Control of Spodoptera

Abstract

Methods for controlling plant pests are disclosed. In particular, insecticidal proteins are provided that are toxic to Spodoptera pests. Polynucleotides encoding the insecticidal proteins, which contain codons optimized for expression in plants, are also provided. Methods for producing the insecticidal proteins and methods for using the insecticidal proteins and polynucleotides encoding the insecticidal proteins of the invention to confer protection from insect pests, for example in transgenic plants, are also disclosed.

Description

Technical Field

[0001] Sequence Listing A sequence listing in ASCII text format submitted under 37 C.F.R.§1.821, created on March 31, 2020, submitted via EFS-Web, having a size of 308 kilobytes and named "81889-WO-REG-ORG-P-1_ST25.txt", is provided in lieu of a paper copy. This sequence listing is incorporated herein by reference for its disclosure.

[0002] The present invention relates to the fields of pest control, protein engineering, and plant molecular biology. More specifically, the present invention relates to methods for controlling pests of the Spodoptera order, particularly Spodoptera frugiperda (fall armyworm; FAW), using the txp40 gene or Txp40 protein or in combination with a second pest control agent. The present invention also relates to methods for protecting crops, particularly corn, against Spodoptera, particularly FAW, and more specifically against Cry and / or Vip3-resistant FAW. The present invention also provides a Txp40 protein toxin having insecticidal activity against FAW and variants thereof, a nucleic acid whose expression results in an insecticidal protein, and methods for producing and using insecticidal proteins and corresponding nucleic acids for controlling FAW pests. The present invention also relates to plants, particularly monocotyledonous plants, specifically corn plants, that can be ectoparasitized by FAW and are transformed with an expressible txp40 gene or are transformed with both an expressible txp40 gene and a gene encoding a second pesticidal agent.

Background Art

[0003] Spodoptera species, including Spodoptera frugiperda (Fall armyworm, FAW), are serious pests of several row crops, including corn, cotton, and soybeans. In Brazil and other South American countries, FAW is the most destructive and consistent pest of corn plants (Zea Mays), resulting in yield reductions of up to 57%. Over time, FAW can cause significant economic damage to corn in other countries, including the United States. The larvae of this insect first feed on leaf tissue and then penetrate deep into the whorl, causing significant damage to the developing tassels. The leaf drop caused by FAW reduces the photosynthetic area, which can inhibit plant growth and reduce yield. FAW larvae also damage the ears by penetrating the sides and feeding on the developing grains, reducing grain quality and yield.

[0004] Historically, control of FAW in corn has been achieved using synthetic insecticides. However, such insecticides are only effective against young larvae and before they burrow deeply into whorls or ears. In addition, in intensive corn cultivation areas, FAW populations have evolved resistance to several insecticides, including lambda-cyhalothrin, chlorpyrifos, spinosad, and lufenuron.

[0005] Other FAW control options, more recently, include the use of transgenic corn plants expressing one or more insecticidal crystalline proteins (Cry proteins) and / or plant insecticidal proteins (VIPs) derived from the bacterium Bacillus thuringiensis (Bt). The most potent FAW-active Bt-derived protein to date is the plant insecticidal protein Vip3. Only a few Bt-derived Cry proteins are active against FAW. Among those with moderate to high activity are Cry1Ab, Cry1Be, Cry1D, Cry1F, Cry1If, Cry1J, and Cry2A. Since approximately 2007, the commercial release of transgenic corn events expressing one or more of these Cry and Vip3 proteins has provided new strategies for FAW management. Currently, products containing these so-called Bt events are cultivated in many major corn growing areas where FAW is a pest. For example, such products are cultivated in over 80 areas of corn cultivation in Brazil (approximately 12.5 million ha / year). The rapid adoption of products containing Bt insecticidal proteins with activity against FAW has contributed to a reduction in insecticide sprays against FAW. However, in some cases, continuous expression of Bt-derived proteins in corn plants exerts strong selective pressure on target FAW pest populations, leading to the evolution of resistance. In Brazil, for example, field populations of FAW have evolved resistance to Cry1F and Cry1Ab proteins expressed in transgenic corn products. FAW has also developed resistance to Cry1F corn in Puerto Rico and the US region. In Brazil, high-frequency product resistance has also been reported in FAW populations, along with stacked products containing Cry1 protein. In contrast, resistance to Vip3A protein has not been detected. [Overview of the project] [Problems that the invention aims to solve]

[0006] There remains an ongoing need to identify new and effective methods for controlling pests in row crops using environmentally acceptable insecticides that also provide economic benefits to farmers. Of particular need are methods for controlling Spodoptera species, especially FAW, using proteins with different mechanisms of action from existing Bt-derived insecticidal proteins, such as Cry protein and / or Vip3 protein, as a means of mitigating the development of resistance. Furthermore, it is most desirable to deliver insect control agents through products that minimize environmental impact, such as transgenic plants. [Means for solving the problem]

[0007] In consideration of these needs, the present invention provides a method for controlling Spodoptera frugiperda (fall armyworm; FAW) and other pests using the Txp40 insecticidal protein and variants of the Txp40 insecticidal protein. The present invention also provides Spodoptera-active Txp40 protein and variant Txp40 protein that are substantially identical to Txp40. Surprisingly, the proteins of the present invention have oral toxicity against FAW, a pest species resistant to numerous types of insecticidal proteins, particularly Bt-derived Cry proteins. The present invention further relates to recombinant codon-optimized polynucleotides encoding the Txp40 protein or variant Txp40 protein.

[0008] The present invention includes expression cassettes and vectors containing the recombinant polynucleotide of the present invention; plants or microorganisms containing such polynucleotides and enabling their expression; plants transformed with such polynucleotides, such as transgenic cone plants; offspring of such plants and / or seeds of such plants and their offspring containing polynucleotides that can be stably incorporated and inherited according to Mendel's laws. The present invention also includes breeding methods for introducing transgenes containing the polynucleotide of the present invention into offspring plants and various cone germplasms.

[0009] The present invention also includes compositions and formulations containing Txp40 or its variants, which can inhibit the ability of FAW to survive, grow and / or reproduce, or limit FAW-related damage or loss to crop plants, for example, by applying Txp40 or its variants as part of a composition or formulation to an exoparasitic area or plant of FAW, or by prophylactically treating a FAW-sensitive area or plant to provide protection against FAW pests.

[0010] The present invention further relates to methods for producing Txp40 or its variants, and to methods for using the polynucleotides of the present invention to control FAW in microorganisms, for example, or to confer protection from FAW damage in transgenic plants.

[0011] Other aspects and advantages of the present invention will become apparent to those skilled in the art from the following description of the invention and the study of non-limiting embodiments. [Modes for carrying out the invention]

[0012] A brief explanation of the sequences in the sequence listing. The nucleotide sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.FR §1.822. The listed nucleotide and amino acid sequences define molecules (i.e., polynucleotides and polypeptides, respectively) having the nucleotides and amino acid monomers as described. The listed nucleotide and amino acid sequences also define the genera of polynucleotides or polypeptides containing the nucleotides and amino acid monomers as described. Considering the redundancy of the genetic code, it will be understood that a nucleotide sequence containing a coding sequence also describes the genera of polynucleotides encoding the same polypeptide as the polynucleotide consisting of the reference sequence. Furthermore, it will be understood that an amino acid sequence also describes the genera of polynucleotide ORFs encoding that polypeptide.

[0013] Only one strand of each nucleic acid sequence is shown, but it is understood that complementary strands are also included by reference to the shown strand. Since the complement and reverse complement of a primary nucleotide sequence are not necessarily disclosed by the primary sequence, complementary and reverse complementary sequences are referenced to their nucleotide sequence unless explicitly stated otherwise (or it is not evident from the context in which the sequence appears). Furthermore, since it is understood in the art that the nucleotide sequence of an RNA strand is determined by the sequence of the DNA from which it is transcribed (apart from the substitution of thymine (T) by the nucleotide base uracil (U)), RNA sequences are included by reference to the DNA sequence that encodes them. In the attached sequence listings: Sequence ID 1 is the Txp40-1 amino acid sequence derived from Phtorhabdus luminescens.

[0014] Sequence ID 2 is the amino acid sequence of the mutant Txp40-1.

[0015] Sequence ID 3 is a natural txp40-1 nucleotide sequence derived from Phtorhabdus luminescens.

[0016] Sequence ID 4 is the E. coli codon-optimized txp40-1 nucleotide sequence.

[0017] Sequence ID 5 is the maize codon-optimized txp40-1 nucleotide sequence.

[0018] Sequence ID 6 is the H1Txp40 amino acid sequence derived from the species Photorhabdus.

[0019] Sequence ID 7 is the H2Txp40 amino acid sequence derived from the species Photorhabdus.

[0020] SEQ ID NO: 8 is the H3Txp40 amino acid sequence derived from the Photorhabdus species.

[0021] SEQ ID NO: 9 is the H5Txp40 amino acid sequence derived from the Photorhabdus species.

[0022] SEQ ID NO: 10 is the Txp40 amino acid sequence derived from Xenorhabdus nematophila.

[0023] SEQ ID NO: 11 is the E. coli codon-optimized txp40-1 K31A mutant nucleotide sequence.

[0024] SEQ ID NO: 12 is the E. coli codon-optimized txp40-1 K48A mutant nucleotide sequence.

[0025] SEQ ID NO: 13 is the E. coli codon-optimized txp40-1 K49A mutant nucleotide sequence.

[0026] SEQ ID NO: 14 is the E. coli codon-optimized txp40-1 K73A mutant nucleotide sequence.

[0027] SEQ ID NO: 15 is the E. coli codon-optimized txp40-1 K75A mutant nucleotide sequence.

[0028] SEQ ID NO: 16 is the E. coli codon-optimized txp40-1 K103A mutant nucleotide sequence.

[0029] SEQ ID NO: 17 is the E. coli codon-optimized txp40-1 K111A mutant nucleotide sequence.

[0030] SEQ ID NO: 18 is the E. coli codon-optimized txp40-1 K119A mutant nucleotide sequence.

[0031] Sequence ID 19 is an E. coli codon-optimized txp40-1 K133A mutant nucleotide sequence.

[0032] Sequence ID 20 is the E. coli codon-optimized txp40-1 K143A mutant nucleotide sequence.

[0033] Sequence ID 21 is an E. coli codon-optimized txp40-1 K170A mutant nucleotide sequence.

[0034] Sequence ID 22 is an E. coli codon-optimized txp40-1 K191A mutant nucleotide sequence.

[0035] Sequence ID 23 is an E. coli codon-optimized txp40-1 K200A mutant nucleotide sequence.

[0036] Sequence ID 24 is an E. coli codon-optimized txp40-1 K209A mutant nucleotide sequence.

[0037] Sequence ID 25 is the E. coli codon-optimized txp40-1 K210A mutant nucleotide sequence.

[0038] Sequence ID 26 is the E. coli codon-optimized txp40-1 K213A mutant nucleotide sequence.

[0039] Sequence ID 27 is an E. coli codon-optimized txp40-1 K221A mutant nucleotide sequence.

[0040] Sequence ID 28 is the E. coli codon-optimized txp40-1 K238A mutant nucleotide sequence.

[0041] Sequence ID 29 is an E. coli codon-optimized txp40-1 K247A mutant nucleotide sequence.

[0042] Sequence ID 30 is an E. coli codon-optimized txp40-1 K250A mutant nucleotide sequence.

[0043] Sequence ID 31 is an E. coli codon-optimized txp40-1 K263A mutant nucleotide sequence.

[0044] Sequence ID 32 is an E. coli codon-optimized txp40-1 K265A mutant nucleotide sequence.

[0045] Sequence ID 33 is an E. coli codon-optimized txp40-1 K271A mutant nucleotide sequence.

[0046] Sequence ID 34 is an E. coli codon-optimized txp40-1 K275A mutant nucleotide sequence.

[0047] Sequence ID 35 is an E. coli codon-optimized txp40-1 K284A mutant nucleotide sequence.

[0048] Sequence ID 36 is an E. coli codon-optimized txp40-1 K296A mutant nucleotide sequence.

[0049] Sequence ID 37 is an E. coli codon-optimized txp40-1 K309A mutant nucleotide sequence.

[0050] Sequence ID 38 is an E. coli codon-optimized txp40-1 K333A mutant nucleotide sequence.

[0051] Sequence ID 39 is an E. coli codon-optimized txp40-1 R10A mutant nucleotide sequence.

[0052] Sequence ID 40 is an E. coli codon-optimized txp40-1 R26A mutant nucleotide sequence.

[0053] Sequence ID 41 is an E. coli codon-optimized txp40-1 R46A mutant nucleotide sequence.

[0054] Sequence ID 42 is an E. coli codon-optimized txp40-1 R167A mutant nucleotide sequence.

[0055] Sequence ID 43 is an E. coli codon-optimized txp40-1 R186A mutant nucleotide sequence.

[0056] Sequence ID 44 is an E. coli codon-optimized txp40-1 R187A mutant nucleotide sequence.

[0057] Sequence ID 45 is an E. coli codon-optimized txp40-1 R208A mutant nucleotide sequence.

[0058] Sequence ID 46 is an E. coli codon-optimized txp40-1 R217A mutant nucleotide sequence.

[0059] Sequence ID 47 is an E. coli codon-optimized txp40-1 R226A mutant nucleotide sequence.

[0060] Sequence ID 48 is an E. coli codon-optimized txp40-1 R240A mutant nucleotide sequence.

[0061] Sequence ID 49 is an E. coli codon-optimized txp40-1 R252A mutant nucleotide sequence.

[0062] Sequence ID 50 is an E. coli codon-optimized txp40-1 R305A mutant nucleotide sequence.

[0063] Sequence ID 51 is an E. coli codon-optimized txp40-1 R311A mutant nucleotide sequence.

[0064] Sequence ID 52 is an E. coli codon-optimized txp40-1 K31A / K49A mutant nucleotide sequence.

[0065] Sequence ID 53 is an E. coli codon-optimized txp40-1 K31A / K333A mutant nucleotide sequence.

[0066] Sequence ID 54 is an E. coli codon-optimized txp40-1 K31A / R208A mutant nucleotide sequence.

[0067] Sequence ID 55 is an E. coli codon-optimized txp40-1 R208A / K333A mutant nucleotide sequence.

[0068] Sequence ID 56 is an E. coli codon-optimized txp40-1 K49A / R208A mutant nucleotide sequence.

[0069] Sequence ID 57 is an E. coli codon-optimized txp40-1 K49A / K333A mutant nucleotide sequence.

[0070] Sequence ID 58 is an E. coli codon-optimized txp40-1 K31A / K49A / K333A mutant nucleotide sequence.

[0071] Sequence ID 59 is an E. coli codon-optimized txp40-1 K31A / K49A / R208A / K333A mutant nucleotide sequence.

[0072] Sequence ID 60 is an E. coli codon-optimized txp40-1 K73A / K213A mutant nucleotide sequence.

[0073] Sequence ID 61 is an E. coli codon-optimized txp40-1 K103A / K213A mutant nucleotide sequence.

[0074] Sequence ID 62 is an E. coli codon-optimized txp40-1 K213A / K284A mutant nucleotide sequence.

[0075] Sequence ID 63 is an E. coli codon-optimized txp40-1 K31A / K49A / K213A mutant nucleotide sequence.

[0076] Sequence ID 64 is an E. coli codon-optimized txp40-1 K119A / K213A mutant nucleotide sequence.

[0077] Sequence ID 65 is the E. coli codon-optimized txp40-1 K213A / R167A mutant nucleotide sequence.

[0078] Sequence ID 66 is an E. coli codon-optimized txp40-1 K119A / R167A mutant nucleotide sequence.

[0079] Sequence ID 67 is an E. coli codon-optimized txp40-1 S11Y / M86I / K119A / K213T mutant nucleotide sequence.

[0080] Sequence ID 68 is an E. coli codon-optimized txp40-1 S11Y / K119A / K213A mutant nucleotide sequence.

[0081] Sequence ID 69 is an E. coli codon-optimized txp40-1 K119A / F169V / K213A mutant nucleotide sequence.

[0082] Sequence ID 70 is an E. coli codon-optimized txp40-1 K119A / P134L / K213A mutant nucleotide sequence.

[0083] Sequence ID 71 is an E. coli codon-optimized txp40-1 I101L / K103T / K119A / K213A / N257T mutant nucleotide sequence.

[0084] Sequence ID 72 is an E. coli codon-optimized txp40-1 H62Q / D99V / K119A / T165I / R167C / K213A mutant nucleotide sequence.

[0085] Sequence ID 73 is an E. coli codon-optimized txp40-1 K119A / K213A / L316V mutant nucleotide sequence.

[0086] Sequence ID 74 is the amino acid sequence of the Txp40-1 K31A mutant.

[0087] Sequence ID 75 is the amino acid sequence of the Txp40-1 K48A mutant.

[0088] Sequence ID 76 is the amino acid sequence of the Txp40-1 K49A mutant.

[0089] Sequence ID 77 is the amino acid sequence of the Txp40-1 K73A mutant.

[0090] Sequence ID 78 is the amino acid sequence of the Txp40-1 K75A mutant.

[0091] Sequence ID 79 is the amino acid sequence of the Txp40-1 K103A mutant.

[0092] Sequence ID 80 is the amino acid sequence of the Txp40-1 K111A mutant.

[0093] Sequence ID 81 is the amino acid sequence of the Txp40-1 K119A mutant.

[0094] Sequence ID 82 is the amino acid sequence of the Txp40-1 K133A mutant.

[0095] Sequence ID 83 is the Txp40-1 K143A mutant nucleotide sequence.

[0096] Sequence ID 84 is the amino acid sequence of the Txp40-1 K170A mutant.

[0097] Sequence ID 85 is the amino acid sequence of the Txp40-1 K191A mutant.

[0098] Sequence ID 86 is the amino acid sequence of the Txp40-1 K200A mutant.

[0099] Sequence ID 87 is the amino acid sequence of the Txp40-1 K209A mutant.

