising, consisting essentially of, or consisting of: any one of SEQ ID NOs: 1-17, or a toxin fragment thereof; or (c) an amino acid sequence encoded by a nucleotide sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-17, or a toxin fragment thereof; NO: 18-40 or a toxin encoding fragment thereof having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity; or (d) an amino acid sequence encoded by a nucleotide sequence comprising, consisting essentially of, or consisting of: SEQ ID NO: 18-40 or a toxin encoding fragment thereof. Those skilled in the art will recognize that the exemplary insecticidal proteins encompassed by the present invention can be modified. Such modifications and the resulting substantially identical nucleic acid or amino acid molecules are encompassed by the present invention.

[0134] The present invention also includes engineered Streptomyces insecticidal proteins (eSMIPs), which may be described as mutant SMIPs or variant SMIPs or modified SMIPs of the present invention. In some embodiments, the modification may include substitution and / or deletion of one or more amino acids in a naturally occurring SMIP sequence and / or insertion of one or more additional amino acids into a naturally occurring SMIP sequence. In other embodiments, the modification may include substitution and / or deletion and / or insertion of one or more amino acids in an engineered SMIP. The substitution and / or insertion may be with naturally occurring amino acids or non-naturally occurring amino acids. In some non-limiting embodiments, the modification comprises, consists essentially of, or consists of: substitution and / or insertion and / or deletion of one or more of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and / or valine at an amino acid position of the SMIP amino acid sequence. Such substitutions and / or insertions and / or deletions can be achieved by changing the codons in the nucleotide sequence encoding the SMIP, thereby generating a modified SMIP nucleotide sequence encoding an eSMIP (which is a mutant SMIP or variant SMIP or modified SMIP of the present invention).

[0135] In some non-limiting embodiments, the SMIP is modified by the following substitutions and / or insertions: (a) one or more amino acids with aliphatic hydrophobic side chains (e.g., alanine, isoleucine, methionine and / or valine; in embodiments, the amino acid is not alanine); or (b) one or more amino acids with aromatic hydrophobic side chains (e.g., phenylalanine, tryptophan and / or tyrosine); or (c) one or more amino acids with polar neutral side chains (e.g., asparagine, cysteine, glutamine, serine and / or threonine); or (d) one or more amino acids with acidic side chains (e.g., aspartic acid and / or glutamic acid); one or more amino acids with basic side chains (e.g., arginine, histidine and / or lysine); or (e) one or more glycine residues; or (f) one or more proline residues; or (g) any combination of (a) to (f).

[0136] In some embodiments, the present invention encompasses an engineered insecticidal protein comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 1, and further comprising at least one mutation at a position corresponding to amino acid positions 1-118 of SEQ ID NO: 1.

[0137] In some embodiments, the mutation is at an amino acid position corresponding to amino acid position 2, 3, 71, 76, 78, 82, 83, 84, 85, 90, or 92 of SEQ ID NO: 1, or any combination thereof. In other non-limiting embodiments, the mutation is at position 2, 3, 71, 76, 78, 82, 83, 84, 85, 90, or 92 of SEQ ID NO: 1. In other embodiments, the mutation is at a position corresponding to amino acids 2 and 3, or 71 and 78, or 71 and 90, or 78 and 92, or 82 and 83 of SEQ ID NO: 1. In further embodiments, the mutation is at amino acid positions 2 and 3, or 71 and 78, or 71 and 90, or 78 and 92, or 82 and 83 of SEQ ID NO: 1. In other embodiments, the mutation at position 2 is A2G or A2V, the mutation at position 3 is A3G or A3V, the mutation at position 71 is A71L, the mutation at position 76 is V76L, the mutation at position 78 is W78F, the mutation at position 82 is Y82F, the mutation at position 83 is I83L, the mutation at position 84 is G84L, the mutation at position 85 is G85L, the mutation at position 90 is A90L, or the mutation at position 92 is C92S or C92L. In other embodiments, the mutations at positions 2 and 3 are A2G / A3G or A2V / A3V, the mutations at positions 71 and 78 are A71L / W78F, the mutations at positions 71 and 90 are A71L / A90L, the mutations at positions 78 and 92 are W78F / C92L, or the mutations at positions 82 and 83 are Y82F / I83L.

[0138] In some embodiments, the protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0139] In some embodiments, the present invention encompasses an engineered insecticidal protein comprising, consisting essentially of, or consisting of an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity to SEQ ID NO: 17, and further comprising at least one mutation at a position corresponding to amino acid positions 33-150 of SEQ ID NO: 17.

[0140] In some embodiments, the mutation is at amino acid position 34, 35, 103, 108, 110, 114, 115, 116, 117, 122, or 124 of SEQ ID NO: 17, or any combination thereof. In other embodiments, the mutation is at amino acid position 34, 35, 103, 108, 110, 114, 115, 116, 117, 122, or 124 of SEQ ID NO: 17, or any combination thereof. In still other embodiments, the mutation is at positions corresponding to amino acids 34 and 35, or 103 and 110, or 103 and 122, or 110 and 124, or 114 and 115 of SEQ ID NO: 17. In still further embodiments, the mutation is at amino acids 34 and 35, or 103 and 110, or 103 and 122, or 110 and 124, or 114 and 115 of SEQ ID NO: 17. In other embodiments, the mutation at position 34 is A34G or A34V, the mutation at position 35 is A35G or A35V, the mutation at position 103 is A103L, the mutation at position 108 is V108L, the mutation at position 110 is W110F, the mutation at position 114 is Y114F, the mutation at position 115 is V115L, the mutation at position 116 is G116L, the mutation at position 117 is S117L, the mutation at position 122 is A122L, or the mutation at position 124 is C124S or C124L. In still other embodiments, the mutations at positions 34 and 35 are A34G / A35G or A34V / A35V, the mutations at positions 103 and 110 are A103L / W110F, the mutations at positions 103 and 122 are A103L / A122L, the mutations at positions 110 and 124 are W110F / C124L, or the mutations at positions 114 and 115 are Y114F / V115L.

[0141] In some non-limiting embodiments, the mutant or variant SMIP of the invention has enhanced digestion by a mammalian digestive protease (e.g., pepsin) compared to a suitable control and / or a parent molecule without the modification of the invention when tested under the same conditions (e.g., enzyme concentration, protein concentration, pH, temperature, and / or time). Methods for evaluating protein digestion by pepsin and other digestive proteases are well known in the art, for example, the simulated gastric fluid (SGF) assay described in Example 2. For example, digestion using pepsin can be performed under the following conditions: about 37° C. and about pH 1.2, optionally with an enzyme concentration of about 10 units (U) of pepsin / microgram of protein. In other embodiments, the digestibility of the SMIP of the invention is enhanced by substitution mutations of amino acids at positions corresponding to amino acid positions 71, 78, 90, or 92 of SEQ ID NO: 1, or any combination thereof, or the digestibility is enhanced by substitution mutations of amino acids at positions corresponding to amino acid positions 103, 110, 122, or 124 of SEQ ID NO: 17, or any combination thereof. In other embodiments, the mutation is at positions corresponding to amino acids 71 ​​and 90 or 78 and 92 of SEQ ID NO: 1. In other embodiments, the mutation is at positions corresponding to amino acids 103 and 122 or 110 and 124 of SEQ ID NO: 17. In still other embodiments, the amino acid corresponding to position 71 of SEQ ID NO: 1 is substituted with Lue (L), the amino acid corresponding to position 78 of SEQ ID NO: 1 is substituted with Phe (F), the amino acid corresponding to position 90 of SEQ ID NO: 1 is substituted with Leu (L), the amino acid corresponding to position 92 of SEQ ID NO: 1 is substituted with Leu (L), or any combination thereof. In still other embodiments, the amino acid corresponding to position 103 of SEQ ID NO: 17 is substituted with Lue (L), the amino acid corresponding to position 110 of SEQ ID NO: 17 is substituted with Phe (F), the amino acid corresponding to position 122 of SEQ ID NO: 17 is substituted with Leu (L), the amino acid corresponding to position 124 of SEQ ID NO: 17 is substituted with Leu (L), or any combination thereof.

[0142] In some embodiments, the digestibility of the SMIPs of the invention is enhanced by substitution mutations of amino acids at amino acid positions 71, 78, 90, or 92 of SEQ ID NO: 1, or any combination thereof. In other embodiments, the mutations are at positions 71 and 90, or 78 and 92 of SEQ ID NO: 1. In other embodiments, the mutation at position 71 of SEQ ID NO: 1 is A71L, the mutation at position 78 of SEQ ID NO: 1 is W78F, the mutation at position 90 of SEQ ID NO: 1 is A90L, or the mutation at position 92 of SEQ ID NO: 1 is C92L, or any combination thereof. In still further embodiments, the mutant or variant SMIP1Aa protein with enhanced digestibility in the SGF assay comprises, consists essentially of, or consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12.

[0143] In some embodiments, the SMIP toxins of the invention, including the eSMIPs of the invention, are active against coleopteran pests. Coleopteran insects include, but are not limited to, any coleopteran insects currently known or later identified, including those of the suborders Protocoleidae, Myxophagata, Carnivora, and Polyphaga, and any combination thereof.

[0144] In some non-limiting embodiments, 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 species of the genus Diabrotica include, but are not limited to: Diabrotica longicornis barberi (northern corn rootworm), Diabrotica nominata (western corn rootworm), Diabrotica eleven-spotted (southern corn rootworm), D. balteata (banded cucumber beetle), D. undecimpunctata undecimpunctata (western spotted cucumber beetle), D. significata (3-spotted leaf beetle), D. speciosa (chrysanthemum beetle), Mexican corn rootworm (Mexican corn rootworm), Beni rootworm (D. beiensis), Christa rootworm (D. cristata), Cove rootworm (D. curvipustulata), two-spotted rootworm (D. dissimilis), elegant rootworm (D. elegantula), Emorsitans, Graminea rootworm (D. graminea), Hispanolae rootworm (D. hispanolae), Lemini Root beetle (D.lemniscata), ochre-legged root beetle (D.linsleyi), miller's root beetle (D.milleri), coin-shaped root beetle (D.nummularis), fan-leaved root beetle (D.occlusa), prai root beetle (D.porracea), snail root beetle (D.scutellata), shin root beetle (D.tibialis), three-line root beetle (D.trifasciata) and micro-green root beetle (D.viridula); and any combination thereof.

[0145] Other non-limiting examples of coleopteran insect pests according to the present invention include Leptinotarsa ​​spp., such as the potato beetle (Colorado potato beetle); Chrysomelas 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 fleabeetle) and E. cucumeris (potato flea beetle): Phyllotreta spp., such as P. cruciferae (crucifer flea beetle) and P. pusilla (western black fleabeetle); 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 seed pod 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), billbug), S. parvulus (bluegrass billbug), and S. callosus (southern corn billbug); Phyllophaga spp. (white grubs); Chaetocnema spp., such as C. pulicaria (corn flea beetle); Popillia spp., such as P. japonensis (Japanese billbug).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 (lister beetles); and any combination of the foregoing.

[0146] The insecticidal proteins of the present invention may also be active against Lepidoptera insects. Such Lepidoptera insects include, but are not limited to, any insect classified as Lepidoptera insects currently known or later identified, including those insect species within the suborders Synaptera, Proboscis, and Heterotrichoptera, 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); Spodoptera spp., such as A. ipsilon (black cutworm), A. segetum (common cutworm), A. gladiaria (clayback cutworm), and A. sedum (black cutworm). 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); Diatraea spp. 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 corn borer); 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); Elasmopalpus spp., such as P. saccharalis (sugarcane borer); Trichoplusia spp., such as T. ni (cabbage looper); Sesamia spp., such as S. nonagroides (Mediterranean corn borer); 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); Elasmopalpus spp., such as P. spp., such as E. lignosellus (lesser cornstalk borer); Pseudoplusia spp., such as P. includens (soybean moth); 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., such as P. nebris (stalk borer); Pseudaletia spp., such as P. nebris (stalk borer); 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.

[0147] The insecticidal proteins of the invention may also be active against Hemiptera, Diptera, Lygus species, and / or other piercing-sucking insects (eg, piercing-sucking insects of the Order Orthoptera or Thysanoptera). Insects of the order Diptera include, but are not limited to, any Diptera insect now known or later 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 (seed corn 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.