[0100] Sequence ID 88 is the amino acid sequence of the Txp40-1 K210A mutant.

[0101] Sequence ID 89 is the amino acid sequence of the Txp40-1 K213A mutant.

[0102] Sequence ID 90 is the amino acid sequence of the Txp40-1 K221A mutant.

[0103] Sequence ID 91 is the amino acid sequence of the Txp40-1 K238A mutant.

[0104] Sequence ID 92 is the amino acid sequence of the Txp40-1 K247A mutant.

[0105] Sequence ID 93 is the amino acid sequence of the Txp40-1 K250A mutant.

[0106] Sequence ID 94 is the amino acid sequence of the Txp40-1 K263A mutant.

[0107] Sequence ID 95 is the amino acid sequence of the Txp40-1 K265A mutant.

[0108] Sequence ID 96 is the amino acid sequence of the Txp40-1 K271A mutant.

[0109] Sequence ID 97 is the amino acid sequence of the Txp40-1 K275A mutant.

[0110] Sequence ID 98 is the amino acid sequence of the Txp40-1 K284A mutant.

[0111] Sequence ID 99 is the amino acid sequence of the Txp40-1 K296A mutant.

[0112] Sequence ID 100 is the amino acid sequence of the Txp40-1 K309A mutant.

[0113] Sequence ID 101 is the amino acid sequence of the Txp40-1 K333A mutant.

[0114] Sequence ID 102 is the amino acid sequence of the Txp40-1 R10A mutant.

[0115] Sequence ID 103 is the amino acid sequence of the Txp40-1 R26A mutant.

[0116] Sequence ID 104 is the amino acid sequence of the Txp40-1 R46A mutant.

[0117] Sequence ID 105 is the amino acid sequence of the Txp40-1 R167A mutant.

[0118] Sequence ID 106 is the amino acid sequence of the Txp40-1 R186A mutant.

[0119] Sequence ID 107 is the amino acid sequence of the Txp40-1 R187A mutant.

[0120] Sequence ID 108 is the amino acid sequence of the Txp40-1 R208A mutant.

[0121] Sequence ID 109 is the amino acid sequence of the Txp40-1 R217A mutant.

[0122] Sequence ID 110 is the amino acid sequence of the Txp40-1 R226A mutant.

[0123] Sequence ID 111 is the amino acid sequence of the Txp40-1 R240A mutant.

[0124] Sequence ID 112 is the amino acid sequence of the Txp40-1 R252A mutant.

[0125] Sequence ID 113 is the amino acid sequence of the Txp40-1 R305A mutant.

[0126] Sequence ID 114 is the amino acid sequence of the Txp40-1 R311A mutant.

[0127] Sequence ID 115 is the Txp40-1 K31A / K49A mutant amino acid sequence.

[0128] Sequence ID 116 is the Txp40-1 K31A / K333A mutant amino acid sequence.

[0129] Sequence ID 117 is the amino acid sequence of the Txp40-1 K31A / R208A mutant.

[0130] Sequence ID 118 is the Txp40-1 R208A / K333A mutant amino acid sequence.

[0131] Sequence ID 119 is the amino acid sequence of the Txp40-1 K49A / R208A mutant.

[0132] Sequence ID 120 is the Txp40-1 K49A / K333A mutant amino acid sequence.

[0133] Sequence ID 121 is the Txp40-1 K31A / K49A / K333A mutant amino acid sequence.

[0134] Sequence ID 122 is the Txp40-1 K31A / K49A / R208A / K333A mutant amino acid sequence.

[0135] Sequence ID 123 is the Txp40-1 K73A / K213A mutant amino acid sequence.

[0136] Sequence ID 124 is the Txp40-1 K103A / K213A mutant amino acid sequence.

[0137] Sequence ID 125 is the Txp40-1 K213A / K284A mutant amino acid sequence.

[0138] Sequence ID 126 is the Txp40-1 K31A / K49A / K213A mutant amino acid sequence.

[0139] Sequence ID 127 is the Txp40-1 K119A / K213A mutant amino acid sequence.

[0140] Sequence ID 128 is the amino acid sequence of the Txp40-1 K213A / R167A mutant.

[0141] Sequence ID 129 is the Txp40-1 K119A / R167A mutant amino acid sequence.

[0142] Sequence ID 130 is the Txp40-1 S11Y / M86I / K119A / K213T mutant amino acid sequence.

[0143] Sequence ID 131 is the Txp40-1 S11Y / K119A / K213A mutant amino acid sequence.

[0144] Sequence ID 132 is the Txp40-1 K119A / F169V / K213A mutant amino acid sequence.

[0145] Sequence ID 133 is the Txp40-1 K119 / P134L / K213A mutant amino acid sequence.

[0146] Sequence ID 134 is the Txp40-1 I101L / K103T / K119A / K213A / N257T mutant amino acid sequence.

[0147] Sequence ID 135 is the Txp40-1 H62Q / D99V / K119A / T165I / R167C / K213A mutant amino acid sequence.

[0148] Sequence ID 136 is the K119A / K213A / L316V mutant amino acid sequence.

[0149] This description is not intended to be a detailed catalog of all the various ways in which the present invention can be carried out or all the features that may be added to the present invention. For example, a feature described in relation to one embodiment may be incorporated into another embodiment, and a feature described in relation to a particular embodiment may be omitted from that embodiment. Accordingly, the present invention is intended to exclude or omit any feature or combination of features described herein in some embodiments of the present invention. Furthermore, numerous variations and additions to the various embodiments suggested herein that do not depart from the present invention will be apparent to those skilled in the art in consideration of this disclosure. Accordingly, the following description is intended to describe some specific embodiments of the present invention and is not intended to exhaustively specify all sorts, combinations and variations thereof.

[0150] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. Technical terms used in the description of the invention herein are for the purpose of describing specific embodiments and are not intended to limit the invention.

[0151] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety with respect to the teachings relating to the texts and / or paragraphs in which they are cited.

[0152] The nucleotide sequences provided herein are presented from left to right in a 5' to 3' direction and are presented using standard codes for representing nucleotide bases, such as adenine (A), cytosine (C), thymine (T), and guanine (G), as described in 37 CFR §§1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25.

[0153] Amino acids are similarly represented 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).

[0154] Unless otherwise indicated in the context, it is particularly intended that the various features of the present invention described herein may be used in any combination. Furthermore, the present invention also assumes that in certain embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted. For illustrative purposes, where this specification states that a composition comprises components A, B, and C, it is particularly intended that any one of A, B, or C, or any combination thereof, may be excluded individually or in any combination and not claimed.

[0155] definition For clarity, the specific terms used herein are defined and presented as follows:

[0156] In use in the specification and appended claims of the present invention, the singular forms "a," "an," and "the" are intended to include the plural form unless otherwise specified in the context.

[0157] As used herein, "and / or" encompasses all possible combinations of one or more of the related enumerated items, and, when interpreted as an alternative ("or"), the absence of any combination.

[0158] As used herein, the term “about” is intended to include variations of ±20, ±10, ±5, ±1, ±0.5, or even ±0.1 from a given amount when referring to a measurable value such as a dosage or duration. As used herein, phrases such as “about X to Y” mean “about X to about Y,” and phrases such as “from about X to Y” mean “from about X to about Y.”

[0159] Unless otherwise specified in the context, phrases such as "from X to Y" should be interpreted as including both X and Y.

[0160] The "activity" of the insecticidal protein of the present invention means that the insecticidal protein, which functions as an orally active insect control agent, has a toxic effect and / or can interfere with or prevent insect feeding, which may or may not cause insect death. When the insecticidal protein of the present invention is delivered to insects, the result is usually the death of the insects or the insects not feeding on the source that makes the insecticidal protein available to the insects. "Pesticide activity" is preferably defined as toxic biological activity that can control harmful organisms such as insects, nematodes, fungi, bacteria or viruses by killing or destroying them. "Insecticidal activity" is preferably defined as toxic biological activity that can control insects by killing them. "Pesticide" is a drug having pesticide activity. "Insecticide" is a drug having insecticidal activity.

[0161] "Associated with / operatably linked to" refers to two nucleic acids that are physically or functionally related. For example, if two sequences are operatably linked, or if a regulatory DNA sequence is positioned in such a way that it can influence the expression level of a coding or structural DNA sequence, then the promoter or regulatory DNA sequence is said to be "associated with" the RNA or protein-coding DNA sequence.

[0162] As used herein, the terms “chimeric construct,” “chimeric gene,” “chimeric polynucleotide,” or “chimeric nucleic acid” (or similar terms) refer to a construct or molecule containing two or more polynucleotides of different origins constructed within a single nucleic acid molecule. The terms “chimeric construct,” “chimeric gene,” “chimeric polynucleotide,” or “chimeric nucleic acid” refer, but are not limited to, any construct or molecule containing (1) a polynucleotide (e.g., DNA) that contains regulatory and coding polynucleotides not found together in nature (i.e., at least one polynucleotide in the construct is heterogeneous with respect to at least one of the other polynucleotides in the construct), or (2) a polynucleotide that codes for a protein that is not contiguous in nature, or (3) a promoter that is not contiguous in nature. Furthermore, a chimeric construct, chimeric gene, chimeric polynucleotide, or chimeric nucleic acid may contain regulatory and coding polynucleotides from different origins, or may contain regulatory and coding polynucleotides from the same origin but sequenced differently from those found in nature. In one embodiment of the present invention, a chimeric construct, chimeric gene, chimeric polynucleotide, or chimeric nucleic acid comprises an expression cassette containing the polynucleotide of the present invention under the control of a regulatory polynucleotide, particularly in plants or bacteria under the control of a functional regulatory polynucleotide.

[0163] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA. Preferably, the RNA is then translated in an organism to produce a protein.

[0164] As used herein, “codon-optimized” sequence means a nucleotide sequence in which codons have been selected to reflect a particular codon bias that a host cell or organism may have. This is typically done in a manner that preserves the amino acid sequence of the polypeptide encoded by the optimized nucleotide sequence. In certain embodiments, the DNA sequence of a recombinant DNA construct includes a sequence that is codon-optimized for the cell in which the construct will be expressed (e.g., an animal, plant, or fungal cell). For example, a construct expressed in a plant cell may have all or part of its sequence (e.g., a first repressor element or gene expression element) that is codon-optimized for expression in plants. See, for example, U.S. Patent No. 6,121,014, which is incorporated herein by reference.

[0165] To “control” insects means to inhibit the ability of pests to survive, grow, feed, and / or reproduce through toxic effects, or to limit insect-related damage or loss to crop plants. To “control” insects may or may not mean killing them, but preferably it means killing them.

[0166] As used herein, the terms “contains” or “includes” specify the presence of the described features, integers, steps, actions, elements, or components, but do not exclude the presence or addition of one or more other features, integers, steps, actions, elements, components, or groups thereof.

[0167] As used herein, the transitional phrase (and grammatical variations) “essentially from” is to be interpreted as encompassing the specified materials or steps described in the claims and any that do not substantially alter the fundamental and novel features of the claimed invention. Therefore, as used in the claims of the invention, the term “essentially from” is not intended to be equivalent to “include.”

[0168] In relation to the present invention, "corresponding to" or "corresponding to" means that when the amino acid sequences of mutant or homologous Cry proteins are aligned with each other, amino acids corresponding to specific enumerated positions in the mutant or homologous protein are aligned with those positions in the reference protein, but do not necessarily have to be at 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: 10, then Gly51 in SEQ ID NO: 1 "corresponds" to Gly42 in SEQ ID NO: 1, or, for example, Val59 in SEQ ID NO: 10 "corresponds" to Ile50 in SEQ ID NO: 1.

[0169] As used herein, the term "Cry protein" means the Bacillus thuringiensis crystalline delta-endotoxin type insecticidal protein. The term "Cry protein" may refer to the protoxin form or any bioactive fragment or toxin thereof, including, for example, partially processed forms and mature toxin forms that do not include the N-terminal peptidyl fragment and / or the C-terminal protoxin tail.

[0170] "Delivering" an insecticidal protein means that the insecticidal protein comes into contact with an insect to produce a toxic effect and control the insect. Insecticidal proteins can be delivered in numerous recognized methods, for example, by transgenic plants expressing the insecticidal protein, formulated protein compositions, sprayable protein compositions, bait substrates, or any other toxin delivery systems recognized in the art.

[0171] The term "domain" refers to a set of amino acids conserved at a specific position along the sequence alignment of evolutionarily related proteins. While amino acids at other positions may differ between homologs, those highly conserved at a particular position indicate amino acids that may be essential to the protein's structure, stability, or function. Because they are identified by their high degree of conservation in the aligned sequence of a protein homolog family, they can be used as identifiers to determine whether any polypeptide of interest belongs to an already identified group of polypeptides.

[0172] "Effective pest control dose" means the concentration of an insecticidal protein that, by its toxic effect, inhibits the ability of insects to survive, grow, feed, and / or reproduce, or limits insect-related damage or loss to crop plants. "Effective pest control dose" may or may not mean killing insects, but preferably it means killing insects.

[0173] As used herein, “expression cassette” means a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell, including a promoter operably linked to the target nucleotide sequence operably linked to a termination signal. An expression cassette typically also includes sequences necessary for the proper translation of the nucleotide sequence. An expression cassette containing the target nucleotide sequence is heterologous in at least one of its components with respect to at least one of the other components. An expression cassette may be naturally occurring but obtained in a recombinant form useful for heterologous expression. However, typically, an expression cassette is heterologous with respect to the host; that is, the specific nucleic acid sequence in the expression cassette does not naturally exist in the host cell but must have been introduced into the host cell or its ancestor by a transformation event. Expression of the nucleotide sequence in an expression cassette may be under the control of a constitutive promoter or an inductive promoter that initiates transcription only when the host cell is exposed to a specific external stimulus. In multicellular organisms such as plants, the promoter may also be specific to a particular tissue or organ or developmental stage.

[0174] An expression cassette containing the target nucleotide sequence can be a chimeric one, meaning that at least one of its components is heterologous with respect to at least one of the other components. An expression cassette may also contain a native promoter that drives its native gene. However, expression cassettes are obtained in recombinant form, which is useful for heterologous expression. Such use of an expression cassette means that it is not naturally present in the cells into which it is introduced.

[0175] The expression cassette may optionally include a transcription and / or translation termination region (i.e., termination region) that is functional in the plant. A variety of transcription terminators are available for use in the expression cassette and are involved in the termination of transcription and precise mRNA polyadenylation across the heterologous nucleotide sequence of interest. The termination region may be native to the transcription start region, native to the operably linked nucleotide sequence of interest, native to the plant host, or of another origin (i.e., exogenous or heterologous to the promoter, the nucleotide sequence of interest, the plant host, or any combination thereof). Suitable transcription terminators include, but are not limited to, the CAMV 35S terminator, the tml terminator, the nopalin synthase terminator, and / or the pea rbcs E9 terminator. These can be used in both monocots and dicots. Furthermore, native transcription terminators of coding sequences can be used. Any available terminator known to be functional in the plant can be used in connection with the present invention.

[0176] When used in relation to polynucleotides such as genes, ORFs or parts thereof, or transgenes in plants, the term “expression” refers to the process of converting the genetic information encoded by a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) by “transcription” of that gene (i.e., via the enzymatic action of RNA polymerase), and, where applicable (e.g., if the gene encodes a protein), converting it into a protein by “translation” of the mRNA. Gene expression can be controlled at many stages of this process. For example, in the case of antisense or dsRNA constructs, expression may refer to the transcription of antisense RNA only or dsRNA only, respectively. In embodiments, “expression” refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. “Expression” may also refer to the production of a protein.

[0177] A "gene" is a defined region located within the genome that contains a coding nucleic acid sequence, as well as other primarily regulatory nucleic acids that are typically involved in the control of the expression of the coding portion, i.e., transcription and translation. A gene may also contain other 5' and 3' untranslated sequences and a termination sequence. Further elements that may be present are, for example, introns. The regulatory nucleic acid sequence of a gene is usually not operably ligated to a naturally occurring related nucleic acid sequence and can therefore be a chimeric gene.

[0178] "Target gene" refers to any nucleic acid molecule that, when introduced into a plant, confers a desired trait to the plant, such as antibiotic resistance, virus resistance, insect resistance, disease resistance or resistance to other pests, herbicide tolerance, abiotic stress tolerance, male sterility, modified fatty acid metabolism, modified carbohydrate metabolism, improved nutritional value, improved performance in industrial processes, or altered reproductive capacity. "Target gene" may also be introduced into a plant for the production of commercially valuable enzymes or metabolites in the plant.

[0179] "Intestinal proteases" are proteases found naturally in the digestive tract of insects. These proteases are typically involved in the digestion of ingested proteins. Examples of intestinal proteases include trypsins, which typically cleave the C-terminal peptide of lysine (K) or arginine (R) residues, and chymotrypsins, which typically cleave the C-terminal peptide of phenylalanine (F), tryptophan (W), or tyrosine (Y).

[0180] A "heterogeneous" nucleic acid sequence or molecule is a nucleic acid sequence or molecule that contains multiple copies of a naturally occurring nucleic acid sequence that are not naturally associated with the host cell into which it is introduced. Heterogeneous nucleic acid sequences or molecules may include chimeric sequences, such as chimeric expression cassettes, in which the promoter and coding regions originate from multiple organismal sources. The promoter sequence may be a constitutive promoter sequence, a tissue-specific promoter sequence, a chemoinducible promoter sequence, a wound-induced promoter sequence, a stress-induced promoter sequence, or a developmental stage-specific promoter sequence.

[0181] A "homologous" nucleic acid sequence is a nucleic acid sequence that is naturally associated with the host cell into which it is introduced.

[0182] "Homologous recombination" is the mutual exchange of nucleic acid fragments between homologous nucleic acid molecules.

[0183] "Identity" or "percent identity" refers to the degree of similarity between two nucleotide or amino acid sequences. For sequence comparison, one sequence typically serves as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the specified program parameters. The phrase "substantially identical" in relation to two nucleotides or two amino acid sequences refers to two or more sequences or subsequences that have identity of at least about 50 nucleotides or amino acid residues when compared and aligned for maximum agreement, as measured using one of the following sequence comparison algorithms or by visual inspection. In a particular embodiment, substantially identical sequences have identity of at least about 60, or at least about 70, or at least about 80, or at least about 85, or even at least about 90 or 95 nucleotides or amino acid residues. In certain embodiments, substantial identity exists over a region of sequences having a length of at least about 50 residues or over a region of at least about 100 residues, or sequences are substantially identical over at least about 150 residues. In further embodiments, sequences are substantially identical if they are identical over the entire length of the coding region.

[0184] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Packag, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see Ausubel et al. below for general information).

[0185] One example of an algorithm suitable for determining sequence identity and sequence similarity percentages is the BLAST algorithm, described in Altschul et al., J.Mol.Biol.215:403-410 (1990). Software for performing BLAST analysis is available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match when aligned with words of the same length in the database sequences or satisfy a threshold score T of some positive value. T is called the adjacent word score threshold (Altschul et al., 1990). These initial adjacent word hits serve as a seed to initiate a search for longer HSPs that contain them. Word hits are then extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Word hit expansion in each direction stops when the cumulative alignment score falls by amount X from its maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments, or when it reaches the end of any sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and rate of alignment. The BLASTN program (for nucleotide sequences) uses a word length of 11 (W), an expected value of 10 (E), a cutoff value of 100, M=5, N=-4, and comparison of both strands as defaults.For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix as defaults (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0186] In addition to calculating the sequence identity percentage, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that the match between two nucleotide or amino acid sequences occurs by chance. For example, if the minimum sum probability in the comparison between the test nucleic acid sequence and 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, the test nucleic acid sequence is considered similar to the reference sequence.

[0187] Another widely used and accepted computer program for performing sequence alignment is CLUSTALW v1.6 (Thompson, et al. Nuc. Acids Res., 22:4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids and multiplied by 100 to obtain the identity percentage. For example, if two 580 base pair sequences have 145 matching bases, they are 25 percent identical. If the two sequences being compared have different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there are 100 matching amino acids between a 200-amino acid protein and a 400-amino acid protein, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids, the number of matches is divided by 150 (for nucleic acid bases) or 50 (for amino acids) and multiplied by 100 to obtain the identity percentage.

[0188] Another indicator that two nucleic acids are substantially identical is that the two molecules hybridize with each other under stringent conditions. The phrase "specifically hybridize" refers to a molecule binding, doubling, or hybridizing to only a specific nucleotide sequence under stringent conditions when the sequence is present in a complex mixture (e.g., whole cell) of DNA or RNA. "Substantially binding" refers to complementary hybridization between the probe nucleic acid and the target nucleic acid, encompassing small discrepancies that can be adapted by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0189] In relation to nucleic acid hybridization experiments such as Southern and Northern hybridization, "stringent hybridization conditions" and "stringent hybridization washing conditions" are sequence-dependent and differ under different environmental parameters. Longer sequences hybridize more specifically at higher temperatures. Extensive guidelines for nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York. Generally, highly stringent hybridization and washing conditions involve the thermal melting point (T) of a particular sequence at a given ionic strength and pH. m The temperature is selected to be approximately 5°C lower than the target temperature. Typically, under "stringent conditions," the probe will hybridize to its target subsequence but not to other sequences.

[0190] T mThis is the temperature at which 50 of the target sequence hybridizes to a probe with a perfect match (under specified ionic strength and pH). Very stringent conditions are the T of a particular probe. m Selected to be equal to . An example of stringent hybridization conditions for hybridization of complementary nucleic acids having more than 100 complementary residues on the filter in Southern or Northern blotting is 50 formamides with 1 mg heparin at 42°C, with this hybridization performed overnight. An example of highly stringent washing conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is washing with 0.2 × SSC at 65°C for 15 minutes (see Sambrook below for a description of SSC buffer). Often, a low-stringency wash is performed before a high-stringency wash to remove background probe signals. For example, an example of a moderate stringency wash for double helix of more than 100 nucleotides is 1 × SSC at 45°C for 15 minutes. For example, an example of low-grade stringency washing for double hemispheres 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 pH of 7.0–8.3 with less than 1.0 M Na ions, a salt concentration of about 0.01–1.0 M Na ions (or other salts), and a temperature of at least 30°C. Stringent conditions can also be achieved by adding destabilizers such as formamide. Generally, a signal-to-noise ratio twice (or higher) that is observed for unrelated probes in a particular hybridization assay indicates the detection of a particular hybridization. Nucleic acids that do not hybridize to one another under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This occurs, for example, when copies of nucleic acids are made using the maximum codon degeneracy permitted by the gene code.

[0191] The following are examples of a set of hybridization / washing conditions that may be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequence of the present invention. The reference nucleotide sequence is preferably washed in 7 sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA at 50°C, with washing in 2XSSC and 0.1 SDS at 50°C; more preferably in 7 sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA at 50°C, with washing in 1XSSC and 0.1 SDS at 50°C; and more preferably further washed in 0.5XSSC and 0.1 SDS at 50°C, with washing in 7 sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA at 50°C. Hybridization to a reference nucleotide sequence occurs in sodium decyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, preferably with washing in 0.1 XSSC and 0.1 SDS at 50°C, and more preferably in sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, preferably with washing in 0.1 XSSC and 0.1 SDS at 65°C.

[0192] A further indicator that two nucleic acids or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross-reactive to or specifically binds to the protein encoded by the second nucleic acid. Therefore, if, for example, two proteins differ only by conservative substitutions, the proteins are usually substantially identical to the second protein.

[0193] When a nucleotide sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by a reference nucleotide sequence, the nucleotide sequence is said to "isocode" with the reference nucleotide sequence.

[0194] "Isolated" nucleic acid molecules, or isolated polynucleotides, or isolated Txp40 proteins are nucleic acid molecules, or polynucleotides, or proteins that exist in a state separate from their natural environment by human intervention and are therefore not natural products. Isolated nucleic acid molecules, or polynucleotides, or proteins may exist in a purified form or, but are not limited to, in a non-natural environment such as recombinant microbial cells, plant cells, plant tissues, or plants.

[0195] The terms "motif," "consensus sequence," or "signature" refer to short, conserved regions within evolutionarily related protein sequences. Motifs are often highly conserved portions of a domain, but may contain only a portion of the domain or may be located outside the conserved domain (if all amino acids of the motif fall outside the defined domain).

[0196] "Natural" or "wild-type" nucleic acids, polynucleotides, nucleotide sequences, polypeptides, or amino acid sequences refer to naturally occurring or endogenous nucleic acids, polynucleotides, nucleotide sequences, polypeptides, or amino acid sequences.

[0197] A "nucleic acid molecule" or "nucleotide sequence" is a segment of single-stranded or double-stranded DNA or RNA that can be isolated from any origin. In relation to the present invention, a nucleic acid molecule is typically a segment of DNA. In some embodiments, the nucleic acid molecule of the present invention is an isolated nucleic acid molecule.

[0198] The terms “nucleic acid molecule” and “polynucleotide” may be used interchangeably herein.

[0199] "Operatively linked" refers to an association of polynucleotides on a single nucleic acid fragment such that the function of one influences the function of the other. For example, a promoter is operationally linked to a coding polynucleotide or functional RNA (i.e., the coding polynucleotide or functional RNA is under the transcriptional control of the promoter) when it can influence the expression of that coding polynucleotide or functional RNA. Sense-oriented or antisense-oriented coding polynucleotides can be operationally linked to regulatory polynucleotides.

[0200] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein.

[0201] "Plant" refers to any plant at any stage of development, especially seed plants.

[0202] A "plant cell" is a structural and physiological unit of a plant, including its protoplast and cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or it may be part of a more highly organized unit, such as plant tissue, a plant organ, or the whole plant.

[0203] "Plant cell cultures" refer to cultures of plant units at various developmental stages, such as protoplasts, cultured cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos.

[0204] "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.

[0205] "Plant organs" are clearly defined, visible, structured, and differentiated parts of a plant, such as roots, stems, leaves, buds, or embryos.

[0206] As used herein, “plant tissue” means a group of plant cells organized into structural and functional units. This includes any tissue of a plant in a plant body or culture. The term includes, but is not limited to, the whole plant, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term with or without any particular type of plant tissue listed above or otherwise included in this definition is not intended to exclude any other type of plant tissue.

[0207] It will be understood that a “part” or “fragment” of the polypeptide of the present invention means an amino acid sequence that is shorter in length than the reference amino acid sequence of the polypeptide of the present invention. Such a part or fragment according to the present invention may, where appropriate, be included in a larger polypeptide (e.g., a tagged or fusion protein) of which it is a component. In embodiments, the “part” or “fragment” substantially retains insecticidal activity (e.g., at least 40, 50, 60, 70, 80, 85, 90, 95 or even 100 of the activity of the full-length protein, or has even greater insecticidal activity than the full-length protein).

[0208] A "promoter" is an uncoding DNA sequence upstream of a coding region that contains an RNA polymerase binding site and initiates DNA transcription. The promoter region may also contain other elements that play a role in regulating gene expression.

[0209] As used herein, the term “recombinant” refers to nucleic acids (e.g., DNA or RNA), proteins, or forms of organisms that are not typically found in nature and are therefore produced by human intervention. As used herein, “recombinant nucleic acid molecule” includes nucleic acid molecules containing combinations of polynucleotides that do not typically coexist in nature and are the result of human intervention, such as nucleic acid molecules composed of a combination of at least two heterogeneous polynucleotides, or artificially synthesized nucleic acid molecules, such as polynucleotides synthesized using constructed nucleotide sequences that deviate from polynucleotides that typically coexist in nature, or nucleic acid molecules containing transgenes artificially incorporated into the genomic DNA of a host cell and related adjacent DNA of the host cell's genome. Another example of a recombinant nucleic acid molecule is a DNA molecule obtained from the insertion of a transgene into the genomic DNA of a plant, which may ultimately result in the expression of recombinant RNA or protein molecules in that organism. As used herein, “recombinant plant” is a plant that does not typically coexist in nature, is the result of human intervention, and contains transgenes or heterogeneous nucleic acid molecules incorporated into its genome. As a result of such genomic modification, the recombinant plant is distinctly different from the related wild-type plant.

[0210] As used herein, the terms “reduce,” “reduced,” “to reduce,” “decrease,” “reduce,” and “suppress” (and their grammatical variations) and similar terms refer to a reduction in the survival, growth, and / or reproduction of plant pests, for example, by contacting plants with the polypeptide of the present invention (e.g., by transgenic expression or topical application). This reduction in survival, growth, and / or reproduction may be at levels observed in the absence of the polypeptide of the present invention (e.g., plants that are not transgenically expressing the polypeptide or have not been topically treated with the polypeptide). Accordingly, in the embodiments, the terms “reduce,” “reduced,” “to reduce,” “decrease,” “reduce,” and “suppress” (and their grammatical variations) and similar terms mean a reduction of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more compared to plants not in contact with the polypeptide of the present invention (e.g., plants not transgenically expressing the polypeptide or not topically treated with the polypeptide). In each embodiment, as a result of the reduction, the detectable survival, growth, and / or reproduction of plant pests is none or substantially none (i.e., a small amount, e.g., less than about 10, less than about 5, or even less than about 1).

[0211] A "regulatory element" refers to a sequence involved in controlling the expression of a nucleotide sequence. Regulatory elements include promoters that are operably ligated to the target nucleotide sequence and termination signals. Regulatory elements typically also include sequences necessary for the proper translation of the nucleotide sequence.

[0212] "Transformation" is a process for introducing a different nucleic acid into a host cell or organism. In certain embodiments, "transformation" means the stable integration of a DNA molecule into the genome (nucleus or plastids) of the organism in question.

[0213] "Transformed / transgenic / recombinant" refers to a host organism, such as bacteria or plants, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host genome, or it can exist as an extrachromosomal molecule. Such extrachromosomal molecules may self-replicate. Transformed cells, tissues, or plants are understood to include not only the final product of the transformation process but also their transgenic offspring. "Untransformed," "non-transgenic," or "non-recombinant" hosts refer to wild-type organisms, such as bacteria or plants that do not contain heterologous nucleic acid molecules.

[0214] As used herein, “Txp40 protein” is a naturally occurring toxin produced by two bacterial genera, Xenorhabdus and Photorhabdus, which are symbiotically associated with the nematode genera Steinernema and Heterrhabditis, respectively. Nematode-bacterial pairs can invade and kill certain insects. The Txp40 protein was first isolated from P. luminescens and shown to possess injectable toxicity against several pests.

[0215] The "mutant Txp40 protein" according to the present invention means a non-naturally occurring modified protein that is orally toxic to Spodoptera pests, particularly the fall armyworm (Spodoptera frugiperda), and has at least 95% identity with SEQ ID NO: 1.

[0216] This invention relates to the Txp40 insecticidal protein, which is shown herein to be remarkably orally toxic to the fall armyworm (FAW), a pest species resistant to numerous types of insecticidal proteins, including the Cry protein derived from Bacillus thuringiensis (Bt). Although the Txp40 protein is known in the art for its natural biology, it has been shown to be insecticidal to certain insects, primarily by direct injection into the hemocoel of insects (see, for example, U.S. Patent No. 6,630,619; Park et al. 2012. J. Agric. Food Chem. 60:4053-4059 and Brown et al. 2006. App. Environ. Microbiol. 72:1653-1662). Such direct injection mimics the natural pathway in which the Txp40 protein is delivered to insects via pathogenic nematodes, such as those of the families Heterorhabditidae and Steinernematiidae, which first invade the hemocoel of the target insect and release pathogenic bacteria, such as Photorhabdus and Xenorhabdus bacteria that produce the Txp40 protein. One group has reported oral toxicity of the Txp40 protein only in insects sensitive to a number of insecticidal proteins, including the greater wax moth, Galleria mellonella (Mathur et al. 2018. Toxicon 154:59-73), insects that are not crop pests, and insects that are highly sensitive to a number of Cry proteins. This invention represents the first known disclosure regarding the oral toxicity of the Txp40 protein against FAW pests. Accordingly, this invention provides a novel method for controlling FAW pest populations using the Txp40 insecticidal protein and variants of the Txp40 protein.The novel method of the present invention is particularly useful for controlling FAW populations that are resistant to or may become resistant to other insect control agents such as Cry protein and / or Vip3 protein.

[0217] Accordingly, in one embodiment, the present invention provides a method for controlling Spodoptera pests, comprising the step of contacting Spodoptera pests with a Txp40 protein that contains, is essentially, or comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 1. In another embodiment, the Txp40 protein contains, is essentially, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1 over its entire length. In another embodiment, the Txp40 protein contains, is essentially, or comprises any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs. 74-136. In another embodiment, the method comprises the step of delivering an effective amount of the Txp40 protein of the present invention to Spodoptera pests or their environment, for example, by oral delivery. Generally, for the Txp40 protein to be effective, it is ingested orally by Spodoptera pests. Examples of methods for orally delivering the Txp40 protein of the present invention to Spodoptera pests include, but are not limited to, (1) providing the protein in a transgenic plant (where the insect eats (ingests) one or more parts of the transgenic plant and thereby ingests the Txp40 polypeptide expressed in the transgenic plant); (2) in a formulated protein composition that can be applied to or incorporated into, for example, an insect growth medium; (3) in a protein composition that can be applied to a surface, for example, sprayed on the surface of a plant part (which is then ingested by the insect when it eats one or more of the sprayed plant parts); (4) in a feed substrate; or (5) providing the protein in any other protein delivery system recognized in the art. Thus, the toxic protein of the present invention can be delivered using any method of oral delivery to Spodoptera pests.In some specific embodiments, the Txp40 insecticidal protein of the present invention is delivered orally to a Spodoptera pest, for example, here the insect ingests one or more parts of the transgenic plant of the present invention. In a further embodiment, the Spodoptera pest is Spodoptera frugiperda (fall armyworm, FAW).

[0218] In other embodiments, the Txp40 insecticidal protein of the present invention is delivered orally to Spodoptera pests, such as the fall armyworm (Spodoptera frugiperda), where the insect ingests one or more parts of a plant sprayed with a composition containing the Txp40 insecticidal protein of the present invention. Delivery of the composition of the present invention to a plant surface can be carried out using any method known to those skilled in the art for applying compounds, compositions, formulations, etc., to a plant surface. Some non-limiting examples of delivery to or contact with a plant or part thereof include spraying, dusting, sprinkling, scattering, misting, atomizing, broadcasting, immersion, soil injection, soil mixing, drenching (e.g., root, soil treatment), dipping, injection, coating, leaf or stem infiltration, lateral application or seed treatment, and combinations thereof. These and other procedures for bringing plants or parts thereof into contact with compounds, compositions, or formulations are well known to those skilled in the art.

[0219] In further embodiments, the present invention provides a method for controlling FAW pests or hazardous organism populations resistant to Cry proteins and / or plant insecticidal proteins, such as Vip3, comprising the step of delivering an effective amount of the Txp40 protein or composition of the present invention to the FAW pest or hazardous organism population or its environment. In one embodiment, the Txp40 protein comprises, essentially comprises, or consists of an amino acid sequence having at least 95% identity with SEQ ID NO: 1. In another embodiment, the Txp40 protein comprises, essentially comprises, or consists of an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1 over its entire length. In another embodiment, the amino acid sequence of the Txp40 protein comprises, essentially comprises, or consists of any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs. 74-136. In another embodiment, Vip3 is the Vip3A protein. In another embodiment, the Vip3A protein is the Vip3Aa20 protein expressed in a corn plant containing the transgenic event MIR162. In yet another embodiment, the Cry protein is the Cry1A, Cry1B, Cry1C, Cry1D, Cry1F, Cry1J, or Cry2A protein. In yet another embodiment, the Cry1F protein is the Cry1Fa protein expressed in a corn plant containing the transgenic event TC1507.