[0148] Insects of the order Orthoptera include, but are not limited to, any Orthoptera insect currently 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.

[0149] Insects of the order Thysanoptera include, but are not limited to, any Thysanoptera 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 combination of the foregoing.

[0150] The insecticidal proteins of the present invention may also be active against nematodes. As used herein, the term "nematode" encompasses any organism currently known or later identified as being classified as an organism of the phylum Nematoda of the animal kingdom, including, but not limited to, nematodes in the class Adenophora (including, for example, Rhizophora, Isopharyngea, Monodonta, Lanceolata, Trichocephala, Trachelata, Muspa, Lactropharyngea, Chromatidae, Taeniformes, Chain Rings, and Monogynae) and / or the class Tubulonema (including, for example, Rhabditis, Strongyloides, Ascaridae, Spirulina, Camelidae, Digastricales, Sphaeroidea, and Sphaeroidea).

[0151] 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 (root-knot nematodes), Heterodera (cyst nematodes), Globodera (cyst nematodes), Pentecostal nematodes (piercing nematodes), Reniform nematodes (kidney-shaped kidney-shaped nematodes), Pratylenchus (rot nematodes), Aphelenchus (leaf nematodes), Heliconematodes (helical nematodes), Nematodes (lance nematodes), Pseudocercosus (short and thick root nematodes), Long-nematodes, Pearl nematodes (pseudo-root-knot nematodes), Subanguina, Stinging nematodes, Microcircular nematodes (microcircular nematodes), nematodes), Angiospermum (angiosperm nematodes), Ditylenchus (dendrum nematodes), Conynematode (conynematodes), Hemicyclenematode (hemicycle nematodes), Sheathnematode (sheath nematodes), Subroot Nematode (subroot nematodes), Hypsoperine, Ditylenchus (large stem nematodes), Melinius, Punctodera, Quinisulcius, Shield Nematode (shield nematodes), Xiphinema (dagger nematodes), Dwarf Nematode (dwarf nematodes), Penelopenetes (piercing nematodes), Bursaphelenchus (roundworms), and any combination thereof.

[0152] 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, Heteroderaschachtii, sugar beet cyst nematode, and more. nematode); Heterodera zeae, corn cyst nematode; Heterodera avenae,cereal cyst nematode), Heterodera carotae, Heterodera trifolii, Hoplolaimus columbus, Hoplolaimus galeatus, Hoplolaimusmagnistylus, 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, Quinisulciusacutus, Radopholus similis, Rotylenchulus reniformis, Tylenchorhynchus dubius), Tylenchulus semipenetrans, Siphinema americanum, X. Mediterraneum, and any combination of the foregoing.

[0153] The present invention also encompasses recombinant vectors and / or recombinant constructs, which may also be referred to as vectors or constructs, which comprise expression cassettes and / or nucleic acid molecules of the present invention. In such vectors, these nucleic acids are preferably in expression cassettes, which comprise regulatory elements for expressing nucleotide molecules in host cells capable of expressing nucleotide molecules. Such regulatory elements generally comprise promoters and termination signals and preferably also comprise elements, which allow effective translation of polypeptides encoded by nucleic acid of the present invention. The vector comprising nucleic acid can replicate (preferably as an extrachromosomal molecule) in specific host cells and can therefore be used to increase nucleic acid of the present invention in these host cells.

[0154] The present invention also encompasses host cells comprising recombinant vectors, expression cassettes or nucleic acid molecules of the present invention. In other embodiments, such vectors are viral vectors and are used to replicate nucleotide sequences in specific host cells (e.g., insect cells or plant cells). Recombinant vectors are also used to transform nucleic acid molecules of the present invention into host cells, whereby these nucleic acid molecules are stably integrated into the DNA of transgenic hosts. In certain embodiments, host cells are bacterial cells or plant cells. In some aspects of these embodiments, bacterial cells are in Bacillus, Clostridium, Xenorhabdus, Photobacterium, Pasteurella, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Sinorhizobium, Sword Pseudomonas, Rhodopseudomonas, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc or Alcaligenes. In some other aspects of these embodiments, the host cell is a plant cell, such as a dicotyledonous plant cell or a monocotyledonous plant cell. In other aspects, the dicotyledonous plant cell is selected from the group consisting of soybean cells, sunflower cells, tomato cells, Brassica crop cells, cotton cells, beet cells and tobacco cells. In still other aspects, the monocotyledonous plant cell is selected from the group consisting of barley cells, maize cells, oat cells, rice cells, sorghum cells, sugarcane cells and wheat cells.

[0155] In some non-limiting embodiments of the present invention, at least one of the nucleic acid molecules of the present invention is inserted into an appropriate expression cassette (comprising a promoter and a termination signal). Expression of the nucleic acid may be constitutive, or an inducible promoter that responds to various types of stimuli to initiate transcription may 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, a bacterium, or a fungus. In yet another embodiment, a virus (such as a baculovirus) contains the nucleic acid of the present invention in its genome and expresses a large amount of the corresponding insecticidal protein after infecting an appropriate eukaryotic cell (suitable for viral replication and expression of the nucleic acid). The insecticidal protein thus produced is used as an insecticide. Alternatively, a baculovirus engineered to include the nucleic acid is used to infect insects in vivo and kill them by expression of the insecticidal toxin or by 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, the method comprising culturing a host cell under conditions where a nucleic acid molecule encoding the polypeptide is expressed.

[0156] Bacterial cells are also hosts for expressing nucleic acids of the present invention. In one embodiment, non-pathogenic symbiotic bacteria (so-called endophytes) that can live and replicate in plant tissues are used, or non-pathogenic symbiotic bacteria (so-called epiphytes) that can settle in the leaf or rhizosphere are used. Such bacteria include the following bacteria: Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Corynebacterium, Enterobacter, Erwinia, Flavobacterium, Klebsiella, Pseudomonas, Rhizobium, Sinorhizobium, Sword Pseudomonas, Serratia, Streptomyces, Sphingomonas, Burkholderia, Candidatus Glomeribacter, Dai Shi Pseudomonas, Grass Spirillum, Bradyrhizobium, Staphylococcus, Methylophilus, Greedy Phage, Streptococcus, Chitinophaga and Xanthomonas. Symbiotic fungi such as Trichoderma and Glucobra are also possible hosts for expressing the nucleic acids of the invention for the same purpose.

[0157] These gene manipulation techniques are specific and known in the art for different available hosts. For example, expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in Escherichia coli (in transcription or translation fusion) after tac or trc promoters. 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)).

[0158] In yet other embodiments, the present invention encompasses a method of controlling insect pests, the method comprising delivering an insecticidal protein of the present invention in an insect control effective amount to the insect pests. In some aspects of these embodiments, the insecticidal protein is delivered by a transgenic plant or by topical application of an insecticidal composition comprising the insecticidal protein. In other aspects, the transgenic plant or the insecticidal composition comprises a second insecticide different from the insecticidal protein. In still other aspects, the second insecticide is a protein, dsRNA or a chemical. In still other aspects, the protein is selected from the group consisting of: Cry protein, VIP toxin, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, pore-forming protein, lectin, engineered antibody or antibody fragment, or chitinase; or the chemical is a carbamate, pyrethroid, organophosphate, friprole, neonicotinoid, organochloride, nereis toxin or a combination thereof; or the chemical comprises an active ingredient selected from the group consisting of: carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cypermethrin, λ-cypermethrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, anthracene, malathion, methyl parathion, phorate, terbufos, tert-butylpyrimidophos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamethoxam and a combination thereof.

[0159] In some embodiments of the invention, at least one of the insecticidal proteins of the invention is expressed in a higher organism, such as a plant. Transgenic plants expressing insecticidal proteins in an insect-controlling effective amount protect themselves from insect pests. When the insect pest begins to feed on the transgenic plant, it also ingests the expressed insecticidal protein. This can prevent the insect from further feeding on plant tissue and / or can even injure or kill the insect. The nucleic acid molecules of the invention are inserted into an expression cassette, and the nucleic acid can then be stably integrated into the genome of the plant. In other embodiments, the nucleic acid molecules are included 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.

[0160] In some embodiments, the present invention encompasses a method of producing a protein that is toxic to an insect pest, i.e., an insecticidal protein, comprising: (a) obtaining a host cell comprising a gene, which gene itself comprises an expression cassette and / or a nucleic acid molecule of the present invention; and (b) growing the host cell or a transgenic host comprising the host cell under conditions where the transgenic host cell produces the protein that is toxic to an insect pest.

[0161] In other embodiments, the present invention encompasses a method of producing a transgenic plant or plant part having enhanced insect resistance compared to a control plant or plant part, the method comprising: (a) introducing a chimeric gene or expression cassette or vector comprising a nucleic acid molecule encoding an insecticidal protein of the present invention into a plant or plant part, wherein the insecticidal protein is expressed in the plant or plant part, thereby producing a plant or plant part having enhanced insect resistance. In other embodiments, the chimeric gene, expression cassette or vector may encode a SMIP insecticidal protein of the present invention comprising, consisting essentially of, or consisting of an amino acid sequence that is 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%, or at least 99% identical or similar to any one of SEQ ID NOs: 1-17. "Enhanced" insect resistance can be measured by any toxic effect of the transgenic plant on insect pests that feed on the transgenic plant. The 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% insecticidal activity compared to a control plant that does not express the insecticidal protein. Plants or plant parts with 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 reproduction methods. Any suitable control plant or plant part can be used, for example, a plant grown under the same environment, with the same or similar genetic background. In an embodiment, the control plant or plant part has the same genetic background and is grown in the same environment as the described plant, but it does not comprise the molecule of the present invention, while the described plant comprises the nucleic acid molecule of the present invention.

[0162] In other embodiments, the present invention encompasses a method for enhancing insect resistance in a plant or plant part compared to a control plant or plant part, the method comprising expressing a nucleic acid molecule or expression cassette of the present invention in the plant or plant part, wherein expression of the heterologous nucleic acid or expression cassette results in the plant or plant part having enhanced insect resistance compared to the control plant or plant part. In some embodiments, the expression cassette or nucleic acid molecule comprises a promoter operably linked to a heterologous nucleic acid molecule comprising a nucleotide sequence comprising, consisting essentially of, or consisting of: (a) a nucleotide sequence of any one of SEQ ID NOs: 18-40; or (b) a nucleotide sequence that is 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%, or at least 99% identical to a nucleotide sequence of any one of SEQ ID NOs: 18-40; or (c) a nucleotide sequence encoding a protein, wherein the amino acid sequence of the protein comprises, consists essentially of, or consists of: any one of SEQ ID NOs: 1-17; or (d) a nucleotide sequence encoding a protein, wherein the amino acid sequence of the protein is identical to that of SEQ ID NOs: 1-18. The amino acid sequence of any one of IDNO:1-17 is 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%, or at least 99% identical; or (e) the nucleotide sequence of any one of the above (a) to (d), which is codon-optimized to be expressed in a transgenic host organism; or (f) a nucleotide sequence complementary to the nucleotide sequence of any one of the above (a) to (e). Nucleic acid molecules or expression cassettes can be introduced into plants. In some 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 the plant parts.

[0163] In certain embodiments, the present invention encompasses methods for producing plants having enhanced insect resistance compared to control plants, the method comprising detecting heterologous nucleic acids comprising nucleic acid molecules or expression cassettes of the present invention in plant parts, and producing plants from the plant parts, thereby producing plants having enhanced insect resistance compared to control plants. In a further embodiment, the present invention encompasses methods for identifying plants or plant parts having enhanced insect resistance compared to control plants or plant parts, the method comprising detecting nucleic acid molecules or expression cassettes of the present invention in the plant or plant parts, thereby identifying plants or plant parts 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, which is unique to the expression cassette of the present invention.

[0164] In other embodiments, the present invention encompasses a method for 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 acid and exhibiting enhanced insect resistance compared to a control plant.

[0165] In some non-limiting embodiments, the method of the present invention gives plants or plant parts the insect resistance of the enhancement for coleopteran insect pests. In the examples, the insect control of coleopteran insect pests has been demonstrated. In a further embodiment, the method of the present invention gives plants or plant parts the insect resistance of the enhancement for rootworm species (including corn rootworms, Barr's rootworms, cucumber eleven-star leaf beetle root-feeding subspecies, Mexican corn rootworms and / or South American leaf beetles (Diabrotica peciosa)) and / or related species. In a further embodiment, the method of the present invention gives plants or plant parts the insect resistance of the enhancement for western corn rootworms, Barr's rootworms and / or cucumber eleven-star leaf beetle root-feeding subspecies.