[0220] In other embodiments, the present invention provides a method for preventing the development of resistance to the Cry protein and / or Vip3 protein expressed in a transgenic plant in a population of Spodoptera pests, such as the fall armyworm, the method comprising the steps of delivering a transgenic plant to a target Spodoptera population, the transgenic plant comprising a polynucleotide containing a nucleotide sequence encoding the Vip3 protein and / or a nucleotide sequence encoding the Cry protein; and a polynucleotide expression cassette or vector of the present invention expressing the Txp40 insecticidal protein of the present invention. In other embodiments, the Vip3 protein is the Vip3A protein. In other embodiments, the Vip3A protein is the Vip3Aa20 protein. In yet another embodiment, the Cry protein is the Cry1A, Cry1B, Cry1C, Cry1D, Cry1F, Cry1J, or Cry2A protein. In yet another embodiment, the Cry1F protein is the Cry1Fa protein. In yet another embodiment, the transgenic plant is a corn plant containing event MIR162 and / or event TC1507. In one embodiment, the Txp40 protein contains, essentially, or consists of an amino acid sequence having at least 95% identity with SEQ ID NO: 1 over its entire length. In another embodiment, the Txp40 protein contains, essentially, or consists of an amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NOs. 74-136. According to the above embodiments, the transgenic plant may include a breeding stack of two or more genes encoding the Txp40 insecticidal protein of the present invention, a molecular stack of two or more genes encoding the Txp40 insecticidal protein of the present invention, or a combination of both.

[0221] The present invention also provides a mutant Txp40 insecticidal protein comprising, essentially, or consisting of an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO: 1, wherein the mutant Txp40 protein has oral activity against Spodoptera pests, particularly Spodoptera frugiperda (Fallopia fulgiperda, FAW) pests. In one embodiment, the mutant Txp40 protein of the present invention comprises, essentially, or consists of an amino acid sequence of SEQ ID NO: 2 or any of SEQ ID NOs. 74-136.

[0222] In one embodiment, the present invention provides a mutant Txp40 insecticidal protein that has at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO: 1 and includes amino acid substitutions compared to SEQ ID NO: 1. In other embodiments, the present invention has at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO: 1, and compared to SEQ ID NO: 1, the amino acid positions 10, 11, 26, 31, 38, 46, 48, 49, 73, 75, 86, 96, 101, 103, 111, 116, 119, 133, 134, 143, 155, 167 The present invention provides a mutant Txp40 insecticidal protein having amino acid substitutions at positions corresponding to 169, 170, 182, 186, 187, 191, 200, 208, 209, 210, 213, 217, 221, 226, 238, 240, 247, 250, 252, 256, 257, 263, 265, 266, 271, 275, 284, 293, 296, 305, 308, 309, 311, 313, 315, 320, 326, 328, or 333, or any combination thereof.

[0223] In one embodiment, the present invention provides a mutant Txp40 insecticidal protein that has at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO: 1, and has amino acid substitutions at positions corresponding to amino acid positions 119 and / or 213 of SEQ ID NO: 1, compared to SEQ ID NO: 1. In another embodiment, the mutant Txp40 protein has A at the position corresponding to amino acid position 119 and / or A at the position corresponding to amino acid position 213 of SEQ ID NO: 1. In another embodiment, the mutant Txp40 protein has the K119A and / or K213A substitution in SEQ ID NO: 1. In another embodiment, the mutant Txp40 protein contains, is essentially derived from, or consists of the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 26, or SEQ ID NO: 127.

[0224] In one embodiment, the present invention provides a mutant Txp40 insecticidal protein that has at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO: 1, and has amino acid substitutions at positions corresponding to amino acid positions 11, 119, and / or 213 of SEQ ID NO: 1, compared to SEQ ID NO: 1. In another embodiment, the mutant Txp40 protein has Y at the position corresponding to amino acid position 11 of SEQ ID NO: 1, A at the position corresponding to amino acid position 119 of SEQ ID NO: 1, and / or A at the position corresponding to amino acid position 213 of SEQ ID NO: 1. In yet another embodiment, the mutant Txp40 protein is SEQ ID NO: 1, where amino acid S at position 11 is substituted with Y, amino acid K at position 119 is substituted with A, and / or amino acid K at position 213 is substituted with A. In other embodiments, the mutant Txp40 protein contains, is essentially, or consists of the amino acid sequence of SEQ ID NO: 131.

[0225] It is recognized that the nucleotide sequence encoding the Txp40 protein of the present invention can be modified by various methods, and that these modifications may result in nucleotide sequences encoding mutant Txp40 proteins having different amino acid sequences from those encoded by the naturally occurring Txp40 protein. The Txp40 protein can be modified by various methods, including amino acid substitutions, deletions, cleavages, and insertions of one or more amino acids in SEQ ID NO: 1, including up to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 17, or 20 amino acid substitutions, deletions, or insertions. Methods of such manipulation are generally known in the art. For example, amino acid sequence mutants of the natural Txp40 protein can be prepared by mutations in the polynucleotide encoding the Txp40 protein. This can be achieved by one of several forms of mutagenesis or by directional evolution. In some embodiments, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such mutants may have the desired insecticidal activity. In other embodiments, the resulting mutant Txp40 protein may have increased insecticidal activity, particularly against Spodoptera pests, compared to the wild-type Txp40 protein. In other embodiments, the resulting mutant protein is encoded by a synthetic mutant polynucleotide. In yet another embodiment, the Txp40 protein of the present invention is encoded by a nucleic acid molecule containing, essentially having, or consisting of any of the nucleotide sequences of SEQ ID NOs: 11-73.

[0226] According to one embodiment, the present invention provides a synthetic polynucleotide comprising, essentially, or consisting of a nucleotide sequence encoding the Txp40 protein of the present invention, which is orally toxic to Spodoptera pests, particularly the fall armyworm, wherein the nucleotide sequence has codons optimized for expression in a transgenic organism. In another embodiment, the transgenic organism is a transgenic bacterium or a transgenic plant. In a further embodiment, the transgenic plant is a transgenic cone plant. Often, microbial genes can be expressed at high levels in plants without modification, but microbial nucleotides having codons that are not selected in plants can result in low expression in transgenic plants. It is known in the art that living organisms have specific selectivity for codon use, and that the codons of the nucleotide sequences described in the present invention can be modified to match plant selectivity while maintaining the amino acids they encode. Furthermore, it is known in the art that high expression in plants, such as corn plants, can be achieved from coding sequences having a GC content of at least about 35, or at least about 45, or at least about 50, or at least about 60. Microbial nucleotide sequences with low GC content may be poorly expressed in plants. Certain nucleotide sequences can be adequately expressed in both monocotyledonous and dicotyledonous plant species, but sequences may be modified to account for the specific codon selectivity and GC content selectivity of monocotyledonous or dicotyledonous plants (as these selectivity has been shown to differ) (Murray et al. Nucl. Acids Res. 17:477-498 (1989)). In addition, in embodiments, nucleotide sequences may be modified to remove non-canonical splice sites that may cause message cleavage.Such modifications to nucleotide sequences can be performed using site-directed mutagenesis, PCR, and well-known techniques for synthetic gene construction using methods described, for example, U.S. Patent Nos. 5,625,136, 5,500,365, and 6,013,523. In one embodiment of the present invention, the codon-optimized nucleotide sequence of the present invention comprises, is essentially derived from, or consists of a nucleotide sequence having a GC content of at least 50, 55, 60, or at least 65. In yet another embodiment, the codon-optimized nucleotide sequence comprises, is essentially derived from, or consists of any of the nucleotide sequences of SEQ ID NOs. 4, SEQ ID NOs. 5, or SEQ ID NOs. 11-73. Those skilled in the art will recognize that such GC content can be achieved in several different ways. For example, such synthetic coding sequences or polynucleotides are constructed according to the procedure disclosed in U.S. Patent No. 5,625,136. This procedure uses a maize-selected codon, i.e., the single codon that most frequently codes for its amino acid in maize. The maize-selected codon for a particular amino acid can be obtained, for example, from a known gene sequence derived from maize. For example, the use of maize codons for 28 genes from maize plants is found in Murray et al., Nucleic Acids Research 17:477-498 (1989). It is recognized that codons optimized for expression in one plant species may function in other plant species, but probably not to the same level as the plant species in which the codons were optimized. In this way, nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of a nucleotide sequence can be optimized or synthesized. That is, a polynucleotide may contain a nucleotide sequence in which part is the natural sequence and part is the codon-optimized sequence.

[0227] In one embodiment of the present invention, a chimeric gene is provided which includes a promoter operably linked to a heterologous nucleic acid molecule containing a nucleotide sequence encoding a Txp40 protein having insecticidal activity against the Spodoptera pest, wherein the nucleotide sequence is (a) any of SEQ ID NOs: 3-5 or 11-73; (b) having at least 95% identity to any of SEQ ID NOs: 3-5 or 11-73; (c) encoding a protein containing an amino acid sequence of any of SEQ ID NOs: 1, 2 or 74-136; (d) encoding a protein containing an amino acid sequence having at least 95% identity to SEQ ID NO: 1; or (e) complementary to any one of the nucleotide sequences of (a) to (d).

[0228] In one embodiment, the nucleic acid molecule comprises a maize codon-optimized nucleotide sequence. In another embodiment, the promoter is a plant-expression promoter, particularly one expressed in maize plants. For example, but are not limited to, plant-expression promoters can be selected from the group of promoters consisting of ubiquitin, cestrum yellow virus, maize TrpA, OsMADS 6, maize H3 histone, bacteriophage T3 gene 9 5' UTR, maize sucrose synthetase 1, maize alcohol dehydrogenase 1, maize light-harvesting complex, maize heat shock protein, maize mtl, pea small subunit RuBP carboxylase, comedin, comediclophyllin, Ti plasmid mannopin synthase, Ti plasmid nopalin synthase, petunia chalcone isomerase, bean glycine-rich protein 1, potato patatin, lectin, CaMV 35S, and S-E9 small subunit RuBP carboxylase promoters.

[0229] Many promoters derived from dicotyledonous plants have been shown to function in monocotyledonous plants, and vice versa; however, in this embodiment, dicotyledonous promoters are selected for expression in dicotyledonous plants, and monocotyledonous promoters are selected for expression in monocotyledonous plants. However, there are no restrictions on the origin of the selected promoters; it is sufficient that they function in promoting the expression of the desired nucleotide sequence in the cell.

[0230] The choice of promoter may vary depending on the temporal and spatial requirements for expression and the host cells being transformed. For example, the expression of the nucleotide sequence of the present invention may occur within any plant and / or plant part (e.g., within leaves, stalks or stems, within inflorescences, within inflorescences (e.g., spikes, panicles, rachis, etc.), within roots, seeds, and / or seedlings). For example, if expression in a specific tissue or organ is desired, a tissue-specific or tissue-selective promoter can be used (e.g., a root-specific / selective promoter). Conversely, if stimulus-responsive expression is desired, a promoter that can be induced by a stimulus or chemical can be used. If a relatively constant level of continuous expression across plant cells is desired, a constitutive promoter can be used.

[0231] Useful promoters in the present invention include, but are not limited to, those that structurally promote the expression of nucleotide sequences, those that promote expression when induced, and those that promote expression in a tissue-specific or embryologically specific manner. Various types of these promoters are known in the art.

[0232] Appropriate constitutive promoters include, for example, the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985); the Arabidopsis At6669 promoter (see PCT Public International Publication No. 04081173); maize Ubi1 (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); comeactin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); and GOS2 (de Pater et al., Plant J This includes November;2(6):837-44, 1992; ubiquitin (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); comecyclophyllin (Bucholz et al., Plant Mol Biol. 25(5):837-43, 1994); maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231:276-285, 1992); actin 2 (An et al., Plant J. 10(1); 107-121, 1996), constitutive root apex CT2 promoter (SEQ ID NO: 1535; see also PCT application Israeli patent application publication No. 2005 / 000627) and synthetic super MAS (Ni et al., The Plant Journal 7:661-76, 1995). Other constituent promoters include those specified in U.S. Patent Nos. 5,659,026, 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, and 5,608,142.

[0233] Tissue-specific or tissue-selective promoters useful for the expression of the polypeptide of the present invention in plants, optionally in maize, include those that direct expression in roots, pith, leaves, or pollen. Appropriate tissue-specific promoters include, but are not limited to, leaf-specific promoters (e.g., those described by Yamamoto et al., Plant J.12:255-265,1997; Kwon et al., Plant Physiol.105:357-67,1994; Yamamoto et al., Plant Cell Physiol.35:773-778,1994; Gotor et al., Plant J.3:509-18,1993; Orozco et al., Plant Mol.Biol.23:1129-1138,1993; and Matsuoka et al., Proc.Natl.Acad.Sci.USA 90:9586-9590,1993), seed-selective promoters (e.g., those derived from seed-specific genes; Simon, et al., Plant Mol.Biol.5.191,1985; Scofield, et al., J.Biol.Chem.262:12202,1987; Baszczynski, et al., Plant Mol.Biol.14:633,1990, Brazil nut albumin (Pearson et al., Plant Mol.Biol.18:235-245,1992), legumin (Ellis, et al., Plant Mol.Biol.10:203-214,1988), glutelin (Takaiwa, et al., Mol.Gen.Genet.208:15-22,1986; Takaiwa, et al., FEBS Letts.221:43-47,1987), zein (Matzke et al., Plant Mol Biol,143).323-32 1990), napA (Stalberg, et al., Planta 199:515-519, 1996), wheat SPA (Albanietal, Plant Cell, 9:171-184, 1997), sunflower oleosin (Cummins, et al., Plant Mol. Biol.(19:873-876,1992), endosperm-specific promoters (e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81-90,1989; NAR 17:461-2), wheat a, b and g gliadin (EMB03:1409-15,1984), barley ltrl promoter, barley B1, C, D hordein (Theor Appl Gen 98:1253-62,1999; Plant J 4:343-55,1993; Mol Gen Genet 250:750-60,1996), barley DOF (Mena et al., The Plant) Journal, 116(1):53-62, 1998), Biz2 (European Patent No. 99106056.7), Synthetic promoter (Vicente-Carbajosa et al., Plant J. 13:629-640, 1998), Rice prolamin NRP33, Rice globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885-889, 1998), Rice alpha-globulin REB / OHP-1 (Nakase et al. Plant Mol. Biol. 33:513-S22, 1997), Rice ADP-glucose PP (Trans Res 6:157-68, 1997), Maize ESR gene family (Plant J 12:235-46, 1997), sorghum gamma-caphirin (Plant Mol. Biol 32:1029-35, 1996), embryo-specific promoters (e.g., rice OSH1; Sato et al., Proc. Nati. Acad. Sci. USA, 93:8117-8122), KNOX (Postma-Haarsma et al., Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et al., J. Biochem., 123:386, 1998), flower-specific promoters, e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al.) al., Mol.Gen Genet.This includes apetala-3 and promoters specific to plant reproductive tissues (e.g., the OsMADS promoter; U.S. Patent Application Publication No. 2007 / 0006344).

[0234] Examples of promoters suitable for selective expression in green tissue include numerous promoters that regulate photosynthetic genes, many of which have been cloned from both monocots and dicots. One such promoter is the maize PEPC promoter derived from the phosphoenolcarboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)). Another promoter for root-specific expression is described by de Framond (FEBS 290:103-106 (1991) or U.S. Patent No. 5,466,785). Another promoter useful in the present invention is a naturally occurring stem-specific promoter that promotes the expression of the maize trpA gene, described in U.S. Patent No. 5,625,136.

[0235] Furthermore, promoters that function in plastids may be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 9 5' UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters useful in the present invention, but not limited to, include the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0236] In one embodiment of the present invention, an inducible promoter may be used. Therefore, for example, a chemically regulated promoter can be used to regulate gene expression in plants by the application of an exogenous chemical regulator. Regulation of the expression of the nucleotide sequence of the present invention by a chemically regulated promoter allows for the synthesis of the polypeptide of the present invention only when the crop plant is treated with an inducible chemical. Depending on the purpose, the promoter may be a chemically inducible promoter if the application of the chemical induces gene expression, or a chemically repressive promoter if the application of the chemical represses gene expression. Examples of such techniques for the chemical induction of gene expression are described in detail in European Patent Application Publication No. 0332104 and U.S. Patent No. 5,614,395.

[0237] Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter activated by benzenesulfonamide herbicide toxicity mitigaters, the maize GST promoter activated by hydrophobic electrophiles used as pre-germination herbicides, and the tobacco PR-1a promoter activated by salicylic acid (e.g., the PR1a system), steroid-responsive promoters (see, for example, glucocorticoid-inducible promoters in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88, 10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257), as well as tetracycline-inducible and tetracycline-inhibiting promoters (e.g., Gatz et al. See al. (1991) Mol. Gen. Genet. 227, 229-237 and U.S. Patent Nos. 5,814,618 and 5,789,156, for examples of Lac repressor system promoters, copper inductive system promoters, salicylate inductive system promoters (e.g., PR1a systems), glucocorticoid inductive promoters (Aoyama et al. (1997) Plant J. 11: 605-612), and ecdysone inductive system promoters.

[0238] Other non-limiting examples of inductive promoters include ABA-inducible and turgor-inducible promoters, auxin-binding protein gene promoters (Schwob et al. (1993) Plant J.4:423-432), UDP-glucose flavonoid glycosyl-transferase promoters (Ralston et al. (1988) Genetics 119:185-197), MPI proteinase inhibitor promoters (Cordero et al. (1994) Plant J.6:141-150), and glyceraldehyde-3-phosphate dehydrogenase promoters (Kohler et al. (1995) Plant Mol.Biol.29:1293-1298; Martinez et al. (1989) J.Mol.Biol.208:551-565; and Quigley et al. Examples include al. (1989) J.Mol.Evol.29:412-421). Also included are benzenesulfonamide-inducible (U.S. Patent No. 5,364,780) and alcohol-inducible (International Patent Application Publications 97 / 06269 and 97 / 06268) systems and glutathione S-transferase promoters. Similarly, 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 can be used. Other chemically inducible promoters useful for promoting the expression of the nucleotide sequence of the present invention in plants are disclosed in U.S. Patent No. 5,614,395. Chemical induction by gene expression is also described in detail in European Patent Application Publication No. 0332104 (granted to Ciba-Geigy) and U.S. Patent No. 5,614,395.

[0239] Another category of promoters useful in the present invention is wound-inducing promoters. Numerous promoters have been described that are expressed at both wound sites and plant pathogen infection sites. Ideally, such promoters should be locally active only at the insect invasion site, so that the insecticidal protein accumulates only in cells that need to synthesize the insecticidal protein to kill the invading pest. Examples of this type of promoter 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 & 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).

[0240] In addition to promoters associated with the nucleotide sequences of the present invention in a functional manner, the expression cassette of the present invention may also include other regulatory elements. Regulatory elements include, but are not limited to, enhancers, introns, translation reader sequences, termination signals, and polyadenylation signal sequences. Examples of suitable transcriptional terminator signals are available and known in the art (e.g., tml from CaMV, E9 from rbcS). Any available terminators known to function in plants can be used in connection with the present invention.

[0241] Numerous other sequences can be incorporated into the chimeric gene described in this invention. These include sequences that have been shown to enhance the expression of intron sequences (e.g., those derived from Adhl and bronzel) and viral reader sequences (e.g., those derived from TMV, MCMV, and AMV).

[0242] For more efficient translation initiation, sequences adjacent to the initiating methionine may be modified. For example, these can be modified by inclusion of sequences known to be effective in plants. Joshi has suggested a suitable consensus for plants (NAR 15:6643-6653 (1987)), and Clonetech has suggested further consensus translation initiators (1993 / 1994 catalog, p. 210). These consensuses are suitable for use with the nucleotide sequences of the present invention. These sequences are incorporated into constructs containing nucleotide sequences up to and including ATG (the second amino acid remains unmodified) or alternatively, nucleotide sequences up to and including the GTC following ATG (which may modify the second amino acid of the transgene).

[0243] In some embodiments, it may be desirable to target the expression of the polypeptide of the present invention to specific cellular sites within plant cells. In some cases, cytosolic localization may be desirable, while in other cases, localization to several intracellular organelles may be desirable. For example, the present invention can be carried out using any mechanism for targeting gene products in plants, such mechanisms are known to exist in plants, and the sequences that control the function of these mechanisms have been characterized in some detail. Sequences that result in the targeting of gene products to other cellular compartments have been characterized. For example, amino-terminal sequences can be involved in the targeting of target proteins to cellular compartments such as vacuoles, mitochondria, peroxisomes, protein granules, endoplasmic reticulum, chloroplasts, starch granules, amyloplasts, apoplasts, or cell walls in plant cells (e.g., Unger et.al. Plant Molec. Biol. 13:411-418 (1989); Rogers et.al. (1985) Proc. Natl. Acad. Sci. USA 82:6512-651; U.S. Patent No. 7,102,057; International Publication No. 2005 / 096704). Optionally, the signal sequence may be a waxy-derived N-terminal signal sequence, a gamma-zein-derived N-terminal signal sequence, a starch-binding domain, a C-terminal starch-binding domain, a chloroplast-targeting sequence for transferring mature proteins into chloroplasts (Comai et al. (1988) J. Biol. Chem. 263:15104-15109; van den Broeck, et al. (1985) Nature 313:358-363; U.S. Patent No. 5,639,949), or an aleurone cell-derived secretory signal sequence (Koehler & Ho, Plant Cell 2:769-783 (1990)). Furthermore, along with the carboxyl-terminal sequence, the amino-terminal sequence is involved in vacuolar targeting of the gene product and can be used in conjunction with the present invention (Shinshi et al. (1990) Plant Molec. Biol. 14:357-368).In one embodiment, the selected signal sequence includes a known cleavage site, and the constructed fusion takes into account any amino acids after the cleavage site required for cleavage. In some cases, this need can be met by the addition of a few amino acids between the cleavage site and the transgene ATG, or alternatively by the substitution of several amino acids within the transgene sequence. These construction techniques are well known in the art and are equally applicable to any cellular compartment.

[0244] It will be recognized that the above mechanisms for cell targeting can be used not only with its own homologous promoters, but also with heterologous promoters, to achieve the objective of specific cell targeting under transcriptional regulation by promoters having different expression patterns from the promoter from which the targeting signal originates.

[0245] The chimeric genes of the present invention may also include nucleotide sequences for selectable markers that can be used to select transformed plants, plant parts, and / or plant cells. Many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0246] Examples of selectable markers include, but are not limited to, nucleotide sequences encoding neo or nptII that confer resistance to kanamycin, G418, etc. (Potrykus et al. (1985) Mol. Gen. Genet. 199:183-188); nucleotide sequences encoding bar that confer resistance to phosphinotricin; nucleotide sequences encoding modified 5-enolpyruvirshikimic acid-3-phosphate (EPSP) synthase that confer resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915-922); and nucleotide sequences encoding nitrilases such as bxn from Klebsiella ozaenae that confer resistance to bromoxyn (Stalker et al. (1988) Science Examples include: 242:419-423); nucleotide sequences encoding modified acetolactic acid synthase (ALS) that confer resistance to imidazolinone, sulfonylurea, or other ALS inhibitory chemicals (European Patent Application Publication No. 154204); nucleotide sequences encoding methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263:12500-12508); nucleotide sequences encoding darapon dehalogenase that confer resistance to darapon; nucleotide sequences encoding mannose-6-phosphate isomerase (also known as phosphomannose isomerase (PMI)) that confers the ability to metabolize mannose (U.S. Patents No. 5,767,378 and No. 5,994,629); nucleotide sequences encoding modified anthranilate synthase that confer resistance to 5-methyltryptophan; or nucleotide sequences encoding hph that confer resistance to hygromycin. Those skilled in the art can select suitable selectable markers for use in the expression cassette of the present invention.

[0247] Further selectable markers include, but are not limited to, nucleotide sequences encoding β-glucuronidase or uidA (GUS), which encode enzymes for which various chromogenic substrates are known; R locus nucleotide sequences encoding products that regulate the production of anthocyanin pigments (red) in plant tissues (Dellaporta et al., “Molecular cloning of the maize R-nj allele by transposon-tagging with Ac” 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); and nucleotide sequences encoding β-lactamase, an enzyme for which various chromogenic substrates are known (e.g., PADAC, chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA). 75:3737-3741); Nucleotide sequence encoding xylE encoding catechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); Nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which aggregate to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); Nucleotide sequence encoding β-galactosidase, an enzyme for which a chromogenic substrate exists; Nucleotide sequence encoding luciferase (lux), which enables bioluminescence detection (Ow et al. (1986) Science 234:856-859); Nucleotide sequence encoding aequorin, which can be used for calcium-sensitive bioluminescence detection (Prasher et al. al. (1985) Biochem. Biophys. Res. Comm. 126: 1259-1268); or nucleotide sequences encoding green fluorescent protein (Niedz et al.(1995) Plant Cell Reports 14:403-406) is an example. Those skilled in the art can select suitable markers for use in the expression cassette of the present invention.

[0248] In some embodiments, the chimeric gene of the present invention may also include a polynucleotide encoding another desired trait in addition to the chimeric insecticidal protein of the present invention. Examples of such other polynucleotides include those encoding polypeptides or dsRNAs for the other desired trait of interest. Such expression cassettes containing "stacked" traits can be used, for example, to produce plants, plant parts, or plant cells having a desired phenotype having the stacked traits (i.e., molecular stacking). Such stacked combinations in plants can also be produced by other methods, including, but not limited to, crossbreeding plants by any conventional method (i.e., breeding stacks). When stacked by genetically transforming plants, the nucleotide sequences of interest can be combined at any point in time and in any order. For example, a transgenic plant containing one or more desired traits can be used as a target for introducing further traits by subsequent transformations. Additional nucleotide sequences can be introduced simultaneously in a simultaneous transformation protocol using the nucleotide sequences, nucleic acid molecules, nucleic acid constructs, or compositions of the present invention provided by any combination of expression cassettes. For example, when introducing two nucleotide sequences, they can be incorporated into separate cassettes (trans) or into the same cassette (cis). Polynucleotide expression can be promoted by the same promoter or different promoters. Furthermore, it is recognized that polynucleotides can be stacked at desired genomic locations using site-directed recombination systems. See, for example, International Patent Application Publications 99 / 25821, 99 / 25854, 99 / 25840, 99 / 25855, and 99 / 25853.

[0249] In each embodiment, the chimeric gene may also include an additional coding sequence for one or more polypeptides or double-stranded RNA molecules (dsRNAs) of interest for agricultural traits (e.g., agricultural traits that are primarily beneficial to seed companies, growers, or grain processors). The polypeptide of interest may be any polypeptide encoded by the nucleotide sequence of interest. Non-limiting examples of polypeptides of interest suitable for production in plants include those that result in agriculturally important traits such as herbicide resistance (sometimes called "herbicide tolerance"), virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, or fungal resistance. See, for example, U.S. Patents No. 5,569,823, 5,304,730, 5,495,071, 6,329,504, and 6,337,431. In embodiments, the target polypeptide may increase the growth potential or yield of plants (including traits that enable plant growth at different temperatures, soil conditions, and levels of sunlight and precipitation) or enable the identification of plants exhibiting the target trait (e.g., selectable markers, seed coat color, etc.). Various target polypeptides and methods for introducing these polypeptides into plants are, for example, U.S. Patents No. 4,761,373, No. 4,769,061, No. 4,810,648, No. 4,940,835, No. 4,975,374, No. 5,013,659, No. 5,162,602, No. 5,276,268, No. 5,304,730, and No. 5 This is described in U.S. Patent Application Publication No. 495,071, No. 5,554,798, No. 5,561,236, No. 5,569,823, No. 5,767,366, No. 5,879,903, No. 5,928,937, No. 6,084,155, No. 6,329,504, and No. 6,337,431, as well as U.S. Patent Application Publication No. 2001 / 0016956. See also lifesci.sussex.ac.uk / home / Neil_Crickmore / Bt / on the World Wide Web.

[0250] Polynucleotides that confer resistance / tolerance to herbicides that inhibit the growth point or meristem, such as imidazarinone or sulfonylurea, may also be suitable in some embodiments of the present invention. Exemplary polynucleotides in this category encode mutant ALS and AHAS enzymes, as described, for example, in U.S. Patents No. 5,767,366 and No. 5,928,937. U.S. Patents No. 4,761,373 and No. 5,013,659 relate to plants resistant to various imidazarinone or sulfonamide herbicides. U.S. Patent No. 4,975,374 relates to plant cells and plants containing nucleic acids encoding mutant glutamine synthetase (GS) resistant to inhibition by herbicides known to inhibit GS, such as phosphinothricin and methionine sulfoximine. U.S. Patent No. 5,162,602 discloses plants resistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. Resistance is conferred by a modified acetyl coenzyme A carboxylase (Accase).

[0251] Polypeptides encoded by nucleotide sequences that confer resistance to glyphosate are also suitable for the present invention. See, for example, U.S. Patent No. 4,940,835 and U.S. Patent No. 4,769,061. U.S. Patent No. 5,554,798 discloses a transgenic glyphosate-resistant maize plant in which resistance is conferred by a modified 5-enolpyruvir-3-phosphoschymate (EPSP) synthase gene.

[0252] Phosphono compounds such as glufosinate ammonium or phosphinotricin, and polynucleotides encoding resistance to pyridinoxy or phenoxypropionic acid and cyclohexone are also suitable. See European Patent Application Publication No. 0242246. See also U.S. Patents Nos. 5,879,903, 5,276,268 and 5,561,236.

[0253] Other suitable polynucleotides include those that encode resistance to photosynthesis-inhibiting herbicides such as triazines and benzonitriles (nitrilases). See U.S. Patent No. 4,810,648. Additional suitable polynucleotides that encode herbicide resistance include those that encode resistance to 2,2-dichlorolopionic acid, cethoxydime, haloxyhop, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides, and bromoxynil. Also suitable are polynucleotides that confer resistance to protox enzymes or enhance resistance to plant diseases; enhance tolerance to unfavorable environmental conditions (abiotic stresses), including, but not limited to, drought, excessive cold, excessive heat, or excessive soil salinity or extreme acidity or alkalinity; and provide changes in plant structure or development, including changes in developmental timing. For example, see U.S. Patent Application Publication No. 2001 / 0016956 and U.S. Patent No. 6,084,155.

[0254] Appropriate additional polynucleotides include those encoding pest-killing (e.g., insecticidal) polypeptides. These polypeptides can be produced in sufficient quantities (i.e., insect control amounts) to control pests, for example. In embodiments, the polypeptides are lepidopteran, coleopteran, hemipteran, and / or dipteran active polypeptides or any combination thereof. It is recognized that the amount of pest-killing polypeptide produced in plants necessary to control insects or other pests may vary depending on the cultivar, type of pest, environmental factors, etc. Polynucleotides useful for additional insect or pest resistance include, for example, those encoding toxins identified in Bacillus organisms. Polynucleotides containing nucleotide sequences encoding the Bacillus thuringiensis (Bt)Cry protein from several subspecies have been cloned, and recombinant clones have been found to be toxic to the larvae of lepidopteran, dipteran, and coleopteran insects. Examples of such Bt insecticidal proteins include Cry proteins such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9B, and Cry9C, as well as plant insecticidal proteins such as Vip1, Vip2, and Vip3, and any combination of the above Bt insecticidal proteins. A complete list of Bt-derived proteins can be found on the World Wide Web in the Bacillus thuringiensis Toxin Nomenclature Database maintained by the University of Sussex (see also Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62: 807-813).

[0255] In embodiments, additional polypeptides include, but are not limited to, alpha-amylase, peroxidase, cholesterol oxidase, patatin, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, modified antibody or antibody fragment, and Bacillus cereus. Insecticidal proteins of *Cereus*, *Xenorhabdus* species (e.g., *X. nematophila* or *X. bovienii*), *Photorhabdus* species (e.g., *P. luminescens* or *P. asymobiotica*), *Brevibacillus* species (e.g., *B. laterosporus*), *Lysinibacillus* species (e.g., *L. sphearicus*), and *Chromobacterium*. This is an insecticidal polypeptide derived from non-Bt sources, containing insecticidal proteins from species such as C. cterium (e.g., C. subtsugae or C. piscinae), Yersinia (e.g., Y. entomophaga), Paenibacillus (e.g., P. propylaea), Clostridium (e.g., C. bifermentans), Pseudomonas (e.g., P. fluorescens), and lignin.

[0256] Polypeptides suitable for production in plants include, for example, those that improve or otherwise promote the conversion of harvested plants or plant parts to commercially useful products, including increasing or altering carbohydrate content or distribution, improving fermentation properties, increasing oil content, increasing protein content, improving digestibility, and increasing nutrient content, such as increasing phytosterol content, increasing tocopherol content, increasing stanol content, or increasing vitamin content. Polypeptides of interest also include, for example, those that result in or contribute to a decrease in the content of unwanted components in the harvested crop, such as phytic acid or sugar-degrading enzymes. "Resulting in" or "contributing" means that the polypeptide of interest directly or indirectly contributes to the presence of the trait of interest (e.g., increased cellulose degradation by the use of heterologous cellulase enzymes).

[0257] In one embodiment, polypeptides contribute to improving the digestibility of food or feed. Xylanase is a hemicellulose-degrading enzyme that improves the breakdown of plant cell walls, thereby allowing animals to better utilize plant nutrients. This results in improved growth rates and feed conversion rates. The viscosity of feed containing xylan may also be reduced. Heterogeneous production of xylanase in plant cells can also promote the conversion of lignocellulose to fermentable sugars in industrial processing.

[0258] Numerous xylanases derived from fungal and bacterial microorganisms have been identified and characterized (see, for example, U.S. Patent No. 5,437,992; Coughlin et al. (1993) “Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases” Espoo; Souminen and Reinikainen, eds. (1993) Foundation for Biotechnical and Industrial Fermentation Research 8:125-135; U.S. Patent Application Publication No. 2005 / 0208178; and PCT International Publication No. 03 / 16654). In particular, three specific xylanases (XYL-I, XYL-II, and XYL-III) have been identified in T. reesei (Tenkanen et al. (1992) Enzyme Microb. Technol. 14:566; Torronen et al. (1992) Bio / Technology 10:1461; and Xu et al. (1998) Appl. Microbiol. Biotechnol. 49:718).

[0259] In other embodiments, polypeptides useful for the present invention may be polysaccharide-degrading enzymes. Plants of the present invention that produce such enzymes may be useful, for example, for producing fermentation raw materials for biological processing. In some embodiments, enzymes useful for fermentation processes include alpha-amylase, protease, pullulanase, isoamylase, cellulase, hemicellulase, xylanase, cyclodextrin tricotransferase, lipase, phytase, laccase, oxidase, esterase, cutinase, granular starch hydrolase, and other glucoamylases.