[0166] In some non-limiting embodiments, the present invention encompasses transgenic plants comprising a heterologous nucleic acid molecule or expression cassette of the present invention, which confers enhanced insect resistance to the transgenic plant when transcribed or translated. In some aspects of these embodiments, the heterologous nucleic acid molecule or expression cassette comprises a nucleotide sequence that is 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: 18-40. In other embodiments, the transgenic plant is a dicot or a monocot. In another aspect, the transgenic plant is alfalfa, 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, Lemon, lime, loblolly pine, mango, melon, mushroom, nut, okra, onion, orange, ornamental, papaya, parsley, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, red chicory, radish, raspberry, rice, rye, sorghum, southern pine, soybean, spinach, squash, strawberry, beet, sunflower, sweet potato, liquidambar, citrus, tea, tobacco, tomato, turf, vine, watermelon, potato or green squash. In still other aspects, the transgenic plant is millet, switchgrass, maize, sorghum, wheat, oats, turf grass, pasture grass, flax, rice, sugarcane, rapeseed or barley.

[0167] In some non-limiting embodiments, the present invention encompasses nucleic acid molecules encoding the insecticidal proteins of the present invention that are modified and optimized for expression in transgenic plants. Although some genes from microbial organisms can be expressed at high levels in plants without modification, low expression in transgenic plants may be due to microbial nucleic acids having 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 in the present invention can be changed to conform to the preferences of plants while maintaining the amino acids encoded thereby, or making certain amino acid changes to the encoded insecticidal proteins. In addition, high expression in plants is best achieved by 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 be poorly expressed in plants due to the presence of ATTTA motifs, which can destabilize the message, and AATAAA motifs, which can cause inappropriate polyadenylation. In embodiments, the sequences may be modified to cater to 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. 17:477-498 (1989)). In addition, the nucleic acids are screened for the presence of irrational splice sites that may cause message shortening. All desired changes within the nucleic acids (such as those described above) may be altered using well-known site-directed mutagenesis, PCR, and synthetic gene construction, for example, using the methods described in published patent applications EP 0385962, EP 0 359472, and WO 93 / 07278.

[0168] In some embodiments of the present invention, the coding sequence of the insecticidal protein of the present invention is made according to the program disclosed in U.S. Patent No. 5,625,136 (incorporated herein by reference). In this program, the preferred codons of maize are used, i.e., the single codons that most frequently encode the amino acids in maize. The preferred codons of maize for specific amino acids can be derived from (for example) known gene sequences from maize. The maize codons for 28 genes from maize plants are found in Murray et al., Nucleic Acids Research [nucleic acid sequences] 17: 477-498 (1989), and its disclosure is incorporated herein by reference. In this way, these nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence can be optimized or synthetic. That is, synthetic or partially optimized sequences can also be used.

[0169] For more effective translation initiation, sequences adjacent to the start methionine can be modified. For example, they can be modified by comprising sequences known to be 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 acids of the present invention. In an embodiment, these sequences are incorporated into the construct comprising the nucleic acid, reaching and including ATG (and not modifying the second amino acid), or alternatively reaching and including GTC after ATG (with the possibility of modifying the second amino acid of the transgenic).

[0170] In transgenic plants, the expression of these nucleic acids is driven by promoters that work in plants. The selection of promoters will vary according to the time and space requirements of 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 (e.g., 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 select monocot promoters for expression in monocots. However, there is no restriction on the origin of the selected promoter; it is sufficient as long as they can effectively drive nucleic acid to be expressed in the desired cell.

[0171] In some non-limiting embodiments, promoters for constitutive expression have been used, including actin or ubiquitin or cmp promoters, or CaMV35S and 19S promoters. Nucleic acid of the present invention can also be expressed under the regulation of promoters regulated by chemical methods. The preferred technology for chemical induction of genetic expression is described in detail in open application EP 0 332 104 (Ciba-Geigy) and United States Patent (USP) 5,614,395. The preferred promoter for chemical induction is the tobacco PR-1a promoter.

[0172] In other embodiments, a class of wound-inducible promoters may be used. Numerous promoters have been described that express at the site of wounding and also at the site of infection by plant pathogens. Ideally, such promoters should be locally active only at the site of infection, and in this way, the insecticidal proteins of the invention accumulate only in cells that need to synthesize these proteins to kill 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).

[0173] Tissue-specific or tissue-preferred promoters for expressing genes encoding 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.

[0174] In addition, promoters that function in plastids can be used. Non-limiting examples of such promoters include the phage T3 gene 9 5'UTR and other promoters disclosed in U.S. Pat. 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).

[0175] In some embodiments of the present invention, inducible promoters can be used. Thus, for example, chemically regulated promoters can be used to regulate the expression of the nucleotide sequence of the present invention by applying exogenous chemical regulators. The expression of the nucleotide sequence of the present invention is regulated by a chemically regulated promoter so that the polypeptide of the present invention can be synthesized only when the crop plants are treated with inducing chemicals. Depending on the purpose, when the expression of the nucleotide sequence of the present invention is induced by the application of chemicals, the promoter can be a chemically inducible promoter, or when the application of chemicals suppresses the expression of the nucleotide sequence of the present invention, the promoter can be a chemically repressible promoter.

[0176] Chemically inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter (which is activated by benzenesulfonamide herbicide safeners), the maize GST promoter (which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides), and the tobacco PR-1a promoter (which is activated by salicylic acid) (e.g., the PR1a system), steroid-responsive promoters (see, e.g., Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant Genetics, Inc., 1996). J. [Plant Journal] 14, 247-257) as well as tetracycline-inducible promoters and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. [Molecular Genetics] 227, 229-237 and U.S. Pat. Nos. 5,814,618 and 5,789,156), Lac repressor system promoters, copper-inducible system promoters, salicylic acid-inducible system promoters (e.g., PR1a system), glucocorticoid-inducible promoters (Aoyama et al. (1997) Plant J. [Plant Journal] 11: 605-612) and ecdysone-inducible system promoters.

[0177] Other non-limiting examples of inducible promoters include ABA-inducible and cell swelling-inducible promoters, the auxin binding protein gene promoter (Schwob et al. (1993) Plant J. 4:423-432), the UDP-glucose flavonoid glycosyltransferase promoter (Ralston et al. (1988) Genetics 119:185-197), the MPI proteinase inhibitor promoter (Cordero et al. (1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphate dehydrogenase promoter (Kohler et al. (1995) Plant J. 10:114-115). Mol. Biol. [Plant Molecular Biology] 29: 1293-1298; Martinez et al. (1989) J. Mol. Biol. [Journal of Molecular Biology] 208: 551-565; and Quigley et al. (1989) J. Mol. Evol. [Journal of Molecular Evolution] 29: 412-421). Also included are benzenesulfonamide-inducible (U.S. Pat. No. 5,364,780) and ethanol-inducible (International Patent Application Publication Nos. WO 97 / 06269 and WO 97 / 06268) systems and glutathione S-transferase promoters. Likewise, any of the inducible promoters described in Gatz (1996) Current Opinion Biotechnol. 7: 168-172 and Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 89-108 may be used. Other chemically inducible promoters suitable for directing expression of the nucleotide sequences of the present invention in plants are disclosed in U.S. Pat. No. 5,614,395, which is incorporated herein by reference in its entirety. Chemical induction of gene expression is also described in detail in published application EP 0 332 104 (granted to Ciba-Geigy) and U.S. Pat. No. 5,614,395. In some embodiments, the promoter for chemical induction may be the tobacco PR-1a promoter.

[0178] In further embodiments, the nucleotide sequences of the invention may be operably associated with a promoter that is inducible by wounding or by infestation with a pest or pathogen (e.g., an insect or nematode plant pest). Numerous promoters have been described that express at sites of wounding and / or at sites of pest attack (e.g., insect / nematode feeding) or plant pathogen infestation. Ideally, such promoters should be locally active only at or adjacent to the site of attack, and in this way, expression of the nucleotide sequences of the invention will be concentrated in the cells invaded or fed. Such promoters include, but are not limited to, those described by Stanford et al., Mol. Gen. Genet. [Molecular and General Genetics] 215:200-208 (1989); Xu et al., Plant Molec. Biol. [Plant Molecular Biology] 22:573-588 (1993); Logemann et al., Plant Cell [Plant Cell] 1:151-158 (1989); Rohrmeier and Lehle, Plant Molec. Biol. [Plant Molecular Biology] 22:783-792 (1993); Firek ...1:151-158 (1993); Firek et al., Plant Cell [Plant Cell] 1:151-158 (1989); Rohrmeier and Lehle, Plant Molec. Biol. [Plant Molecular Biology] 1:151-158 (1993); Firek et al., Plant Cell [Plant Cell] 1:151-158 (1993); 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.

[0179] In some embodiments of the present invention, a "minimal promoter" or "basic promoter" is used. Minimal promoters are capable of recruiting and binding RNA polymerase II complexes and their accessory proteins to allow transcription initiation and extension. In some embodiments, minimal promoters are constructed to contain only nucleotides / nucleotide sequences of selected promoters necessary for the binding of transcription factors and the transcription of target nucleotide sequences, and this target nucleotide sequence is operably associated with a minimal promoter including but not limited to a TATA box sequence. In other embodiments, minimal promoters lack cis sequences that recruit and bind transcription factors, and these transcription factors regulate (e.g., enhance, repress, confer tissue specificity, confer induction or repression) transcription. Minimal promoters are usually placed upstream (i.e., 5') of the nucleotide sequence to be expressed. Therefore, nucleotides / nucleotide sequences from any promoter available with the present invention can be selected to be used as minimal promoters.

[0180] Many 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).

[0181] The targeted expression of nucleic acid of the present invention in plants for different cell localizations may be more preferred. In some cases, the localization in the cytosol may be desirable, and in other cases, the localization in a certain subcellular organelle may be preferred. The subcellular localization of the transgenic of the encoding enzyme is carried out using technology well known in the art. Typically, the DNA encoding the target peptide from the gene product of the known organelle targeting is operated and fused to the upstream of the nucleic acid. Many such target sequences for chloroplasts are known and have been demonstrated to function in heterologous constructs. The expression of nucleic acid of the present invention is also targeted to the endoplasmic reticulum or vacuole of the host cell. The technology to realize it is well known in the art.

[0182] Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are suitable, and for direct gene transfer, any vector is suitable, and only the linear DNA containing the purpose construct may be preferred. In the case of direct gene transfer, conversion or co-transformation with a single DNA species can be used (Schocher et al., Biotechnology [biotechnology] 4: 1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transformation, conversion is usually (but not necessarily) carried out with a selective marker, which can provide resistance to antibiotics (kanamycin, hygromycin or methotrexate) or herbicides (basta). The plant transformation vector comprising the nucleic acid molecule of the present invention may also include the following genes (e.g., phosphomannose isomerase; PMI), which provide positive selection of transgenic plants, as disclosed in U.S. Patents 5,767,378 and 5,994,629 (incorporated herein by reference). However, the selection of selective markers is not essential for the present invention.

[0183] In some embodiments, 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 are usually able to 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 involves, for example, using biolistic or protoplast transformation (e.g., calcium chloride or PEG-mediated transformation), the cloned plastid DNA region flanking the selective marker is introduced into a suitable target tissue together with the target gene. 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, JM, and Maliga, P. (1992) Plant Cell [Plant Cell] 4, 39-45). This produces stable homoplasmic transformants at a frequency of about 1 per 100 target leaf bombardments. The presence of a cloning site between these markers allows the establishment of plastid targeting vectors for the introduction of foreign genes (Staub, JM and Maliga, P. (1993) EMBO J. [European Journal of Molecular Biology] 12, 601-606). A substantial increase in transformation frequency was obtained 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 [Proceedings of the National Academy of Sciences of the United States of America] 90, 913-917). Previously, this marker has been successfully used for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. [Nucleic Acids Research] 19: 4083-4089).Other selective markers useful for plastid transformation are known in the art and are encompassed 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 all of the thousands of copies of the circular plastid genome 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 proteins. 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 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.