[0260] Polysaccharide-degrading enzymes include starch-degrading enzymes, such as alpha-amylase (EC3.2.1.1) and glucuronidase (EC3.2.1.131); exo-1,4-alpha-D-glucanases, such as amyloglucosidase and glucoamylase (EC3.2.1.3), beta-amylase (EC3.2.1.2), alpha-glucosidase (EC3.2.1.20) and other exo-amylases; and starch debranching enzymes, such as a) isoamylase (EC3.2.1.6 8) pullulanase (EC3.2.1.41), etc.; b) cellulase, e.g., exo-1,4-3-cellobiohydrolase (EC3.2.1.91), exo-1,3-beta-D-glucanase (EC3.2.1.39), beta-glucosidase (EC3.2.1.21); c) L-arabinase, e.g., endo-1,5-alpha-L-arabinase (EC3.2.1.99), alpha-arabinosidase (EC3.2.1.55), etc.; d) galactanase, e.g. e) Mannanases, e.g., endo-1,4-beta-D-galactanase (EC3.2.1.89), endo-1,3-beta-D-galactanase (EC3.2.1.90), alpha-galactosidase (EC3.2.1.22), beta-galactosidase (EC3.2.1.23), etc.; e) Mannanases, e.g., endo-1,4-beta-D-mannanase (EC3.2.1.78), beta-mannosidase (EC3.2.1.25), alpha-mannosidase (EC3.2.1. 24) etc; f) xylanases, e.g., endo-1,4-beta-xylanase (EC3.2.1.8), beta-D-xylosidase (EC3.2.1.37), 1,3-beta-D-xylanase, etc.; and g) other enzymes, e.g., alpha-L-fucosidase (EC3.2.1.51), alpha-L-rhamnosidase (EC3.2.1.40), revanase (EC3.2.1.65), inulanase (EC3.2.1.7), etc. In one embodiment, the alpha-amylase is the synthetic alpha-amylase Amy797E described in U.S. Patent No. 8,093,453.

[0261] Further enzymes that may be used in conjunction with the present invention include proteases, such as fungal and bacterial proteases. Fungal proteases include, but are not limited to, those obtained from Aspergillus, Trichoderma, Mucor, and Rhizopus, such as A. niger, A. awamori, A. oryzae, and M. miehei. In one embodiment, the polypeptide of the present invention may be a cellobiohydrolase (CBH) enzyme (EC3.2.1.91). In one embodiment, the cellobiohydrolase enzyme may be CBH1 or CBH2.

[0262] Other enzymes useful in conjunction with the present invention include, but are not limited to, hemicellulases, e.g., mannase and arabinofuranosidase (EC 3.2.1.55); ligninases; lipases (e.g., EC 3.1.1.3); glucose oxidases, pectinases, xylanases, transglucosidases, alpha-1,6-glucosidases (e.g., EC 3.2.1.20); esterases, e.g., ferulate esterase (EC 3.1.1.73) and acetylxylan esterase (EC 3.1.1.72); and cutinases (e.g., EC 3.1.1.74).

[0263] Useful double-stranded RNA (dsRNA) molecules in conjunction with the present invention include, but are not limited to, those that repress genes of target pests (e.g., insects). In embodiments, the dsRNA targets genes of lepidopteran, coleopteran, hemipteran, or dipteran pests, or any combination thereof. As used herein, the term “gene repression” is intended to mean, collectively, any well-known method for reducing the level of protein produced as a result of gene transcription to mRNA and subsequent translation of mRNA. Gene repression is also intended to mean a reduction in protein expression from a gene or coding sequence, including post-transcriptional gene repression and transcriptional repression. Post-transcriptional gene repression is mediated by homology between all or part of the mRNA transcribed from the gene or coding sequence targeted for repression and the corresponding double-stranded RNA used for repression, and refers to a substantial and measurable reduction in the amount of mRNA available in the cell for ribosome binding. The transcribed RNA may be oriented in either sense orientation to produce so-called co-repression, antisense orientation to produce so-called antisense repression, or both orientations to produce dsRNA to produce so-called RNA interference (RNAi). Transcriptional repression is mediated by the presence of gene repressors in the cell, such as dsRNA exhibiting substantial sequence identity to a promoter DNA sequence or its complement to produce so-called promoter-trans repression. Gene repression may be effective against native plant genes related to traits, for example, to provide plants that have reduced levels of proteins encoded by native genes or increased or decreased levels of affected metabolites. Gene repression may also be useful against target genes in plant pests that can ingest or come into contact with plant material containing gene repressors designed to inhibit or repress the expression of one or more homologous or complementary sequences in the cells of the pest.Such genes targeted for suppression can encode essential proteins whose predictive functions are selected from the group consisting of muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, digestive enzyme synthesis, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, spermatogenesis, pheromone synthesis, pheromone sensing, antenna formation, wing formation, leg formation, development and differentiation, oogenesis, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration, and apoptosis.

[0264] In one embodiment, the polynucleotide of the present invention further comprises, essentially comprises, or can comprise a vector. In another embodiment, the polynucleotide and expression cassette of the present invention are contained within a vector. Vectors for use in the transformation of plants and other organisms are well known in the art. Non-limiting examples of common types of vectors include plasmids, phage vectors, phagemide vectors, cosmid vectors, fosmids, bacteriophages, artificial chromosomes, or viral vectors. In one embodiment, the vector is, for example, a plant vector for use in the transformation of plants. In one embodiment, the vector is, for example, a bacterial vector for use in the transformation of bacteria. Vectors suitable for plants, bacteria, and other organisms are known in the art.

[0265] The present invention also encompasses transgenic non-human host cells comprising the polynucleotides, nucleic acid molecules, expression cassettes, vectors, or polypeptides of the present invention. Transgenic non-human host cells may include, but are not limited to, plant cells (including monocotyledonous and / or dicotyledonous plant cells), yeast cells, bacterial cells, or insect cells. Therefore, in some embodiments, the present invention may include genera such as Bacillus, Brevibacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteuria, Escherichia, Pseudomonas, Erwinia, Serratia, Klebsiella, Salmonella, Pasteurella, and Xanthomonas. The present invention provides bacterial cells selected from the genera (Xanthomonas), Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, or Alcaligenes. Thus, for example as a biological insect control agent, the Txp40 insecticidal protein of the present invention can be produced by the expression of the polynucleotide encoding it in bacterial cells. For example, in one embodiment, Bacillus thuringiensis cells containing the polynucleotide encoding the Txp40 insecticidal protein of the present invention are provided.

[0266] In one embodiment, the transgenic plant cells are dicotyledonous plant cells or monocotyledonous plant cells. In an additional embodiment, the dicotyledonous plant cells are soybean cells, sunflower cells, tomato cells, cole crop cells, cotton cells, sugar beet cells, or tobacco cells. In a further embodiment, the monocotyledonous plant cells are barley cells, maize cells, oat cells, rice cells, sorghum cells, sugarcane cells, or wheat cells. In an embodiment, the present invention provides a plurality of dicotyledonous plant cells or monocotyledonous plant cells comprising polynucleotides expressing the chimeric insecticidal protein of the present invention. In another embodiment, the plurality of cells are juxtaposed to form an apoplast and grown in sunlight. In another embodiment, the transgenic plant cells cannot regenerate an entire plant.

[0267] In one embodiment of the present invention, the Txp40 insecticidal protein of the present invention is expressed in a higher organism, such as a plant. In one embodiment, the plant may be exoparasitized by a Spodoptera pest, particularly by the fall armyworm. In another embodiment, the plant that may be exoparasitized by the fall armyworm is a corn plant. In this case, the transgenic plant expressing an effective amount of the Txp40 insecticidal protein protects itself at least from Spodoptera pests, particularly the fall armyworm. When an insect begins to feed on such a transgenic plant, the insect ingests the expressed Txp40 insecticidal protein. This may prevent the insect from further gnawing on the plant tissue or may further harm or kill the insect. In one embodiment, the polynucleotide of the present invention is inserted into an expression cassette, which is then stably integrated into the plant genome. In another embodiment, polynucleotides are contained within a non-pathogenic, self-regenerating virus.

[0268] In one embodiment of the present invention, the transgenic plant cells comprising the nucleic acid molecule or polypeptide of the present invention are, but are not limited to, cells of roots, leaves, seeds, flowers, fruits, pollen, organs or plant cultures, or cells of plant parts, plant organs or plant cultures (each as described herein), including callus cells or cultures.

[0269] Transgenic plants or plant cells according to the present invention may be, but are not limited to, monocotyledonous or dicotyledonous plants or plant cells, including corn, soybeans, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugar beet, cotton, sugarcane, rapeseed, alfalfa, tobacco, peanuts, vegetables (sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, carrot, eggplant, cucumber, radish, spinach, potato) The present invention includes crop plants or plant cells such as corn, sorghum, wheat, sunflower, tomato, pepper, celery, pumpkin, zucchini, etc., fruits (including lignon, pear, quince, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, etc.), special plants or plant cells (e.g., Arabidopsis), or woody plants or plant cells (e.g., conifers and / or deciduous trees). In embodiments, the plants or plant cells of the present invention are crop plants or plant cells such as corn, sorghum, wheat, sunflower, tomato, Brassicaceae, pepper, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, Brassica plants, or plant cells.

[0270] The present invention provides a transgenic plant part of the present invention. Optionally, the plant part further comprises the chimeric insecticidal protein and / or nucleic acid encoding it of the present invention.

[0271] The present invention further provides seeds of the transgenic plant of the present invention or seeds that give rise to the transgenic plant of the present invention. Optionally, the seeds contain the chimeric insecticidal protein and / or nucleic acid encoding it of the present invention.

[0272] Additional embodiments of the present invention include harvested products produced from the transgenic plants, plant parts, or seeds of the present invention, and processed products produced from the harvested products. The harvested products may be the whole plant or any plant part, as described herein. In some embodiments, non-limiting examples of harvested products include seeds, fruits, flowers or parts thereof (e.g., anthers, stigmas, etc.), leaves, stems, etc. In other embodiments, processed products may include, but are not limited to, flowers, coarse flour, oil, starch, grains, etc., produced from the harvested seeds or other plant parts of the present invention. Optionally, the harvested products or processed products may contain the chimeric insecticidal protein and / or nucleic acids encoding it of the present invention.

[0273] In other embodiments, the present invention provides extracts from transgenic plants or plant parts of the present invention, optionally comprising the chimeric insecticidal protein and / or nucleic acid encoding it. Extracts from plants or plant parts can be prepared according to procedures well known in the art (see de la Torre et al., Food, Agric. Environ. 2(1):84-89 (2004); Guidet, Nucleic Acids Res. 22(9):1772-1773 (1994); Lipton et al., Food Agric. Immun. 12:153-164 (2000)).

[0274] Chimeric insecticidal proteins can function as insect control agents in plant parts, plant cells, plant organs, seeds, harvested products, processed products, or extracts. In other words, chimeric insecticidal proteins can continue to perform the insecticidal function they had in the transgenic plant. Nucleic acids can perform the function of expressing chimeric insecticidal proteins. As an alternative to encoding the insecticidal proteins of the present invention, nucleic acids can perform the function of identifying the transgenic plant parts, plant cells, plant organs, seeds, harvested products, processed products, or extracts of the present invention.

[0275] In embodiments, the transgenic plant, plant part, plant cell, plant organ, or seed of the present invention is hemizygous to the polynucleotide or expression cassette of the present invention. In embodiments, the transgenic plant, plant part, plant cell, plant organ, or seed of the present invention is homozygous to the polynucleotide or expression cassette of the present invention.

[0276] In embodiments, transgenic plants, plant parts, plant cells, plant organs, seeds, harvested products, processed products, or extracts exhibit increased resistance to one or more pests (e.g., lepidopteran pests such as the fall armyworm) compared to suitable controls that do not contain nucleic acids encoding the insecticidal protein of the present invention.

[0277] Procedures for transforming plants are well known, routine, and described throughout the literature. Non-limiting examples of methods for transforming plants include bacterial-mediated nucleic acid delivery (e.g., by Agrobacterium), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle impaction, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and transformation by any other electrical, chemical, physical (mechanical) or biological mechanism (including any combination thereof) resulting in the introduction of nucleic acids into plant cells. General guides to various plant transformation methods known in this field include Miki et al. ("Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and Biotechnology, Glick, BRand Thompson, JE, Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0278] For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA boundary sequence are generally suitable, while for direct gene transfer (e.g., particle impact), any vector is suitable, and linear DNA containing only the target construct can be used. For direct gene transfer, transformation with a single DNA species or simultaneous transformation can be used (Schocher et al., Biotechnology 4:1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transformation, transformation is usually (but not always) carried out with a selectable marker (e.g., phosphomannose isomerase) which may be a positive selection, providing resistance to antibiotics (e.g., kanamycin, hygromycin, or methotrexate) or herbicides (e.g., glyphosate or glufosinate). However, the selection of the selectable marker is not important to the present invention.

[0279] Agrobacterium-mediated transformation is a commonly used method for transforming plants due to its high transformation efficiency and versatility across numerous different species. Agrobacterium-mediated transformation typically involves introducing a binary vector carrying the target exogenous DNA into a suitable Agrobacterium strain, which may depend on the complement of the vir gene carried by the host Agrobacterium strain, either on a coexisting Ti plasmid or on a chromosome (Uknes et al. (1993) Plant Cell 5:159-169). The introduction of recombinant binary vectors into Agrobacterium can be achieved by a tripearmal mating procedure using Escherichia coli carrying the recombinant binary vector and a helper E. coli strain carrying a plasmid capable of recruiting the recombinant binary vector to the target Agrobacterium strain. Alternatively, recombinant binary vectors can be introduced into Agrobacterium by nucleic acid transformation (Hoefgen & Willmitzer (1988) Nucleic Acids Res. 16:9877).

[0280] Dicotyledonous and monocotyledonous plants can be transformed using Agrobacterium. Methods for Agrobacterium-mediated transformation of rice include well-known methods for transforming rice, such as those described in any of the following: European Patent Application Publication No. 1198985, Aldemita and Hodges (Planta 199:612-617, 1996); Chan et al. (Plant Mol Biol 22(3):491-506, 1993); Hiei et al. (Plant J 6(2):271-282, 1994) (these disclosures are incorporated herein by reference as if they were fully expressed). For the transformation of cone, preferred methods are as described in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1):13-22, 2002) (these disclosures are incorporated herein by reference as if they were fully expressed). The methods are further described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. SDKung and R. Wu, Academic Press (1993) 128-143 and Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The expressed nucleic acid or construct is preferably cloned into a vector suitable for transforming Agrobacterium tumefaciens, such as pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711).Agrobacteria transformed by such vectors can then be used in known ways for the transformation of plants, such as crop plants like tobacco, or, for example, plants used as model plants like Arabidopsis, by immersing damaged or chopped leaves in an agrobacteria solution and then culturing them in a suitable medium. Plant transformation using Agrobacterium tumefaciens is described, for example, by Hagen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877, or in particular by FF White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. SDKung and R. Wu, Academic Press, 1993, pp. 15-38.

[0281] Plant transformation with recombinant Agrobacterium typically involves co-culturing Agrobacterium with plant explants, following methods well known in the art. Transformed tissue is regenerated in a selective medium containing antibiotic or herbicide resistance markers between the T-DNA boundaries of a binary plasmid.

[0282] As previously mentioned, another method for transforming plants, plant parts, and plant cells involves propagating inactive or bioactive particles into plant tissues and cells. See, for example, U.S. Patent No. 4,945,050; No. 5,036,006 and No. 5,100,792. Generally, this method involves propagating inactive or bioactive particles into plant cells under conditions effective in penetrating the outer surface of the cell and providing uptake into its interior. When inactive particles are used, the vector can be introduced into the cell by coating the particles with a vector containing the nucleic acid of interest. Alternatively, one or more cells can be surrounded by a vector so that the vector is carried into the cell by the path the particles have taken. Bioactive particles (e.g., dried yeast cells, dried bacteria, or bacteriophages, each containing one or more nucleic acids to be introduced) can also be propagated into plant tissues.

[0283] In other embodiments, the polynucleotides of the present invention can be directly transformed into plastid genomes. A major advantage of plastid transformation is that plastids generally have the ability to express bacterial genes with substantially no modification, and that plastids have the ability to express a large number of open reading frames under the control of a single promoter. Plastiid transformation techniques are extensively described in U.S. Patents Nos. 5,451,513, 5,545,817, and 5,545,818, PCT International Publication No. 95 / 16783, and McBride et al. (1994) Proc. Nati. Acad. Sci. USA 91,7301-7305. Basic techniques for chloroplast transformation involve introducing a region of cloned plastid DNA adjacent to a selectable marker along with the gene of interest into a suitable target tissue, for example, using biolistec or protoplast transformation (e.g., calcium chloride or PEG-mediated transformation). A 1-1.5 kb adjacent region called a targeting sequence promotes homologous recombination by the plastid genome, thus enabling substitution or modification of specific regions of the plastome. Initially, point mutations in chloroplast 16S rRNA and the rps12 gene that confer resistance to spectinomycin or streptomycin can be used as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, JM, and Maliga, P. (1992) Plant Cell 4, 39-45). The presence of cloning sites between these markers makes it possible to construct plastid-targeted vectors for the introduction of foreign genes (Staub, JM, and Maliga, P. (1993) EMBO J. 12, 601-606).A substantial increase in transformation frequency can be achieved by replacing recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin detoxification enzyme aminoglycoside-3'-adenylate (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). This marker has been used without issue for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089). Other selectable markers useful for plastid transformation are known in the art and are included within the scope of this invention. Typically, approximately 15–20 cell division cycles are required after transformation to reach a homoplastidic state. Plastidal expression, in which genes are inserted by homologous recombination into all thousands of copies of the circular plastid genome present in each plant cell, leverages the advantage of a vast number of copies compared to nuclear expression genes, enabling expression levels that can easily exceed 10 times the total expression level of soluble plant proteins. In one embodiment, the polynucleotide of the present invention can be inserted into a plastid-targeting vector and transformed into the plastid genome of a desired plant host. Thus, a plant homoplastic to a plastid genome containing the nucleotide sequence of the present invention can be obtained, enabling high expression of the polynucleotide.

[0284] Methods for selecting transformed transgenic plants, plant cells, or plant tissue cultures are routine in the art and can be used in the methods of the present invention provided herein. For example, the recombinant vector of the present invention may also include an expression cassette containing a nucleotide sequence for a selectable marker that can be used to select transformed plants, plant parts, or plant cells.

[0285] Furthermore, as is well known in the art, intact transgenic plants can be regenerated from transformed plant cells, plant tissue cultures, or cultured protoplasts using any of the various known techniques. Regeneration of plants from plant cells, plant tissue cultures, or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co., New York (1983)) and Vasil IR (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)).