[0184] In other embodiments, the transgenic plant of the present invention may comprise a heterologous nucleic acid molecule encoding at least one other desired proterties. Other proterties may be encoded on a heterologous nucleic acid molecule identical to the nucleic acid molecule of the present invention, or may be encoded on a second heterologous nucleic acid molecule. Other 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 an industrial process, or the reproductive capacity of a change. Other desired proterties may also induce commercially valuable enzymes or metabolites to produce in plants.

[0185] In some embodiments, the desired additional trait is a second pesticide.The second pesticide can 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., N. virescens or N. cincticeps (green leafhopper), or N. nigropictus (rice leafhopper); spp.) (e.g., B.leucopterusleucopterus (chinch bug)); Scotinophora spp. (e.g., S.vermidulate, rice blackbug); Acrosternum spp. (e.g., A.hilare (green stink bug); Parnara spp. (e.g., P.guttata, rice skipper); Chilo spp. (e.g., C.suppressalis (rice striped stem borer), C.auricilius (gold-fringed stem borer); borer), or C. polychrysus (dark-headed stem 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), Spodoptera exigua, beet armyworm, S. littoralis, climbing cutworm or S. praefica, western yellow striped armyworm); Mythimna species (e.g. Mythmnaseperata, Pseudaletia seperata); Spodoptera species (e.g. corn earworm (corn ear moth)); Colaspis species (e.g. C. brunnea, grape colaspis); Lissorhoptrus 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); Rootworm species (e.g., O. nubilalis (Western corn rootworm), O. barnetta (Northern corn rootworm), O. eleven-spotted (Southern corn rootworm), O. mexicana (Mexican corn rootworm); Cucumis sativus (banded cucumber beetle); Ostrinia spp. (e.g., O. nubilalis, European corn borer); Spodoptera species (e.g., Agrotissima (black cutworm); Elasmopalpus spp. (e.g., E. lignosellus (lesser cornstalk borer); Cyclocephalus spp. (wireworm); Cyclocephala spp. (e.g., C. borealis, northern masked rhinoceros beetle); chafer or southern masked chafer (C. immaculata); Popillia spp. (e.g., Japanese beetle (P. japonica); Chaetocnema spp. (e.g., corn flea beetle (C. pulicaria); Sphenophorus spp. (e.g., maize billbug (S. maidis); Rhopalosiphum spp. (e.g., corn aphid (R.maidis (corn leaf aphid); Anuraphis spp. (e.g., A. maidiridicis, corn root aphid); Melanoplus spp. (e.g., M. femurrubrum (redlegged grasshopper), M. differentialis (differential grasshopper), or M. sanguinipes (migratory grasshopper); Hylemya spp. (e.g., H. platura, seed corn maggot); Anaphothrips spp. (e.g., A. obscrurus, grass thrips); Solenopsis spp. (e.g., S. milesta, thief ant); ant); Tetranychus spp. (e.g., twospotted spider mite (T. urticae), carmine spider mite (T. cinnabarinus); Spodoptera spp. (e.g., corn earworm (cotton bollworm), or H. armigera (American bollworm); Pectinophora spp. (e.g., pink bollworm (P. gossypiella); Earias spp. (e.g., spotted bollworm (E. vittella); Heliothis spp. (e.g., tobacco budworm (H. virescens) budworm); Asterix spp. (e.g. A. grandis (boll weevil); Pseudatomoscelis spp. (e.g. P. seriatus (cotton fleahopper); Trialeurodes spp. (e.g. T. abutiloneus (banded-winged whitefly), T.vaporariorum (greenhouse whitefly); Bemisia spp. (e.g., B. argentifolii (silverleaf whitefly); Aphis spp. (e.g., A. gossypii (cotton aphid); Lygus spp. (e.g., L. lineolaris (tarnished plant bug) or L. hesperus (western tarnished plant bug); Euschistus spp. (e.g., E. conspersus (consperse spp. (e.g., F. fusca (tobacco thrips) or F. occidentalis (western flower thrips); Leptinotarsa ​​spp. (e.g., L. decemlineata (Colorado potato beetle)); beetle), L. juncta (false potato beetle), or L. texana (Texan false potato beetle); Lema species (e.g. three-lined potato beetle); Epitrix spp. (e.g. potato flea beetle), E. hirtipennis (flea beetle), or tuber flea beetle (E.tuberis, tuberflea beetle); Eryngium spp. (e.g., E. vittata, striped blister beetle); Phaedon spp. (e.g., P. cochleariae (mustard leaf beetle); Epilachna spp. (e.g., E. varivetis (Mexican bean beetle); Acheta spp. (e.g., A. domesticus (house cricket); Empoasca spp. (e.g., E. fabae (potato leafhopper); Myzus spp. spp.) (e.g., M. persicae (green peach aphid)); Paratrioza 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 spp. (e.g., T. Pallidovirens (red shouldered stink bug)); stinkbug); P. operculella (e.g., potato tuberworm); Spodoptera species (e.g., tomato fruitworm); P. spp. (e.g., tomato pinworm); Limonius spp. (wireworms); Manduca spp., such as M. sexta (tobacco hornworm) and M.quinquemaculata, tomato hornworm; Liriomyza spp. (e.g., L. sativae, L. trifolli, or L. huidobrensis (leaf miner)); Drosophilla spp. (e.g., Drosophila melanogaster, D. yakuba, D. Pseudoobscura, or D. simulans); Carabus spp. (e.g., C. granulatus); Chironomus spp. (e.g., C. tentanus); Ctenocephalus spp. (e.g., C. felis, cat flea); Diaprepes spp. (e.g., D. abbreviation, root weevil); weevil); Ips spp. (e.g. I. pini (pine engraver); Tribolium spp. (e.g. T. innotata, red floor beetle); Glossina spp. (e.g. G. Morsitans, tsetse fly); Anopheles spp. (e.g. A. gambiae (malaria mosquito)); Helicoverpa spp. (e.g. H. armigera (African Bollworm); Acyrthosiphon spp. (e.g. A. pisum, pea aphid); Apis spp. (e.g. A. melifera (honey bee)); Homalodisca spp. 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 (African migratory locust)).migratoria (migratory locust); Boophilus spp. (e.g., B. microplus (cattle tick); Acanthoscurria spp. (e.g., A. Gomesiana (red-haired chololate bird 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 butterfly); weevil); Plutella spp. (e.g., P. xylostella, diamondback moth); Amblyomma spp. (e.g., A. variegatum, cattle tick); Anteraea spp. (e.g., A. Yamamai (silkmoth); and Armige spp. (e.g., A. subalbatus).

[0186] The SMIP toxins of the present invention can be used in combination with other insecticides to increase the target range of pests. In addition, the SMIP toxins of the present invention are used in combination with a second insecticide (which has a different mode of action or targets a different receptor in the insect gut) for prevention and / or management of insect resistance. In some embodiments, the SMIP toxins of the present invention are combined with a second insecticide protein selected from the group consisting of Cry1A, Cry1Ab, Cry1Ac, Cry1A.105, Cry1Ae, Cry1B, Cry1C, Cry1C variants, Cry1D, Cry1E, Cry1F, Cry1A / F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B, Cry3C, Cry3D, Cry3E, Cry1F, Cry1A / F chimeras, Cry1G, Cry1H, Cry1I, Cry1J, Cry1K, Cry1L, Cry2A, Cry2Ab, Cry2Ae, Cry3, Cry3A variants, Cry3B ...3F, Cry3A variants, Cry3B, Cry3D, Cry3E, Cry3F, Cry3A variants, Cry3B, Cry3B, Cry3E, Cry3F, Cry3A variants, Cry3B, Cry3B, Cry 4B, Cry6, Cry7, Cry8, Cry9, Cry15, Cry34, Cry35, Cry43A, Cry43B, Cry46A, Cry51Aa1, PtIP-96, PtIP-83, PHI-4, MP467, MP81 , PS149B1, DIG-3, DIG-5, DIG-10, DIG-11, DIG-17, DIG-657, IRDIG28688.1, IRDIG28688.1, IRDIG28684.1, IRDIG28682.1, I RDIG28680.1, IRDIG28674.1, IRDIG28672.1, IRDIG27642, IRDIG28688.1, IRDIG28686.1, IRDIG28684.1, IRDIG28682.1, IR DIG28680.1, IRDIG28674.1, IRDIG28672.1, IRDIG27642, IRDIG28678.2, IRDIG28678.1, IRDIG31125.1, IRDIG28696.1, IRD IG29781.1, IRDIG29779.1, IRDIG30844.1, IRDIG30850.1, IRDIG30852.1, IRDIG30854.1, IRDIG30856.1, IRDIG30858.1, IRDIG30862.1, IRDIG30860.1, IRDIG30848.1, RETIRE2021, VIP3A, VIP3B, VIP3Ab, binary VIP1 and VIP2, or other vegetative insecticidal proteins, mCry3A, eCry3.1Ab, AXMI-001, AXMI-002, AXMI-030, AXMI-035, AXMI-036, AXMI-045, AXMI52, AXMI58, AXMI88, AXMI97, AX MI102, AXMI112, AXMI113, AXMI115, AXMI117, AXMI100, AXMI-115, AXMI-113, and AXMI-005, AXMI134, AXMI-15 0, AXMI171, AXMI-184, AXMI196, AXMI204, AXMI207, AXMI209, AXMI205, AXMI218, AXMI220, AXMI221z, AXMI222z, AXMI223z, AXMI224z and AXMI225z, AXMI238, AXMI270, AXMI279, AXMI345, AXMI-R1 and its variants, IP3 and its variants, ET29, E T33, ET34, ET35, ET66, ET70, TIC400, TIC407, TIC417, TIC431, TIC800, TIC807, TIC834, TIC836, TIC844, TIC853, TIC860 or variants thereof, TIC867 or variants thereof, TIC868 or variants thereof, TIC869, TIC900 or related proteins, TIC901, TIC1100, TIC1201, TIC1362, TIC1414, TIC1415, TIC1422, TIC1497, TIC1498, TIC1885, TIC1886, TIC1922, TIC1925, TIC1974, TIC2032, TIC2120, TIC2160, TIC3131, TIC3244, TIC6757, TIC7243, TIC7472, and TIC7473 proteins, or hybrid proteins or chimeras made from any of the foregoing insecticidal proteins. The second insecticide may also be an agent selected from the group consisting of alpha amylase, peroxidase, cholesterol oxidase, potato glycoprotein, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, engineered antibody or antibody fragment, Bacillus cereus insecticidal protein, Xenorhabdus species (such as X. nematophila or Xenorhabdus bovienii) insecticidal protein, Photorhabdus species (such as P. luminescens or P. asymobiotica) insecticidal protein, Brevibacillus species (such as B. laterosporous) insecticidal protein, Lysinibacillus spp.In some embodiments, the second agent may be an insecticidal protein derived from at least one insecticide toxin complex (Tc) from Photorhabdus, Xenorhabus, Serratia, or Yersinia. In still other embodiments, the second insecticidal protein may be a binary toxin derived from an insecticide bacterium, such as ISP1A and ISP2A from Brevibacillus laterosporus or BinA and BinB from Bacillus sphaericus. The combination of the SMIP toxin of the present invention and the second pesticide can be expressed in a transgenic plant by expression of the two proteins. In some embodiments, the transgenic plant is a transgenic corn plant. In other embodiments, the combination in the transgenic corn plant is a SMIP toxin of the present invention and mCry3A and / or eCry3.1Ab and / or Cry3Bb1 and / or Cry34 / Cry35. .

[0187] In some embodiments, the transgenic plants of the present invention may include at least one non-protein second pesticidal agent. In a preferred embodiment, the second pesticidal agent is an interfering RNA molecule. Interfering RNA molecules typically include at least one RNA fragment for a target gene, a spacer sequence, and a second RNA fragment complementary to the first RNA fragment, so that a double-stranded RNA structure can be formed. 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 about 19-24 nucleotides in length, which is referred to as small interfering RNA (siRNA). These siRNAs are then diffused or 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), and the effector strand (or "guide strand") of RNA is located in the complex. This guide strand serves as a recognition and destruction template for duplex sequences. 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 specific genes transcribed from the mRNA. It is known in the art that interfering RNA can be used for insect control (see, for example, publication WO 2013 / 192256, which is incorporated herein by reference). Interfering RNA designed for insect control produces non-naturally occurring 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 result in death of insect pests. Interfering RNA molecules can confer insect resistance to the same target pests as the proteins of the present invention, or different pests can be targeted. Target insect plant pests can be fed by chewing, sucking or piercing. It is known in the art that interfering RNA can be used for insect control. In other embodiments, interfering RNA can confer resistance to non-insect plant pests (such as nematode pests or viral pests).

[0188] Co-expression of more than one pesticide in the same transgenic plant can be achieved by making a single recombinant vector (containing the coding sequence of more than one pesticide in a so-called molecular stack) and genetically engineering the plant so that all the pesticides are contained and expressed in the transgenic plant. Such molecular stacks can also be made by using mini-chromosomes, as described in (for example) U.S. Patent 7,235,716. Alternatively, a transgenic plant containing a nucleic acid encoding a first pesticide can be re-transformed with a different nucleic acid encoding a second pesticidal agent, 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, a progeny plant expressing all the genes introduced into parent 1 and parent 2 is obtained.

[0189] Transgenic plants or seeds comprising the insecticidal proteins of the invention may also be treated with an insecticide or insecticidal seed coating as described in U.S. Pat. Nos. 5,849,320 and 5,876,739, incorporated herein by reference. 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 (i) is useful in methods for further enhancing the activity of the composition of the invention against the target insect and (ii) is useful 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, the invention provides a method for enhancing control of a rootworm insect population 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.

[0190] Even where the insecticidal seed coating is active against different insects, the insecticidal seed coating is useful for expanding the spectrum of insect control, for example by adding an insecticidal seed coating active against Lepidoptera insects to transgenic seeds of the invention (active against Coleoptera and some Lepidoptera in some embodiments), the resulting coated transgenic seeds control both Lepidoptera and Coleoptera pests.

[0191] Examples of such insecticides and / or insecticide seed coatings include, but are not limited to, carbamates, pyrethroids, organophosphates, friprole, neonicotinoids, organochlorides, nereis toxins, 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, chloroxyfos, dimethoate, ethoprophos, malathion, methyl parathion, phorate, terbufos, tert-butyl pyrimidine phosphorus (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), (aminonaphthalene), (Bifenthrin), (Tefluthrin), (Cypermethrin), (Cypermethrin), Delta (Deltamethrin), (λ-cyhalothrin), (Permethrin), (Permethrin), (Bifenthrin), (Bifenthrin), (tefluthrin)), (λ-cyhalothrin), (Chlorpyrifos), (Chlorophene Oxygen), (Methoate), (Phorate), (Phorate, flucythinate), (Phorate), (terbufos), (Dimethoate), isochlorophos, (fipronil)), (Thiamethoxam), (Imidacloprid), (Imidacloprid), (thiamethoxam) and (Cyfluthrin, Cypermethrin).

[0192] In some embodiments, the present invention also encompasses a composition comprising an insecticidal protein of the present invention in an effective amount for controlling insects. In further embodiments, the composition comprises a suitable agricultural carrier and an insecticidal protein of the present invention. The agricultural carrier may include adjuvants, mixtures, enhancers, etc. that are beneficial to the application of active ingredients (such as proteins of the present invention, including proteins comprising, consisting essentially of, or consisting of: an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to any one of SEQ ID NO: 1-17). Suitable carriers should not be phytotoxic to valuable crops (especially 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., polypeptides of the present invention or other composition ingredients). 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 before 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 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, isopropanol, 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 other embodiments, the proteins 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 for delivery of the insect control agent to the insect and results in toxic effects to the insect pest.

[0193] In other embodiments, the composition of the present invention can be a powder, dust, pill, granule, spray, emulsion, colloid or solution. The composition of the present invention can be prepared by dehydrating, freeze-drying, homogenizing, extracting, filtering, centrifuging, settling or concentrating a culture of bacterial cells. The composition of the present invention can contain at least 1%, about 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. The composition of the present invention can contain at least a second pesticide (which can be insecticidal, nematicidal, fungicidal, bactericidal). At least the second pesticide can have an insecticidal effect on the same insect as the polypeptide of the present invention or a different insect. The second pesticide can be a polypeptide. The pesticide can be an interfering RNA. The second pesticide can be a microorganism (such as bacteria) comprising a nucleic acid molecule encoding a pesticide and / or comprising a pesticide (such as a polypeptide or interfering RNA). The microorganism can be attenuated, heat-inactivated or freeze-dried. The microorganism may die or be 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, dimethoate, ethoxyproline, malathion, methyl parathion, phorate, terbufos, tert-butyl pyrimidine phosphorus (tebupirimiphos), fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamic acid, and a combination thereof, or a commercial product containing an insecticide and an insecticide seed coating as described above.

[0194] The composition of the present invention (e.g., a composition comprising a protein 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 a composition comprising a polypeptide of the present invention to a plant or seed. 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 by any means, such as an aircraft spray barrel, irrigation equipment, direct injection spray equipment, backpack spray barrel, livestock dip tank, farm equipment used in ground spraying (e.g., spray tube sprayer, hand sprayer), etc. The desired place can be soil, plant, etc.

[0195] The composition 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 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 seeds or plant propagules using conventional coating techniques and machines (such as fluidized bed technology, drum grinding method, static rotation (rotostatic) seed processor and drum coater).

[0196] In some embodiments, the present invention also includes a method of controlling a population of coleopteran pests, the method comprising contacting the pest population with an insect control effective amount of an insecticidal protein of the present invention, wherein the protein comprises, consists essentially of, or consists of an amino acid sequence that is 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: 1-17. Contacting includes ingestion or ingestion of the insecticidal protein by members of the pest population. The insecticidal protein may be incorporated into an insect dietary food, or may be expressed or present in plant tissues that are then ingested by the insect population. In further embodiments, controlling a population of coleopteran pests comprises killing the insects by contacting the insects with an insect control effective amount of an insecticidal protein of the present invention.

[0197] The present invention further comprises a method for increasing plant yield, the method comprising 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.

[0198] 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.

[0199] In some embodiments, the present invention encompasses a method of providing a corn grower with a means of controlling a rootworm pest population in a corn crop, the method comprising (a) selling or providing to the grower a transgenic corn seed comprising a nucleic acid molecule, expression cassette, vector or chimeric gene of the present invention; and (b) advertising to the grower that the transgenic corn seed produces a transgenic corn plant that controls a rootworm pest population.

[0200] In some embodiments, the present invention also encompasses a method of identifying an insecticidal protein comprising, consisting essentially of, or consisting of a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to any one of SEQ ID NOs: 1-17, or a toxin fragment thereof, the method comprising the steps of: (a) generating a primer pair that will amplify the nucleotide sequence of SEQ ID NOs: 1-17 from a nucleic acid sample or a complementary sequence thereof; NO:18-40, (b) amplifying the orthologous polynucleotide from the nucleic acid sample, (c) identifying the nucleotide sequence of the orthologous polynucleotide, (d) producing a protein encoded by the orthologous polynucleotide, and (e) determining that the protein of step (d) has insecticidal activity against insect pests.

[0201] Examples

[0202] Embodiments of the present invention can be better understood by reference to the following examples. The aforementioned and following embodiments of the present invention and the description of various embodiments are not intended to limit the claims, but are illustrative thereof. Therefore, it should be understood that the claims are not intended to be limited to the specific details of these examples. It should be understood by those skilled in the art that other embodiments of the present invention can be practiced without departing from the spirit and scope of the present disclosure, and the scope of the present disclosure is limited by the appended claims. Art-recognized recombinant DNA and molecular cloning techniques can be found, for example, in 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, FM 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 Co., Ltd. Press [World Scientific Press] (1992), and Schultz et al., Plant Molecular Biology Manual [Plant Molecular Biology Manual], Kluwer Academic Publishers [Kluwer Academic Publishers] (1998).

[0203] Example 1: Identification of insecticidal proteins.

[0204] Based on a proprietary algorithm, candidate nucleotide sequences encoding proteins described in the art as hypothetical proteins were identified in the genomes of Gram-negative bacteria belonging to the Actinomycetes family. Two candidate sequences were selected for expression and testing against insect pests. Two candidate nucleotide sequences were identified in the genomes of Streptomyces species strain F-5053 (SEQ ID NO: 18) and Streptomyces species strain 8K308 (SEQ ID NO: 19). Surprisingly, it was found that a protein comprising the amino acid sequence of SEQ ID NO: 1 encoded by SEQ ID NO: 18 and a protein comprising the amino acid sequence of SEQ ID NO: 17 encoded by SEQ ID NO: 19 had insecticidal activity against western corn rootworm (WCR; corn rootworm). Produced the codon optimized version of F-5053 and 8K308 nucleotide sequence of Escherichia coli, respectively SEQ ID NO:20 and SEQ ID NO:36, and each was introduced into pET29a bacterial expression vector to produce the albumen called pET-Strept5053 and pET-Strept8K308 respectively.Construct was transformed into Escherichia coli BL21*(DE3) and lysate was made from the culture (producing protein overnight at about 18 ℃) induced by isopropyl β-D-1-thiogalactopyranoside (IPTG).In the feed incorporation bioassay experiment, the insecticidal activity of lysate to WCR was detected.In brief, the Escherichia coli lysate was mixed with the artificial insect feed (Bioserv company, Frenchtown, New Jersey (NJ)) of equal volume of heating in 1.5mL centrifuge tube, and then applied to a small culture dish. After the diet-sample mixture cooled and solidified, 12 WCR larvae were added to each plate. The plates were sealed and maintained under ambient laboratory conditions with respect to temperature, light, and relative humidity. Buffer without lysate, lysate from E. coli BL21*(DE3) culture (containing an empty pET29a vector), and artificial insect diet alone served as negative controls. The results are shown in Table 1. Percent mortality and growth inhibition observations were obtained at 4 and 7 days post-infection, where s=small larvae, m=medium larvae, and l=large larvae.

[0205] Table 1: Insecticidal activity of Streptomyces proteins against WCR.

[0206]

[0207] As shown in Table 1, lysates from E. coli cultures expressing Streptomyces species F-5053 protein (SEQ ID NO: 1) or Streptomyces species 8K308 protein (SEQ ID NO: 17) have insecticidal effects on western corn rootworm, Diabrotica zeae. The Streptomyces species F-5053 insecticidal protein is designated Strept5053CRW or SMIP1Aa, and the Streptomyces species 8K308 protein is designated Strept8K308CRW or SMIP2Aa. SMIP1Aa has 118 amino acids, is 12.8 kDa, and includes an aegerolysin-like pore-forming domain from about amino acid position 20 to about amino acid position 82 of SEQ ID NO: 1. SMIP2Aa has 150 amino acids, is 16.2 kDa, and includes an aegerolysin-like pore-forming domain from about amino acid position 52 to about amino acid position 114 of SEQ ID NO: 17. Compared to SMIP1Aa, the SMIP2Aa protein appears to have an N-terminal peptide extension of 32 amino acids (amino acids 1-32 of SEQ ID NO: 17). The apparent core toxin of the SMIP2Aa protein, i.e., a fragment comprising amino acids 33-150 of SEQ ID NO: 17, has 71% identity with SEQ ID NO: 1 as shown in the alignment in Table 2, wherein "." indicates the same amino acid at the aligned position.

[0208] Table 2. Alignment of SMIP1Aa and SMIP2Aa.

[0209]

[0210] Example 2. Fate of SMIPs in simulated gastric fluid assay

[0211] Experimental results testing SMIP1Aa and SMIP2Aa in a simulated gastric fluid (SGF) assay showed that the time for complete digestion of the natural insecticidal protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 17 was delayed, with undigested bands remaining for a period of approximately 30 minutes. The SGF assay is used to estimate the digestibility of proteins in the mammalian intestine and is a standard composition for evaluating any new insecticidal protein for regulatory approval. Therefore, SMIP1Aa mutants were prepared and then tested in the SGF assay to determine their digestibility.