[0286] Furthermore, the genetic characteristics designed within the transgenic seeds and plants, plant parts, or plant cells of the present invention can be transmitted by sexual reproduction or vegetative growth, and thus maintained and propagated in offspring plants. Generally, maintenance and propagation are carried out using known agricultural methods developed to suit specific purposes such as harvesting, sowing, or tilling.

[0287] Therefore, as described above, polynucleotides can be introduced into plants, plant parts, or plant cells by any number of methods known in the art. Thus, rather than relying on a specific method for introducing one or more polynucleotides into a plant, any method can be used that allows one or more polynucleotides to be stably incorporated into the plant genome. When two or more polynucleotides are introduced, each polynucleotide can be constructed as part of a single nucleic acid molecule or as separate nucleic acid molecules, and can be located in the same or different nucleic acid molecules. Thus, polynucleotides can be introduced into target cells in a single transformation event, in separate transformation events, or, for example, as part of a breeding protocol in plants.

[0288] Once a desired polynucleotide is transformed into a specific plant species, it can be propagated within that species using conventional breeding techniques, or transferred to other varieties of the same species (especially commercial varieties).

[0289] In one embodiment, the present invention provides an insecticidal composition comprising the chimeric insecticidal protein of the present invention in an agrochemically acceptable carrier. As used herein, “agrochemically acceptable carrier” may include natural or synthetic organic or inorganic materials which are combined with an active protein to facilitate application to or within a plant or its parts. Examples of agrochemically acceptable carriers include, but are not limited to, powders, dusts, pellets, granules, sprays, emulsions, colloids, and solutions. Agrochemically acceptable carriers may further include, but are not limited to, inert components, dispersants, surfactants, auxiliaries, tackifiers, stickers, binders, or combinations thereof that can be used in agrochemical formulations. Such compositions can be applied in any way that brings the insecticidal protein or other pest control agent into contact with a pest. Thus, the compositions can be applied to the surface of a plant or plant part, including seeds, leaves, flowers, stems, tubers, roots, etc. In another embodiment, a plant that produces the insecticidal protein of the present invention within its body is an agrochemically acceptable carrier of the expressed insecticidal protein. In embodiments, the composition of the present invention and the agriculturally acceptable carriers exclude transgenic plants.

[0290] In further embodiments, the insecticidal composition comprises bacterial cells or transgenic bacterial cells of the present invention, wherein the bacterial cells or transgenic bacterial cells produce the insecticidal proteins of the present invention. Such insecticidal compositions can be prepared by drying, freeze-drying, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of Bacillus thuringiensis (Bt) containing a transgenic Bt culture. In embodiments, the composition of the present invention may comprise, by weight, at least about 1, at least about 5, at least about 10, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 95, at least about 97, or at least 99 polypeptides of the present invention. In additional embodiments, the composition comprises, by weight, about 1 to about 99 insecticidal proteins of the present invention.

[0291] The insecticidal proteins of the present invention can be used in combination with other pest control agents to expand the target range of pests and / or to prevent or control insect resistance. Furthermore, the use of the insecticidal proteins of the present invention in combination with insecticides having different mechanisms of action or targeting different receptors in the intestines of insects has particular utility for preventing and / or controlling insect resistance.

[0292] Thus, in one embodiment, the present invention provides a composition for controlling one or more phytophagous pests (e.g., pests such as Lepidoptera pests, Coleoptera pests, Hemiptera pests and / or Diptera pests), wherein the composition comprises a first pest control agent which is a chimeric insecticidal protein of the present invention and a second pest control agent which is at least different from the first pest control agent. In other embodiments, the composition is a formulation for topical application to plants. In still other embodiments, the composition is a transgenic plant. In further embodiments, the composition is a combination of formulations topically applied to a transgenic plant. In one embodiment, when the transgenic plant comprises a second pest control agent, the formulation comprises a first pest control agent which is a chimeric insecticidal protein of the present invention. In other embodiments, when the transgenic plant comprises a first pest control agent which is a chimeric insecticidal protein of the present invention, the formulation comprises a second pest control agent.

[0293] In one embodiment, the second pest control agent can be one or more of a chemical pesticidal agent, such as an insecticide, a Bacillus thuringiensis (Bt) insecticidal protein and / or, without limitation, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Brevibacillus laterosporus insecticidal protein, a Bacillus sphaericus insecticidal protein, a protease inhibitor (both serine and cysteine types), a lectin, an alpha - amylase, a peroxidase, a cholesterol oxidase or a double - stranded RNA (dsRNA) molecule, a non - Bt pesticidal agent.

[0294] In other embodiments, the second pest control agent is one or more chemical pesticides, optionally a seed coating. Non-limiting examples of chemical pesticides include pyrethroids, carbamates, neonicotinoids, neuronal sodium channel blockers, insecticidal macrocyclic lactones, gamma-aminobutyric acid (GABA) antagonists, insecticidal ureas, and juvenile hormone mimetic compounds.In other embodiments, the chemical pesticides include abamectin, acephate, acetamiprid, amidoflumeth (S-1955), avermectin, azadirachtin, azinophos-methyl, bifenthrin, vinphenazate, buprofezin, carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole, phenoticarb, phenoxycarb, fenpropathrin, fenprok Simate, fenvalerate, fipronil, flonicamide, flucitrinate, tau-fluvalinate, fluphenerim (UR-50701), flufenoxuron, fonofos, halofenozide, hexaflumuron, imidacloprid, indoxacarb, isofenphos, lufenuron, malathion, metaldehyde, methamidophos, methidathion, methomyl, methoprene, methoxychlor, monoclotophos, methoxyfenozide, nithiazine, novaron, nobiflumulon (XDE-007), oxamyl, parathion, parathion-methyl, permethrin, phorate, fosalon, fosmet, phosphamidone, pyrimicarb, profenofos, pymetrozine, pyridaryl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060) One or more of the following: sulproos, tebufenozide, teflubenzuron, tefluthrin, terbuphos, tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin, trichlorfon and triflumulone, aldicarb, oxamyl, phenamiphos, amitraz, quinomethionate, chlorobenzylate, cyhexatin, dicofol, dienochlor, etoxazole, phenazaquin, fenbutasin oxide, fenpropatrin, fenpyroximate, hexythiazox, propargit, pyridaben, and tebufenpyrad.In yet another embodiment, the chemical pesticide is selected from one or more of cypermethrin, cyhalothrin, cyfluthrin and beta-cyfluthrin, esfenvalerate, fenvalerate, tralomethrin, phenoticarb, methomyl, oxamyl, thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb, spinosad, abamectin, avermectin, emamectin, endosulfan, ethiprole, fipronil, flufenoxuron, triflumulon, diofenolan, pyriproxyfen, pimetrozine and amitraz.

[0295] In an additional embodiment, the second pest control agent may be one or more insecticidal chimeras of any number of Bacillus thuringiensis insecticidal proteins containing Cry protein, plant insecticidal proteins (VIPs), and any of the aforementioned insecticidal proteins. In other embodiments, the second pest control agent is Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Af, Cry1Ag, Cry1Ah, Cry1Ai, Cry1Aj, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Bd, Cry1Be, Cry1Bf, Cry1Bg, Cry1Bh, Cry1Bi, Cry1Ca, Cry1Cb, Cry1Da, Cry1Db, Cry1Dc, Cry1Dd, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Gb, Cry1Gc, Cry1Ha, Cry1Hb, Cry1Hc, Cry1Ia, Cry1Ib, Cry1Ic, Cry1Id, Cry1Ie, Cry1If, Cry1Ig, C ry1Ja, Cry1Jb, Cry1Jc, Cry1Jd, Cry1Ka, Cry1La, Cry1Ma, Cry1Na, Cry1Nb, Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cr y2Ag, Cry2Ah, Cry2Ai, Cry2Aj, Cry2Ak, Cry2Al, Cry2Ba, Cry3Aa, Cry3Ba, Cry3Bb, Cry3Ca, Cry4Aa, Cry4Ba, Cry4Ca, Cry4Cb, Cry 4Cc, Cry5Aa, Cry5Ab, Cry5Ac, Cry5Ad, Cry5Ba, Cry5Ca, Cry5Da, Cry5Ea, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry7Ac, Cry7Ba, Cry7 Bb, Cry7Ca, Cry7Cb, Cry7Da, Cry7Ea, Cry7Fa, Cry7Fb, Cry7Ga, Cry7Gb, Cry7Gc, Cry7Gd, Cry7Ha, Cry7Ia, Cry7Ja, Cry7Ka, Cry7K b, Cry7La, Cry8Aa, Cry8Ab, Cry8Ac, Cry8Ad, Cry8Ba, Cry8Bb, Cry8Bc, Cry8Ca, Cry8Da, Cry8Db, Cry8Ea, Cry8Fa, Cry8Ga, Cry8Ha,Cry8Ia、Cry8Ib、Cry8Ja、Cry8Ka、Cry8Kb、Cry8La、Cry8Ma、Cry8Na、Cry8Pa、Cry8Qa、Cry8Ra、Cry8Sa、Cry8Ta、Cry9Aa、Cry9Ba、Cry9Bb、Cry9Ca、Cry9Da、Cry9Db、Cry9Dc、Cry9Ea、Cry9Eb、Cry9Ec、Cry9Ed、Cry9Ee、Cry9Fa、Cry9Ga、Cry10Aa、Cry11Aa、Cry11Ba、Cry11Bb、Cry12Aa、Cry13Aa、Cry14Aa、Cry14Ab、Cry15Aa、Cry16Aa、Cry17Aa、Cry18Aa、Cry18Ba、Cry18Ca、Cry19Aa、Cry19Ba、Cry19Ca、Cry20Aa、Cry20Ba、Cry21Aa、Cry21Ba、Cry21Ca、Cry21Da、Cry21Ea、Cry21Fa、Cry21Ga、Cry21Ha、Cry22Aa、Cry22Ab、Cry22Ba、Cry22Bb、Cry23Aa、Cry24Aa、Cry24Ba、Cry24Ca、Cry25Aa、Cry26Aa、Cry27Aa、Cry28Aa、Cry29Aa、Cry29Ba、Cry30Aa、Cry30Ba、Cry30Ca、Cry30Da、Cry30Db、Cry30Ea、Cry30Fa、Cry30Ga、Cry31Aa、Cry31Ab、Cry31Ac、Cry31Ad、Cry32Aa、Cry32Ab、Cry32Ba、Cry32Ca、Cry32Cb、Cry32Da、Cry32Ea、Cry32Eb、Cry32Fa、Cry32Ga、Cry32Ha、Cry32Hb、Cry32Ia、Cry32Ja、Cry32Ka、Cry32La、Cry32Ma、Cry32Mb、Cry32Na、Cry32Oa、Cry32Pa、Cry32Qa、Cry32Ra、Cry32Sa、Cry32Ta、Cry32Ua、Cry33Aa、Cry34Aa、Cry34Ab、Cry34Ac、Cry34Ba、Cry35Aa、Cry35Ab、Cry35Ac、Cry35Ba、Cry36Aa、Cry37Aa、Cry38Aa、Cry39Aa、Cry40Aa、Cry40Ba、Cry40Ca、Cry40Da、Cry41Aa、Cry41Ab、Cry41Ba、Cry42Aa、Cry43Aa、A Cry protein selected from Cry43Ba, Cry43Ca, Cry43Cb, Cry43Cc, Cry44Aa, Cry45Aa, Cry46Aa, Cry46Ab, Cry47Aa, Cry48Aa, Cry48Ab, Cry49Aa, Cry49Ab, Cry50Aa, Cry50Ba, Cry51Aa, Cry52Aa, Cry52Ba, Cry53Aa, Cry53Ab, Cry54Aa, Cry54Ab, Cry54Ba, Cry55Aa, Cry56Aa, Cry57Aa, Cry57Ab, Cry58Aa, Cry59Aa, Cry59Ba, Cry60Aa, Cry60Ba, Cry61Aa, Cry62Aa, Cry63Aa, Cry64Aa, Cry65Aa, Cry66Aa, Cry67Aa, Cry68Aa, Cry69Aa, Cry69Ab, Cry70Aa, Cry70Ba, Cry70Bb, Cry71Aa, Cry72Aa, Cry73Aa or any combination of the foregoing. In an embodiment, the Cry protein is, for example, Cry1Fa as represented by the maize event TC1507.,

[0296] In a further embodiment, the second pest control agent is Vip3Aa1, Vip3Aa2, Vip3Aa3, Vip3Aa4, Vip3Aa5, Vip3Aa6, Vip3Aa7, Vip3Aa8, Vip3Aa9, Vip3Aa10, Vip3Aa11, Vip3Aa12, Vip3Aa13, Vip3Aa14, Vip3Aa15, Vip3Aa16, Vip3Aa17, Vip3Aa18, Vip3Aa19, Vip3Aa20, Vip3Aa21, Vip3Aa22, Vip3Aa2, Vip3Aa24, Vip3Aa25, Vip3Aa26, Vip3 Aa27, Vip3Aa28, Vip3Aa29, Vip3Aa30, Vip3Aa31, Vip3Aa32, Vip3Aa33, Vip3Aa34, Vip3Aa35, Vip3Aa36, Vip3Aa37, Vip3Aa38, Vip3Aa39, Vip3Aa40, Vip3 Aa41, Vip3Aa42, Vip3Aa43, Vip3Aa44, Vip3Ab1, Vip3Ab2, Vip3Ac1, Vip3Ad1, Vip3Ad2, Vip3Ae1, Vip3Af1, Vip3Af2, Vip3Af3, Vip3Ag1, Vip3Ag2, Vip3Ag3 The Vip3 plant insecticidal protein is one or more Vip3 proteins selected from HM117633, Vip3Ag4, Vip3Ag5, Vip3Ah1, Vip3Ba1, Vip3Ba2, Vip3Bb1, Vip3Bb2, Vip3Bb3, or any combination thereof. In embodiments, the Vip3 protein is, for example, Vip3Aa (US Patent No. 6,137,033) as represented by Corn Event MIR162 (US Patent No. 8,232,456; US Patent No. 8,455,720; and US Patent No. 8,618,272).

[0297] In embodiments, the second pest control agent may be obtained from sources other than B. thuringiensis. For example, the second pest control agent may be alpha-amylase, peroxidase, cholesterol oxidase, patatin, protease, protease inhibitor, urease, alpha-amylase inhibitor, pore-forming protein, chitinase, lectin, modified antibody or antibody fragment, Bacillus cereus insecticidal proteins of *Cereus* species, *Xenorhabdus* species (e.g., *X. nematophila* or *X. bovienii*), *Photorhabdus* species (e.g., *P. luminescens* or *P. asymobiotica*), *Brevibacillus* species (e.g., *B. laterosporus*), *Lysinibacillus* species (e.g., *L. sphearicus*), *Chromobacteria* These may be insecticidal proteins from Chromobacterium species (e.g., C. subtsugae or C. piscinae), Yersinia species (e.g., Y. enterophaga), Paenibacillus species (e.g., P. propylaea), Clostridium species (e.g., C. bifermentans), Pseudomonas species (e.g., P. fluorescens), and lignin. In other embodiments, the second control agent may be at least one insecticidal protein obtained from an insecticidal toxin complex (Tc) derived from Photorhabdus, Xenorhabus, Serratia, or Yersinia.In other embodiments, the insecticidal protein may be an ADP-ribosyltransferase derived from an insecticidal bacterium such as the species Photorhabdus. In other embodiments, the insecticidal protein may be a VIP protein such as VIP1 and / or VIP2 derived from B. cereus. In yet another embodiment, the insecticidal protein may be a binary toxin derived from an insecticidal bacterium, such as ISP1A and ISP2A derived from B. laterosporus or BinA and BinB derived from L. sphaericus. In yet another embodiment, the insecticidal protein may be manipulated or may be a hybrid or chimera of any of the aforementioned insecticidal proteins.

[0298] In one embodiment, the second biocide may be a non-proteinogenic interfering RNA molecule, such as dsRNA, which can be expressed by gene transfer or applied as part of a composition (e.g., by topical methods). Interfering RNA typically comprises at least an 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. RNA interference (RNAi) occurs when an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them. The resulting hydrolysis product is a small RNA fragment about 19-24 nucleotides long, called small interfering RNA (siRNA). siRNA is then diffused or transported throughout the organism (including through cell membranes), where it hybridizes to mRNA (or other RNA) and causes RNA hydrolysis. Interfering RNA is recognized by the RNA interference silencing complex (RISC), on which an effector strand (or "guide strand") of RNA is loaded. This guide strand acts as a template for the recognition and disruption of the double-stranded sequence. This process is repeated each time an siRNA hybridizes to its complementary RNA target, effectively preventing the translation of those mRNAs and thus "silencing" the expression of the specific gene to which the mRNA is transcribed. Interfering RNAs are known in the art to be useful in insect control (see, for example, International Publication No. 2013 / 192256, incorporated herein by reference). Interfering RNAs designed for use in insect control produce double-stranded RNAs that do not exist in nature, which, by utilizing the insect's natural RNAi pathway, cause downregulation of target genes that can lead to cessation of feeding and / or growth, potentially resulting in pest death. Interfering RNA molecules can confer insect resistance to the same target pests as the proteins of the present invention, or they can target different pests. Targeted insect and plant pests can be ingested by chewing, sucking, or burrowing. Interfering RNAs are known in the art to be useful in insect control.In embodiments, dsRNAs useful for insect control are described in U.S. Provisional Patent Application No. 62 / 371,259, No. 62 / 371,261, or No. 62 / 371,262, filed on August 5, 2016. In embodiments, dsRNAs useful for insect control are described in U.S. Patent No. 9,238,8223, No. 9,340,797, or No. 8,946,510. In embodiments, dsRNAs useful for insect control are described in U.S. Patent Publication No. 12 / 868,994, No. 13 / 831,230, No. 14 / 207,313, or No. 14 / 207318. In other embodiments, interfering RNAs can confer resistance to non-insect plant pests, such as nematode pests or viral pests.