[0212] Mutants were generated by making single or double amino acid substitutions in the native SMIP1Aa sequence (SEQ ID NO: 1). The mutants made included the following: A71 residue was changed to L (SMIP1Aa-A71L; SEQ ID NO:2), A90 residue was changed to L (SMIP1Aa-A90L; SEQ ID NO:3), C92 residue was changed to L or S (SMIP1Aa-C92L; SEQ ID NO:4 and SMIP1Aa-C92S; SEQ ID NO:5), G84 residue was changed to L (SMIP1Aa-G84L; SEQ ID NO:6), G85 residue was changed to L (SMIP1Aa-G85L; SEQ ID NO:7), V76 residue was changed to L (SMIP1Aa-V76L; SEQ ID NO:8), W78 residue was changed to F (SMIP1Aa-W78F; SEQ ID NO:9), and both A71 and A90 residues were changed to L (SMIP1Aa-A71L / A90L; SEQ ID NO:10). NO:10), A71 and W78 residues were changed to L and F, respectively (SMIP1Aa-A71L / W78F; SEQ ID NO:11), W78 and C92 residues were changed to F and L, respectively (SMIP1Aa-W78F / C92L; SEQ ID NO:12), Y82 and I83 residues were changed to F and L, respectively (SMIP1Aa-Y82F / I83L; SEQ ID NO:13), and A2 and A3 residues were both changed to G (SMIP1Aa-A2G / A3G; SEQ ID NO:14) or V (SMIP1Aa-A2V / A3V; SEQ ID NO:15).

[0213] The simulated gastric fluid (SGF) assay measures the in vitro digestibility of a test protein under strictly controlled conditions representative of the mammalian upper digestive tract. Briefly, bacterially produced test SMIP1Aa proteins and mutants (at a concentration of 0.5-5 mg / ml) were exposed to pepsin (from porcine gastric mucosa, dissolved in 2 mg / ml NaCl, pH 1.2) at a rate of 10 units of pepsin activity / μg test protein at 37°C over a one hour period. Samples were removed at 1, 2, 5, 10, 30 and 60 minutes and immediately quenched by adding preheated (95°C - 2 minutes) stop buffer (65% 0.5 M sodium bicarbonate (pH 11), 35% Tricine loading buffer) to immediately inactivate pepsin and returned to heating for an additional 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 visualized down to 1 kDa) to follow the kinetics and level of digestion by pepsin.

[0214] The results of the SGF assay showed that the mutants SMIP1Aa-A71L / A90L (SEQ ID NO: 10) and SMIP1Aa-W78F / C92L (SEQ ID NO: 12) were degraded very rapidly compared to the wild-type SMIP1Aa protein. Although the mutants SMIP1Aa-A71L (SEQ ID NO: 2), SMIP1Aa-A90L (SEQ ID NO: 3), SMIP1Aa-C92S (SEQ ID NO: 5) and SMIP1Aa-W78F (SEQ ID NO: 9) were not completely degraded, they did show a significant improvement in digestibility compared to the wild-type SMIP1Aa protein. The digestibility of mutants SMIP1Aa-C92L (SEQ ID NO:4), SMIP1Aa-G85L (SEQ ID NO:7), SMIP1Aa-V76L (SEQ ID NO:8), and SMIP1Aa-Y82F I83L (SEQ ID NO:13) was no different from that of the wild-type SMIP1Aa protein under these SGF conditions.

[0215] These results provide evidence that the Streptomyces insecticidal proteins (SMIPs) of the invention can be mutated to improve digestibility in a standard SGF assay.

[0216] Example 3. Solubility and insecticidal activity of SMIP variants against WCR.

[0217] The above SMIP1Aa mutants were tested for solubility and insecticidal activity against western corn rootworm (WCR) in a diet incorporation bioassay experiment similar to that described in Example 1. The results of the bioassay are shown in Tables 3 to 6. Percent mortality and growth inhibition observations were taken at 3, 4 and / or 6 days after infestation, where s = small larvae, m = medium larvae, and l = large larvae. All mutants were highly soluble, except SMIP1Aa-A71L / W78F (SEQ ID NO: 11), which had very low solubility.

[0218] Table 3. Insecticidal activity of SMIP1Aa mutants against WCR.

[0219]

[0220] Table 4. Insecticidal activity of SMIP1Aa-A71L mutant against WCR.

[0221]

[0222]

[0223] Table 5. Insecticidal activity of SMIP1Aa-A2G / A3G mutants against WCR.

[0224]

[0225] Table 6. Insecticidal activity of SMIP1Aa--W78F / C92L and -A71L / A90L mutants against WCR.

[0226]

[0227]

[0228] Example 4. Insecticidal activity of SMIP1Aa against Cry-resistant WCR.

[0229] To determine whether the toxicity of SMIPs is through a mode of action different from that of Cry3-related proteins, SUMO-tagged SMIP1Aa and SIMP1Aa-W78F / C92L variants were purified as described in Example 2 and tested for their efficacy against WCR strains resistant to modified Cry3A (mCry3A) toxins (mCry3A-R) and / or against WCR strains resistant to eCry3.1Ab toxins (eCry3.1Ab-R). Feed incorporation assays were performed essentially as described in Example 2, and mortality and growth inhibition observations were obtained at 4 and / or 6 days after infection, where s = small larvae, m = medium larvae, and l = large larvae. SMIP1Aa and SMIP1Aa-W78F / C92L were tested at different concentrations. The negative control was 1xPBS. A WCR strain that is not resistant to mCry3A or eCry3.1Ab, i.e., wild type, was used as a positive control in the SMIP1Aa assay. As shown in Tables 7 and 8, SMIPs have insecticidal activity against Cry-resistant WCR strains, indicating that these proteins have a unique mode of action compared to Cry proteins from Bacillus thuringiensis. Therefore, the combination of SMIPs (e.g., SMIP1Aa or SMIP1Aa-W78F / C92L) of the present invention and Cry proteins will effectively mitigate the development of resistance to Cry proteins or SMIPs.

[0230] Table 7. Insecticidal activity of SMIP1Aa against Cry-resistant WCR

[0231]

[0232] Table 8. Insecticidal activity of SMIP1Aa-W78F / C92L variants against eCry3.1Ab-R WCR.

[0233]

[0234] Example 5. Transformation of maize with SMIP1Aa

[0235] Nucleotide sequences encoding SMIP1Aa proteins or mutants, such as any of SEQ ID NOs: 18-40, or maize optimized nucleotide sequences, such as any of SEQ ID NOs: 37-40, which can be generated as described in U.S. Pat. No. 6,051,760, are transformed into corn to control corn rootworms.

[0236] Two plant expression cassettes were constructed to introduce the SMIP1Aa-W78F / C92L (SEQ ID NO:39) or SMIP1Aa-A71L / A90L (SEQ ID NO:38) coding sequences into Zea mays. The first cassette contains the Zea mays ubiquitin 1 (Ubi1) promoter operably linked to the SMIP1Aa-W78F / C92L or SMIP1Aa-A71L / A90L coding sequence, which is operably linked to the Zea mays Ubi361 terminator. The second cassette contains the Zea mays Ubi1 promoter operably linked to the pmi coding sequence encoding the selectable marker phosphomannose isomerase (PMI), and the pmi coding sequence is operably linked to the Zea mays Ubi1 terminator. A recombinant plant transformation binary vector containing the two expression cassettes was generated for Zea mays transformation experiments.

[0237] The binary vector was transformed into Agrobacterium tumefaciens using standard molecular biology techniques. To prepare Agrobacterium for transformation, the cells were cultured overnight at 28 °C and 220 rpm in liquid YPC medium.

[0238] Agrobacterium-mediated transformation of immature Zea mays embryos was performed essentially as described in Negrotto et al., 2000 (Plant Cell Reports 19:798 - 803). For this example, all media components were essentially as described in Negrotto et al. above. However, various media components known in the art can be substituted.

[0239] Briefly, the Agrobacterium strain LBA4404 (pSB1) containing the binary vector plant transformation vector was grown on solid YEP (yeast extract (5 g / L), peptone (10 g / L), NaCl (5 g / L), 15 g / l agar, pH 6.8) medium at 28 °C for 2 - 4 days. Approximately 0.8X 10 9 Agrobacterium cells were suspended in LS-inf medium supplemented with 100 μM As (Negrotto et al., ibid.). The bacteria were pre-induced in this medium for 30 to 60 minutes.

[0240] The immature embryos from the genotype that is suitable for are excised in the liquid LS-inf+100 μM As from the big ear of 8-12 days. Rinse these embryos with fresh infection culture medium. Add agrobacterium solution then, and these embryos are vortexed for 30 seconds and allowed to settle 5 minutes with bacteria. Then these embryo scutels are transferred upwards in the LSA culture medium, and cultivated two to three days in the dark. Subsequently, embryos between 20 and 25 of every petri plate (petri plate) are transferred to the LSDc culture medium that is supplemented with cefotaxime (250mg / l) and silver nitrate (1.6mg / l), and cultivated 10 days at 28 ℃ in the dark.

[0241] The immature embryos that produce embryonic callus are transferred to the LSD1M0.5S culture medium. On this culture medium, the selection that culture continues for about 6 weeks is carried out, and there is the subculture step of about 3 weeks. The callus that survives is transferred to the Reg1 culture medium that is supplemented with mannose. After (16 hours of illumination / 8 hours of dark scheme) cultivation in illumination, then green tissue is transferred to the Reg2 culture medium that does not have growth regulator and hatches about 1-2 week. These plantlets are transferred to the Magenta GA-7 box (Magenta Corp, Chicago, Illinois) that contains Reg3 culture medium and it is grown in illumination.

[0242] After transformation, selection and regeneration, use The analysis was used to determine whether the pmi gene and SMIP1Aa-W78F / C92L (SEQ ID NO: 39) or SMIP1Aa-A71L / A90L (SEQ ID NO: 38) maize codon optimized coding sequence were present in the plants. The presence of the vector backbone was also tested in the plants. Plants that were negative for the vector backbone and contained a copy of the transgenic from the binary vector were transferred to the greenhouse and tested for insecticidal activity against WCR.

[0243] Example 6. SMIP toxins in combination with a second insecticide.

[0244] SMIP or variant SMIP as described above is purified as in Example 2. Double-stranded RNA (dsRNA) complementary to the essential target and known to have insecticidal activity is also prepared. In a non-limiting example, the dsRNA can target harmful insect genes encoding vacuolar ATP synthase, β-tubulin, 26S proteosome subunit p28 protein, EF1α48D, troponin I, transmembrane tetraprotein, clathrin heavy chain, γ-coat body, β-coat body and / or juvenile hormone epoxide hydrolase (PCT Patent Application No. PCT / US 17 / 044825; PCT / US 17 / 044831; PCT / US 17 / 044832; U.S. Patent No. 7,812,219; each incorporated herein by reference). The efficacy of dsRNA and purified protein against WCR was tested in a dietary incorporation assay performed essentially as described in Example 1.

[0245] Example 7. In situ genome editing in plant cells to generate modified SMIP toxins.

[0246] The following examples illustrate the use of genome editing of plant cell genomes in situ to incorporate mutations described herein (including but not limited to the mutations described in Example 2) into the coding sequence of native SMIPs, including SMIP1Aa (SEQ ID NO: 18), or into the coding sequence of already modified SMIP1Aa or SMIP2Aa proteins.

[0247] Directed genome modification (also referred to as genome editing) can be used to introduce mutations in a specific DNA sequence. These genome editing techniques, including zinc finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly spaced short palindromic repeats (CRISPRs), have been successfully applied to more than 50 different organisms including crop plants. See, for example, Belhaj, K. et al., Plant Methods [Plant Methods] 9, 39 (2013); Jiang, W. et al., Nucleic Acids Res [Nucleic Acids Research] 41, e188 (2013)). The CRISPR / Cas system for genome editing is based on the transient expression of a single guide RNA (sgRNA) engineered with a Cas9 nuclease and a specified target polynucleotide sequence.

[0248] Cas9 is a large monomeric DNA nuclease that is guided to a DNA target sequence by a complex of two 20-nucleotide (nt) noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), which are functionally available as a single synthetic RNA chimera. The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cuts the complementary DNA strand, while the RuvC-like domain cuts the non-complementary strand, thus introducing a blunt cut in the target DNA.

[0249] When Cas9 and sgRNA are transiently expressed in living maize cells, double-strand breaks (DSBs) in specific target DNA are generated in transgenic maize cells. Mutations at the break sites are introduced by non-homologous end joining and homology-directed DNA repair pathways.

[0250] By using a recombinant plasmid expressing Cas9 nuclease and sgRNA target (codon optimized for SMIP1Aa or mutant SMIP1Aa sequence) in transgenic maize, specific mutations (such as those described in Example 2 above) are introduced into the coding sequence of the native SMIP1Aa insecticidal protein (SEQ ID NO: 1) or mutant SMIP1Aa protein. The method is implemented by agroinfiltration of Agrobacterium tumefaciens carrying a binary plasmid containing a specified target sequence of interest. After the sgRNA binds to the target SMIP1Aa or mutant SMIP1Aa coding sequence, the Cas9 nuclease specifically cuts the coding sequence and introduces the desired mutation or mutations during the DNA repair process. Thus, the mutated SMIP1Aa coding sequence will now encode a modified SMIP1Aa variant protein, such as the variant described in Example 2, for example, wherein the mutation at position 71 replaces alanine (A) with leucine (L), or wherein the mutation at position 90 replaces alanine (A) with leucine (L), or wherein the mutation at position 92 replaces cysteine ​​(C) with leucine (L) or serine (S), or wherein the mutation at position 84 replaces glycine (G) with leucine (L), or wherein the mutation at position 85 replaces glycine (G) with leucine (L), or wherein the mutation at position 76 replaces valine (V) with leucine (L), or wherein the mutation at position 78 replaces tryptophan (W) with phenylalanine (F), or any combination thereof.

[0251] Plant cells containing the genome-edited SMIP1Aa coding sequence were screened by PCR and sequencing. Calli with genome-edited mutations in SMIP1Aa or modified SMIP1Aa coding sequences were induced to regenerate plants for phenotypic evaluation and to evaluate the insecticidal activity of the expressed SMIP1Aa protein against the following: western corn rootworm (Diplodia zeae), northern corn rootworm (Diplodia barnetta), southern corn rootworm (Diplodia elevenspotted root-feeding subspecies) and / or Mexican corn rootworm (Diplodia mexicana).

[0252] It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes based on the description thereof will suggest themselves to those skilled in the art and will be included within the spirit and scope of the present application and the scope of the appended claims.

[0253] All publications and patent applications mentioned in this specification are indicative of the technical level of those skilled in the art to which the invention pertains. All of these publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was expressly and individually indicated to be incorporated by reference. Sequence Listing <110> Syngenta Crop Protection AG REYNOLDS, Clarence Michael <120> Insecticide protein <130> 81720-WO-REG-ORG-P-1 <150> US 62 / 780,808 <151> 2018-12-17 <160> 40 <170> PatentIn version 3.5 <210> 1 <211> 118 <212> PRT <213> Streptomyces species <400> 1 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 2 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa - A71L <400> 2 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Leu Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 3 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa - A90L <400> 3 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Leu Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 4 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa-C92L <400> 4 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Leu Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 5 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa-C92S <400> 5 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Ser Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 6 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa-G84L <400> 6 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Leu Gly Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa-G85L <400> 7 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Leu Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 8 <211> 118 <212> PRT <213> Artificial sequence <220> <223> SMIP1Aa-V76L <400> 8 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Leu Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 9 <211> 118 <212> PRT <213> Artificial sequence <220> <223> SMIP1Aa-W78F <400> 9 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Phe Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 10 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa - A71L / A90L <400> 10 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Leu Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Leu Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 11 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa - A71L / W78F <400> 11 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Leu Gly Asp Phe Arg Val Asp Phe Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 12 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa-W78F / C92L <400> 12 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Phe Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Leu Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa-Y82F / I83L <400> 13 Met Ala Ala Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Phe Leu Gly Gly Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 14 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa - A2G / A3G <400> 14 Met Gly Gly Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 15 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> SMIP1Aa - A2V / A3V <400> 15 Met Val Val Arg Ser Tyr Glu Ile Ala Ile Val Asn Leu Thr Asp Val 1 5 10 15 Glu Phe Thr Arg Lys Glu Ala His Leu Asp His Gly Val Trp Ser Lys 20 25 30 Asp Gly Asn Tyr Thr Pro Pro Asp Lys Ile Ser Thr Gly Gln Thr Ala 35 40 45 His Phe Gly Ser Glu Ser Gln Gly Val Ala Thr Gly Thr Glu Gly His 50 55 60 Val Ile Tyr Ser Ser Ser Ala Gly Asp Phe Arg Val Asp Trp Asp Asn 65 70 75 80 Pro Tyr Ile Gly Gly Asp Ser Ser Ser Ala Lys Cys Pro Pro Ser Tyr 85 90 95 Glu Lys Val Leu Ser Asp Ser Lys Gly Asn Asp Ala Thr Leu Lys Val 100 105 110 Val Phe Tyr Lys Lys Ser 115 <210> 16 <211> 237 <212> PRT <213> Artificial Sequence <220> <223> SUMO - SMIP1Aa <400> 16 Met Gly Ser Ser His His His His His His Gly Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser Ala Ser Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys 20 25 30 Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys 35 40 45 Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr 50 55 60 Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu 65 70 75 80 Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp 85 90 95 Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala 100 105 110 His Arg Glu Gln Ile Gly Gly Met Ala Ala Arg Ser Tyr Glu Ile Ala 115 120 125 Ile Val Asn Leu Thr Asp Val Glu Phe Thr Arg Lys Glu Ala His Leu 130 135 140 Asp His Gly Val Trp Ser Lys Asp Gly Asn Tyr Thr Pro Pro Asp Lys 145 150 155 160 Ile Ser Thr Gly Gln Thr Ala His Phe Gly Ser Glu Ser Gln Gly Val 165 170 175 Ala Thr Gly Thr Glu Gly His Val Ile Tyr Ser Ser Ser Ala Gly Asp 180 185 190 Phe Arg Val Asp Trp Asp Asn Pro Tyr Ile Gly Gly Asp Ser Ser Ser 195 200 205 Ala Lys Cys Pro Pro Ser Tyr Glu Lys Val Leu Ser Asp Ser Lys Gly 210 215 220 Asn Asp Ala Thr Leu Lys Val Val Phe Tyr Lys Lys Ser 225 230 235 <210> 17 <211> 150 <212> PRT <213> Streptomyces species <400> 17 Met Ser Glu Asn Val Thr Gly Ala His Pro Tyr Gly Ser Ile His Trp 1 5 10 15 Val Pro Glu Ile Phe Val Asp Pro Ala Lys Ser Arg Gly Glu Gln Asp 20 25 30 Met Ala Ala Arg Ser Tyr Asp Ile Ser Ile Val Asn Leu Thr Asn Met 35 40 45 Glu Leu Thr Arg Glu Lys Ser His Leu Asp His Gly Val Trp Ser Gly 50 55 60 Glu Gly Ser Glu Thr Pro Pro Asp Lys Ile Ala Pro Gly Glu Thr Val 65 70 75 80 His Phe Gly Ser Glu Ser Gln Gly Phe Met Thr Gly Thr Glu Gly Tyr 85 90 95 Val Thr Tyr Gly Ser Pro Ala Gly Asp Phe Thr Val Tyr Trp Asp Asn 100 105 110 Pro Tyr Val Gly Ser Asp Ser Ser Ser Ala Thr Cys Pro Ser Gly Tyr 115 120 125 Glu Lys Val Lys Ser Asp Ser Ala Gly Asn Asn Ala Thr Leu Lys Val 130 135 140 Val Phe Tyr Glu Asn Ser 145 150 <210> 18 <211> 357 <212> DNA <213> Streptomyces species <400> 18 atggccgcaa ggtcgtacga aatcgcgatc gtcaacctta ccgacgtgga gttcacgcgc 60 aaagaggcac atctcgatca tggggtgtgg agcaaggacg ggaactacac gcctccggac 120 aagatcagca ccggtcagac agcgcatttc ggaagtgaat cccagggagt ggcgaccgga 180 accgaggggc atgtgatcta ttcgtccagc gccggggatt ttcgcgtcga ctgggacaac 240 ccctacatcg ggggtgactc atcctccgcg aagtgcccgc cctcttacga aaaggtgctc 300 agtgactcca aaggtaacga cgcaaccctg aaggtggtct tctacaagaa gagctga 357 <210> 19 <211> 453 <212> DNA <213> Streptomyces species <400> 19 atgtctgaaa acgtgactgg tgcccatcca tatgggtcta tacattgggt cccggaaatc 60 ttcgtagatc cggcgaaaag ccgaggggag caggacatgg ccgcgagatc ctacgatatc 120 tcgatcgtca acctcaccaa tatggaactg acccgggaga agtcgcacct ggaccacggg 180 gtgtggagcg gggaaggcag cgaaacaccc ccggacaaga tagcccccgg agaaacggtg 240 catttcggca gcgaatccca gggattcatg acgggaaccg aagggtacgt cacctacggc 300 tcgcccgccg gagacttcac cgtctactgg gacaacccgt atgtcggttc ggattcgtcg 360 tccgcgacgt gcccgtcggg ttacgaaaag gtcaagagcg actccgcggg caacaacgcg 420 accctgaagg tggtcttcta cgagaacagc tga 453 <210> 20 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Escherichia coli-optimized SMIP1Aa. <400> 20 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcagggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttgggataat 240 ccatacattg gtggcgactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 21 <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-A71L. <400> twenty one atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc ttaggggact tccgcgtcga ttgggataat 240 ccatacattg gtggcgactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> twenty two <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-A90L. <400> twenty two atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttgggataat 240 ccatacattg gtggcgactc tagcagctta aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 23 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Escherichia coli-optimized SMIP1Aa-C92L. <400> 23 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcagggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttgggataat 240 ccatacattg gtggcgactc tagcagcgct aaactgccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 24 <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-C92C. <400> twenty four atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttgggataat 240 ccatacattg gtggcgactc tagcagcgct aaatccccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 25 <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-G84L. <400> 25 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttgggataat 240 ccatacattt taggcgactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 26 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Escherichia coli-optimized SMIP1Aa-G85L. <400> 26 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcagggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttgggataat 240 ccatacattg gtttagactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 27 <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-V76L. <400> 27 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcttaga ttgggataat 240 ccatacattg gtggcgactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 28 <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-W78F. <400> 28 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttttgataat 240 ccatacattg gtggcgactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 29 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Escherichia coli-optimized SMIP1Aa-A71L / A90L. <400> 29 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcagggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc ttaggggact tccgcgtcga ttgggataat 240 ccatacattg gtggcgactc tagcagctta aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 30 <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-A71L / W78F. <400> 30 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc ttaggggact tccgcgtcga ttttgataat 240 ccatacattg gtggcgactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 31 <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-W78F / C92L. <400> 31 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttttgataat 240 ccatacattg gtggcgactc tagcagcgct aaactgccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 32 <211> 357 <212> DNA <213> Artificial sequence <220> <223> Escherichia coli-optimized SMIP1Aa-Y82F / I83L. <400> 32 atggccgccc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcagggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttgggataat 240 ccattcctgg gtggcgactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 33 <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-A2G / A3G. <400> 33 atgggcggtc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttgggataat 240 ccatacattg gtggcgactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 34 <211> 357 <212> DNA <213> Artificial sequence <220> <223> E. coli optimized SMIP1Aa-A2V / A3V. <400> 34 atggtggtcc gtagttatga aattgcaatt gttaacttaa cggatgtaga atttacccgg 60 aaagaagccc atctcgacca cggagtttgg agcaaggatg gtaattacac tccgccggat 120 aaaatctcca ccgggcagac ggctcatttt ggtagcgaga gtcaggggagt tgctaccggt 180 accgaaggtc atgtaattta ctcctccagc gcgggggact tccgcgtcga ttgggataat 240 ccatacattg gtggcgactc tagcagcgct aaatgcccac cgtcgtacga aaaagttctg 300 tccgattcca agggtaacga tgcaacctta aaagtggtct tttataaaaa gtcctaa 357 <210> 35 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli-optimized SUMO-SMIP1Aa. <400> 35 atgggcagca gccatcatca tcatcatcac ggcagcggcc tggtgccgcg cggcagcgct 60 agcatgtcgg actcagaagt caatcaagaa gctaagccag aggtcaagcc agaagtcaag 120 cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt cttcaagatc 180 aaaaagacca ctcctttaag aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa 240 atggactcct taagattctt gtacgacggt attagaattc aagctgatca gacccctgaa 300 gatttggaca tggaggataa cgatattatt gaggctcaca gagaacagat tggtggtatg 360 gccgcccgta gttatgaaat tgcaattgtt aacttaacgg atgtagaatt tacccggaaa 420 gaagcccatc tcgaccacgg agtttggagc aaggatggta attacactcc gccggataaa 480 atctccaccg ggcagacggc tcattttggt agcgagagtc agggagttgc taccggtacc 540 gaaggtcatg taatttactc ctccagcgcg ggggacttcc gcgtcgattg ggataatcca 600 tacattggtg gcgactctag cagcgctaaa tgcccaccgt cgtacgaaaa agttctgtcc 660 gattccaagg gtaacgatgc aaccttaaaa gtggtctttt ataaaaagtc ctaa 714 <210> 36 <211> 453 <212> DNA <213> Artificial Sequence <220> <223> Escherichia coli-optimized SMIP2Aa. <400> 36 atgtctgaga atgtgacggg ggcgcatccc tatggctcta tccactgggt ccccgaaatt 60 tttgtagatc cagcgaagtc ccgtggtgag caggacatgg ctgcgcgtag ttacgatatt 120 tcgattgtta atctgactaa catggagctt acccgtgaga aaagtcactt agatcatggc 180 gtctggtctg gagagggaag tgaaacacct cccgacaaga tcgctcccgg tgagactgtc 240 cactttgggt cagagtcgca aggatttatg acgggaacgg agggatatgt cacatacgga 300 tccccggcgg gtgattttac cgtatattgg gataatcctt acgttggttc ggattcttca 360 agtgcgacat gcccctcggg atacgaaaag gttaaatcgg acagtgcagg gaacaacgca 420 acgctgaaag ttgtgttcta tgaaaatagc taa 453 <210> 37 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Maize-optimized SMIP1Aa. <400> 37 atggccgcca ggtcctacga gatcgccatc gtgaacctga ccgacgtgga gttcaccagg 60 aaggaggccc acctggacca cggcgtgtgg tccaaggacg gcaactacac cccgccggac 120 aagatctcca ccggccagac cgcccacttc ggctccgagt cccagggcgt ggccaccggc 180 accgagggcc acgtgatcta ctcctcctcc gccggcgact tcagggtgga ctgggacaac 240 ccgtacatcg gcggcgactc ctcctccgcc aagtgcccgc cgtcctacga gaaggtgctg 300 tccgactcca agggcaacga cgccaccctg aaggtggtgt tctacaagaa gtcc 354 <210> 38 <211> 354 <212> DNA <213> Artificial sequence <220> <223> Maize-optimized SMIP1Aa-W78F / A92L. <400> 38 atggccgcca ggtcctacga gatcgccatc gtgaacctga ccgacgtgga gttcaccagg 60 aaggaggccc acctggacca cggcgtgtgg tccaaggacg gcaactacac cccgccggac 120 aagatctcca ccggccagac cgcccacttc ggctccgagt cccagggcgt ggccaccggc 180 accgagggcc acgtgatcta ctcctcctcc gccggcgact tcagggtgga cttcgacaac 240 ccgtacatcg gcggcgactc ctcctccgcc aagctgccgc cgtcctacga gaaggtgctg 300 tccgactcca agggcaacga cgccaccctg aaggtggtgt tctacaagaa gtcc 354 <210> 39 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Maize-optimized SMIP1Aa-A71L / A90L. <400> 39 atggccgcca ggtcctacga gatcgccatc gtgaacctga ccgacgtgga gttcaccagg 60 aaggaggccc acctggacca cggcgtgtgg tccaaggacg gcaactacac cccgccggac 120 aagatctcca ccggccagac cgcccacttc ggctccgagt cccagggcgt ggccaccggc 180 accgagggcc acgtgatcta ctcctcctcc ctgggcgact tcagggtgga ctgggacaac 240 ccgtacatcg gcggcgactc ctcctccctg aagtgcccgc cgtcctacga gaaggtgctg 300 tccgactcca agggcaacga cgccaccctg aaggtggtgt tctacaagaa gtcc 354 <210> 40 <211> 453 <212> DNA <213> Artificial Sequence <220> <223> Zea mays-optimized SMIP2Aa. <400> 40 atgtccgaga acgtgaccgg cgcccacccg tacggctcca tccactgggt gccggagatc 60 ttcgtggacc cggccaagtc caggggcgag caggacatgg ccgccaggtc ctacgacatc 120 tccatcgtga acctgaccaa catggagctg accagggaga agtcccacct ggaccacggc 180 gtgtggtccg gcgagggctc cgagaccccg ccggacaaga tcgccccggg cgagaccgtg 240 cacttcggct ccgagtccca gggcttcatg accggcaccg agggctacgt gacctacggc 300 tccccggccg gcgacttcac cgtgtactgg gacaacccgt acgtgggctc cgactcctcc 360 tccgccacct gcccgtccgg ctacgagaag gtgaagtccg actccgccgg caacaacgcc 420 accctgaagg tggtgttcta cgagaactcc tga 453

Claims

1. A nucleic acid molecule consisting of a nucleotide sequence encoding a protein toxic to insect pests, wherein the nucleotide sequence (a) encodes a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 2-10 or 12-15; or (b) consists of any one of SEQ ID NOs: 21-29, 31-34 or 38-39.

2. The nucleic acid molecule of claim 1, wherein the protein toxic to insect pests consists of the amino acid sequence of any one of SEQ ID NOs: 2-10 or 12-15.

3. The nucleic acid molecule of claim 1, wherein the nucleotide sequence consists of any one of SEQ ID NOs: 21-29, 31-34 or 38-39.

4. A chimeric gene, comprising the nucleic acid molecule according to any one of claims 1 to 3 and a heterologous promoter operably linked thereto. The chimeric gene of claim 4 , wherein the heterologous promoter is a plant-expressible promoter.

6. The chimeric gene of claim 4, wherein the insect pest is a coleopteran insect pest.

7. The chimeric gene of claim 6, wherein the coleopteran insect pest is a Diabrotica insect pest.

8. The chimeric gene of claim 7, wherein the insect pest of the genus Diabrotica is selected from the group consisting of: corn rootworm, barker's rootworm, cucumber rootworm subsp. undemmaculata, and maize rootworm.

9. A protein toxic to insect pests, wherein the protein consists of the following amino acid sequence: (a) an amino acid sequence consisting of any one of SEQ ID NOs: 2-10 or 12-15; or (b) an amino acid sequence encoded by a nucleotide sequence consisting of any one of SEQ ID NOs: 21-29, 31-34 or 38-39.

10. The protein of claim 9, wherein the insect pest is a coleopteran insect pest.

11. The protein of claim 10, wherein the coleopteran insect pest is a Diabrotica insect pest.

12. The protein of claim 11, wherein the rootworm insect pest is selected from the group consisting of: corn rootworm, barker's rootworm, cucumber rootworm subsp. undemmaculata, and maize rootworm.

13. A recombinant vector comprising the chimeric gene according to claim 4.

14. A host cell comprising the recombinant vector according to claim 13, wherein the host cell is a bacterial cell.

15. The host cell of claim 14, wherein the host cell is in the genus Escherichia.

16. An engineered insecticidal protein, wherein the protein consists of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:

9.

17. An engineered insecticidal protein, wherein the protein consists of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO:

15.

18. The engineered insecticidal protein of any one of claims 16-17, wherein the protein is active against coleopteran insect pests.

19. The engineered insecticidal protein of claim 18, wherein the coleopteran insect pest is a Diabrotica insect pest.

20. The engineered insecticidal protein of claim 19, wherein the Diabrotica insect pest is selected from the group consisting of: corn rootworm, barker's rootworm, cucumber rootworm subsp. undecemlineata, and maize rootworm.

21. An insecticidal composition comprising the protein toxic to insect pests according to claim 9 and an agriculturally acceptable carrier.

22. The composition of claim 21, wherein the agriculturally acceptable carrier is selected from the group consisting of dusts, pellets, granules, sprays, emulsions, colloids and solutions.

23. The composition of claim 21, wherein the composition is prepared by dehydrating, freeze-drying, homogenizing, extracting, filtering, centrifuging, sedimenting or concentrating a culture of an E. coli strain.

24. The composition of claim 21, comprising from 1% to 99% by weight of the protein toxic to insect pests.

25. The composition of any one of claims 21-24, wherein the composition further comprises a second pesticide.

26. The composition of claim 25, wherein the second pesticide is a biological agent or a chemical agent.

27. The composition of claim 26, wherein (a) the biological agent is or is derived from a Bacillus thuringiensis insecticidal protein, a Bacillus cereus insecticidal protein, a Xenorhabdus species insecticidal protein, a Photorhabdus species insecticidal protein, a Brevibacillus laterosporus insecticidal protein, a Lysinibacillus sphaericus insecticidal protein, a Chromobacterium species insecticidal protein, a Yersinia entomophaga insecticidal protein, a Bacillus lactis insecticidal protein, or a Clostridium species insecticidal protein; (b) the biological agent is or is derived from dsRNA, a Cry protein, a Vip protein, a potato glycoprotein, a protease, a protease inhibitor, a urease, an α-amylase inhibitor, a pore-forming protein , lectins, engineered antibodies or antibody fragments, or chitinases; (c) the chemical agent is a carbamate, a pyrethroid, an organophosphate, friprole, a neonicotinoid, an organochloride, a nereis toxin, or a combination thereof; or (d) the chemical agent comprises an active ingredient selected from the group consisting of carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, lambda-cyfluthrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, ethoxyphos, malathion, methyl parathion, phorate, terbufos, tert-butylpyrimidophos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamethoxam, and a combination thereof.

28. A method for producing an insecticidal protein, the method comprising culturing the host cell of claim 14 or an organism comprising the host cell under conditions whereby the host cell produces the insecticidal protein.

29. A method of producing a transgenic maize plant or maize plant part having enhanced insect resistance compared to a control maize plant or maize plant part, the method comprising: (a) introducing into a maize plant or maize plant part The chimeric gene of claim 4, wherein the protein toxic to an insect pest is expressed in the maize plant or maize plant part, thereby producing a maize plant or maize plant part having enhanced insect resistance.

30. The method of claim 29, wherein the introducing step is accomplished by: (a) transforming the maize plant or maize plant part; or (b) crossing a first maize plant comprising the mosaic gene with a second, different maize plant.

31. The method of claim 30, wherein the chimeric gene encodes an insecticidal protein consisting of the amino acid sequence of any one of SEQ ID NOs: 2-10 or 12-15.

32. A method of controlling insect pests, the method comprising delivering an effective amount of a protein toxic to insect pests according to claim 9 to the insect pests or their environment.

33. The method of claim 32, wherein the protein toxic to insect pests is delivered via a transgenic maize plant or by topical application of an insecticidal composition comprising the protein toxic to insect pests.

34. The method of claim 33, wherein the transgenic maize plant or the insecticidal composition comprises a second insecticide different from the protein that is toxic to an insect pest.

35. The method of claim 34, wherein the second insecticide is a protein, a dsRNA, or a chemical.

36. The method of claim 35, wherein (a) the protein is selected from the group consisting of Cry protein, Vip protein, potato glycoprotein, protease, protease inhibitor, urease, α-amylase inhibitor, pore-forming protein, lectin, engineered antibody or antibody fragment, or chitinase; (b) the chemical is a carbamate, pyrethroid, organophosphate, friprole, neonicotinoid, organochloride, nereistoxin or a combination thereof; or (c) the chemical comprises an active ingredient selected from the group consisting of carbofuran, carbaryl, methomyl, bifenthrin, tefluthrin, permethrin, cyfluthrin, λ-cyfluthrin, cypermethrin, deltamethrin, chlorpyrifos, chloroxyfos, dimethoate, ethoxyphos, malathion, methyl parathion, phorate, terbufos, tert-butylpyrimidophos, fipronil, acetamiprid, imidacloprid, thiacloprid, thiamethoxam, endosulfan, sulfamic acid and a combination thereof.

37. The method of any one of claims 32-36, wherein the insect pest is a coleopteran insect pest.

38. The method of claim 37, wherein the coleopteran insect pest is a species of the genus Diabrotica.

39. The method of claim 38, wherein the species of the genus Diabrotica is selected from the group consisting of: Corn Rootworm, Barker's Rootworm, Cucumis sativus ssp. rhizophagous, and Maize Rootworm.

40. A method for reducing resistance development in a root beetle population to a protein toxic to insect pests as claimed in claim 9, the method comprising expressing the protein toxic to insect pests and an interfering RNA molecule in a transgenic corn plant ingested by the root beetle population, wherein the interfering RNA molecule inhibits expression of a target gene in larval and adult root beetle insects, thereby reducing resistance development in the root beetle population compared to a root beetle population exposed only to the protein toxic to insect pests.

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