[0299] In further embodiments, the first insecticide and the second pesticide, which are chimeric insecticidal proteins of the present invention, are co-expressed in a transgenic plant. This co-expression of two or more pesticides in the same transgenic plant can be achieved by genetically engineering the plant to contain and express nucleic acid sequences encoding insecticides. For example, the co-expression of two or more pesticides in the same transgenic plant can be achieved by creating a single recombinant vector containing the coding sequences of two or more pesticides in a "molecular stack," and then genetically engineering the plant to contain and express all of the pesticides in the transgenic plant. Such a molecular stack can be created, for example, using minichromosomes as described in U.S. Patent No. 7,235,716. Alternatively, a plant, parent 1, can be genetically engineered for the expression of the chimeric insecticidal protein of the present invention. A second plant, parent 2, can be genetically engineered for the expression of the second pesticide. By crossing parent 1 and parent 2, offspring plants expressing both insecticides from parent 1 and 2 can be obtained.

[0300] In other embodiments, the present invention provides stacked transgenic plants resistant to ectoparasitic plant pests, comprising a nucleic acid (e.g., DNA) sequence encoding a dsRNA for suppression of essential genes in target pests, and a nucleic acid (e.g., DNA) sequence encoding the chimeric insecticidal protein of the present invention exhibiting insecticidal activity against target pests. It has been reported that dsRNA is ineffective against certain lepidopteran pests, possibly due to high midgut pH which destabilizes the dsRNA (Rajagopol et al. 2002. J. Biol. Chem. 277:468-494). Therefore, in some embodiments where the target pest is a lepidopteran pest, the chimeric insecticidal protein of the present invention acts to transiently lower the midgut pH, which helps stabilize the co-ingested dsRNA and enable the dsRNA in silencing the target gene.

[0301] Transgenic plants or seeds containing and / or expressing the insecticidal protein of the present invention may also be treated with insecticides or insecticidal seed coatings as described in U.S. Patent No. 5,849,320 and U.S. Patent No. 5,876,739. In embodiments where both the insecticide or insecticidal seed coating and the transgenic plants or seeds of the present invention are active against the same target insect, e.g., lepidopteran pests (e.g., fall armyworm), this combination is useful in (i) a method for further enhancing the activity of the composition of the present invention against the target insect, and / or in (ii) a method for preventing the development of resistance to the composition of the present invention by providing yet another mechanism of action against the target insect. Accordingly, in embodiments, the present invention provides transgenic plants or seeds of the present invention and a method for enhancing control of lepidopteran insect populations, comprising the step of applying an insecticide or insecticidal seed coating to the plants or seeds of the present invention.

[0302] Even if the insecticide or insecticidal seed coating is active against different insects, by adding an insecticide or insecticidal seed coating having activity against, for example, Coleopteran insects to the transgenic seeds of the present invention, the insecticide or insecticidal seed coating is useful to expand the scope of insect control. In one embodiment, the transgenic seeds of the present invention have activity against Lepidopteran insects, and the produced coated transgenic seeds control both Lepidopteran and Coleopteran pests.

[0303] The present invention also encompasses methods for producing insect-resistant (e.g., lepidopteran insect-resistant) transgenic plants. In each embodiment, the method includes the steps of introducing a polynucleotide, expression cassette, or vector of the present invention (including toxin fragments and modified forms substantially identical to the polypeptide specifically disclosed herein) containing a nucleotide sequence encoding the chimeric insecticidal protein of the present invention into a plant (wherein the nucleotide sequence is expressed in the plant to produce the chimeric insecticidal protein of the present invention and thereby confer to the plant resistance to pests), and producing an insect-resistant transgenic plant (e.g., compared to a suitable control plant, e.g., a plant that does not contain the polynucleotide, expression cassette, or vector of the present invention and / or does not express the polypeptide of the present invention).

[0304] In one embodiment, a method for introducing the polynucleotide, expression cassette, or vector of the present invention into a plant comprises the steps of first transforming plant cells with the polynucleotide, expression cassette, or vector, and then regenerating a transgenic plant therefrom, wherein the transgenic plant contains the polynucleotide, expression cassette, or vector and expresses the chimeric insecticidal protein of the present invention.

[0305] Alternatively or additionally, the introduction step may include the step of crossing a first plant containing polynucleotides, an expression cassette, or a vector with a second plant (e.g., a plant different from the first plant, e.g., a plant that does not contain polynucleotides, an expression cassette, or a vector), and optionally the step of producing offspring plants containing polynucleotides, an expression cassette, or a vector, expressing the chimeric insecticidal protein of the present invention, thereby resulting in increased resistance to at least one pest. Thus, the transgenic plants of the present invention encompass plants and their offspring (any generation) that are the direct result of a transformation event, containing polynucleotides, an expression cassette, or a vector, optionally expressing the chimeric insecticidal protein, and resulting in increased resistance to at least one pest.

[0306] The present invention further provides a method for identifying the transgenic plants of the present invention, the method comprising the step of detecting the presence of the polynucleotides, expression cassettes, vectors, or chimeric insecticidal proteins of the present invention in a plant (or plant cells, plant parts, etc., obtained therefrom), thereby identifying the plant as a transgenic plant of the present invention based on the presence of the polynucleotides, expression cassettes, vectors, or chimeric insecticidal proteins of the present invention.

[0307] The present invention further provides a method for producing transgenic plants with increased resistance to at least one pest (e.g., at least one lepidopteran pest), the method comprising the steps of sowing seeds containing the polynucleotide, expression cassette, or vector of the present invention, and growing transgenic plants from the seeds, wherein the transgenic plants contain the polynucleotide, expression cassette, or vector and produce chimeric insecticidal proteins.

[0308] In embodiments, the transgenic plant produced by the method of the present invention comprises the polynucleotide, expression cassette, or vector of the present invention. In embodiments, the transgenic plant produced by the method of the present invention comprises the chimeric insecticidal protein of the present invention and optionally has increased resistance to at least one pest.

[0309] The method for producing transgenic plants described herein optionally includes a further step of harvesting seeds from the transgenic plants, wherein the seeds contain polynucleotides, expression cassettes or vectors and produce chimeric insecticidal proteins. Optionally, the seeds produce further transgenic plants, which contain polynucleotides, expression cassettes or vectors and produce chimeric insecticidal proteins, thereby increasing resistance to at least one pest.

[0310] The present invention further provides plant parts, plant cells, plant organs, plant cultures, seeds, plant extracts, harvested products, and processed products of transgenic plants produced by the method of the present invention.

[0311] In a further embodiment, the present invention also provides a method for producing seeds, the method comprising the steps of providing a transgenic plant comprising the polynucleotide, expression cassette or vector of the present invention, and harvesting seeds from the transgenic plant, wherein the seeds comprise the polynucleotide, expression cassette or vector and produce a chimeric insecticidal protein. Optionally, the seeds produce further transgenic plants, which comprise the polynucleotide, expression cassette or vector and produce a chimeric insecticidal protein, thereby increasing resistance to at least one pest. In each embodiment, the step of providing a transgenic plant comprises sowing seeds that produce transgenic plants.

[0312] The present invention further provides a method for producing hybrid plant seeds, the method comprising the step of crossing a first inbred plant, which is a transgenic plant comprising the polynucleotide, expression cassette, or vector of the present invention and optionally expressing the chimeric insecticidal protein of the present invention, with a different inbred plant (e.g., an inbred plant that does not contain the polynucleotide, expression cassette, or vector of the present invention) to form hybrid seeds. Optionally, the method further comprises harvesting the hybrid seeds. In embodiments, the hybrid seeds may comprise the polynucleotide, expression cassette, or vector of the present invention and, in embodiments, further comprise the chimeric insecticidal protein of the present invention, and may have increased resistance to pests. In embodiments, the hybrid seeds produce a transgenic plant comprising the polynucleotide, expression cassette, or vector of the present invention and expressing the chimeric insecticidal protein of the present invention, and having increased resistance to at least one pest.

[0313] In one embodiment, the transgenic plant of the present invention is resistant to at least one lepidopteran pest (as described herein). In an embodiment, the transgenic plant controls fall armyworm pests or colonies resistant to Vip3A (e.g., Vip3Aa protein, e.g., expressed in maize event MIR162) and / or Cry1F protein (e.g., Cry1Fa protein, e.g., expressed in maize event TC1507).

[0314] In a further embodiment, a method for controlling at least one pest (e.g., at least one lepidopteran pest such as the fall armyworm) comprises the step of providing the chimeric insecticidal protein of the present invention. In embodiments, the method comprises the step of delivering an effective amount of the chimeric insecticidal protein of the present invention to the pest or its environment (e.g., by oral delivery). Generally, polypeptides are ingested orally by insects to be effective. However, chimeric insecticidal proteins can be delivered to insects in many recognized ways. Methods for orally delivering proteins to insects include, but are not limited to, providing the protein in (1) a transgenic plant (wherein the insect ingests one or more parts of the transgenic plant and thereby ingests polypeptides expressed in the transgenic plant); (2) a formulated protein composition that can be applied to or incorporated into, for example, an insect growth medium; (3) a protein composition that can be applied to a surface, for example, sprayed onto the surface of a plant part (which is then ingested by the insect when it eats one or more of the sprayed plant parts); (4) a feed substrate; or (5) any other protein delivery system recognized in the art. Thus, the toxic proteins of the present invention can be delivered using any method of orally delivery to insects. In some specific embodiments, the chimeric insecticidal proteins of the present invention are delivered orally to insects, for example, wherein the insect ingests one or more parts of the transgenic plant of the present invention.

[0315] In other embodiments, the pesticidal protein of the present invention is delivered orally to an insect, where the insect ingests one or more parts of a plant sprayed with a composition containing the pesticidal protein of the present invention. Delivery of the composition of the present invention to the plant surface can be carried out using any method known to those skilled in the art for applying compounds, compositions, formulations, etc. to the plant surface. Some non-limiting examples of delivering to or contacting a plant or a part thereof include spraying, dusting, sprinkling, scattering, misting, atomizing, broadcast spreading, dipping, soil injection, soil mixing, perfusion (e.g., root, soil treatment), dipping, injection, coating, leaf or stem infiltration, side dressing or seed treatment, etc., and combinations thereof. These and other procedures for contacting a plant or a part thereof with a compound, composition or formulation are well known to those skilled in the art.

[0316] In certain embodiments, the present invention encompasses a method of providing to a farmer a means for controlling Spodoptera pests, the method including supplying or selling to the farmer a plant material such as a seed, the plant material including a Txp40 polynucleotide, expression cassette or vector capable of expressing the Txp40 pesticidal protein of the present invention. In other embodiments, the Spodoptera pest is a Spodoptera frugiperda pest, the plant material contains the Txp40 pesticidal protein of the present invention, and optionally, at least the resistance to Spodoptera frugiperda is increased. In other embodiments, the plant material is a corn seed, and the corn plant grown from the seed contains the Txp40 polynucleotide, expression cassette or vector of the present invention capable of expressing the Txp40 pesticidal protein of the present invention, and at least the resistance to Spodoptera frugiperda is increased.

Examples

[0317] The present invention will be further described with reference to the following detailed examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. The standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in J. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press (2001), T. S. Silhavy, M. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984), 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 Press (1992), and Schultz et al., Plant Molecular Biology Manual, Kluwer Academic Publishers (1998).

[0318] Example 1. Insecticidal activity of Txp40 protein against cry-resistant fall armyworm The insecticidal activity of the Txp40 protein (SEQ ID NO: 1) obtained from Photorhabdus lumimescens was determined using an artificial feed bioassay in which the surface of the artificial insect feed was coated with E. coli extract containing the Txp40 protein. The FAW strains used in these bioassays were obtained from field populations of FAW resistant to Cry1F and Cry1Ab insecticidal proteins. The Vip3 protein was used as a positive control. E. coli clones containing empty vectors, i.e., vectors that do not contain the txp40 coding sequence, were used as negative controls. The results of the bioassays were recorded on day 7 as mortality and effective mortality percentages. Here, effective mortality takes into account the growth stage achieved by the FAW larvae during the test; i.e., treated larvae that have growth inhibition and are in a mortal state are considered to be effectively dead.

[0319] The results are shown in Table 1. When E. coli extracts containing Txp40 were tested by insect bioassay, Txp40 surprisingly showed significant insecticidal activity against Cry1F-resistant FAW. These results not only demonstrate the remarkable oral activity of the Txp40 protein against FAW, but also indicate that Txp40 has a different mechanism of action than the Cry1F and Cry1Ab insecticidal proteins derived from B. thuringiensis.

[0320] [Table 1]

[0321] To test the efficacy of the Txp40 protein, dilutions of Txp40 E. coli extract were prepared and added to the surface of artificial insect feed as described above. Undiluted extracts were also tested for both Txp40 treatment and Vip3 positive control treatment.

[0322] The results shown in Table 2 demonstrate that the Txp40 protein exhibits significant activity even at a 1:32 dilution, suggesting that Txp40 expression in transgenic cone plants may be a viable FAW control option, particularly against cry-resistant FAW.

[0323] [Table 2]

[0324] Example 2. FAW activity of the Txp40 mutant Mutations were introduced into Txp40-1 (SEQ ID NO: 1), and the protein stability and insecticidal activity of bacterial lysates from each Txp40 mutant were assayed. Mutations included amino acid changes at various residues, as well as insertions and / or deletions of amino acid residues. Using a feed overlay assay essentially performed as described in Example 1, mutant Txp40 proteins with one or more mutations were tested for insecticidal activity against two strains of fall armyworm: a Brazilian strain (BR-FAW; Spodoptera frugiperda) and a North American strain (NA-FAW; Spodoptera frugiperda). The plant insecticidal protein Vip3D, wild-type Txp40 protein, and / or Bacillus thuringiensis strain C0756 were used as positive controls. The pET29 empty vector was used as a negative control. Tables 3-4 show the results of expression, solubility, and / or insecticidal activity assays of mutant Txp40 proteins with targeted mutations.

[0325] [Table 3-1] [Table 3-2]

[0326] [Table 4]

[0327] The results in Table 3 demonstrate that mutant Txp40 proteins with equivalent or superior insecticidal activity against fall armyworm (FAW) compared to wild-type Txp40 protein can be produced. For example, mutant proteins with single mutations in K73A, K191A, K209A, K238A, K309A, R10A, R26A, R46A, R186A, and R226A had nearly the same insecticidal activity against NA-FAW as wild-type Txp40, and mutant proteins with single mutations in K49A, K200A, K221A, R26A, R167A, R208A, and R217A had nearly the same insecticidal activity against BR-FAW as wild-type Txp40.

[0328] Some mutations in Txp40 produced mutant proteins with increased or significantly increased (by 20%) insecticidal activity against NA-FAW compared to the wild-type Txp40 protein. For example, all mutant proteins with a single mutation in K31A, K49A, K73A, K75A, K103A, K111A, K119A, K170A, K191A, K200A, K210A, K213A, K221A, K284A, K333A, R167A, R208A, and R252A exhibited superior activity against NA-FAW compared to wild-type Txp40. Of these mutants, K31A, K49A, K75A, K111A, K119A, K200A, and K333A showed an increase of more than 20% in insecticidal activity against NA-FAW compared to wild-type Txp40. Some mutations in Txp40 produced mutant proteins with increased or significantly increased (by 14%) insecticidal activity against BR-FAW compared to wild-type Txp40 protein. For example, all mutant proteins with single mutations in K31A, K49A, K73A, K75A, K103A, K111A, K119A, K200A, K213A, and K333A exhibited superior activity against BR-FAW compared to wild-type Txp40, and among these, K73A, K103A, and K213A showed a significant increase in insecticidal activity against BR-FAW compared to wild-type Txp40 protein.

[0329] The results further demonstrate that specific mutations in Txp40 (SEQ ID NO: 1) produced mutant proteins with remarkably different toxicity to NA-FAW compared to BR-FAW. Several mutants, e.g., K31A, K48A, K75A, K111A, K119A, K133A, K170A, K209A, K210A, K238A, K333A, R10A, R208A, and R226A, were more toxic to NA-FAW than to BR-FAW. Several other mutants, e.g., K73A, K103A, K213A, K247A, K271A, K284A, R26A, R46A, R187A, R217A, and R240A, were more toxic to BR-FAW than to NA-FAW, suggesting that these locations are important for different FAW activity.

[0330] Most mutant Txp40 proteins containing multiple mutations exhibited reduced insecticidal activity against NA-FAW compared to wild-type Txp40, and some mutations completely knocked out the activity, again demonstrating the importance of these amino acid positions to the biological activity of Txp40. Some mutants with multiple mutations had the same activity as wild-type Txp40, while one mutant (SEQ ID NO: 127) with a mutation at position K119A / K213A significantly increased insecticidal activity compared to wild-type Txp40.

[0331] Using degenerate oligonucleotide random mutagenesis technique in full-length Txp40-K119A / K213A DNA, performed by GenScript® (Piscataway, NJ), DNA encoding the mutant Txp40-K119A / K213A protein (SEQ ID NO: 127) was used as a template to induce further mutations. A library of mutants containing random mutations was subcloned into the pET29a vector. Hundreds of bacterial lysates containing the mutant Txp40 protein were tested for insecticidal activity against NA-FAW, BR-FAW, and soybean looper (SBL; Chrysodeixis includens) as described above. Results for specific mutants are shown in Table 5.

[0332] [Table 5]

[0333] As described above, wild-type Txp40 (SEQ ID NO: 1) and mutant Txp40 proteins exhibit different toxicity to NA-FAW and BR-FAW. The objective of the randomized experiment was to identify one or more Txp40 mutants containing mutations that confer high insecticidal activity against both NA- and BR-FAW strains, as well as other lepidopteran species. The Txp40 mutant containing the K119A / K213A mutation (SEQ ID NO: 127), used as a template for random mutagenesis, was highly active against NA-FAW but showed significantly lower activity against BR-FAW and SBL (see Table 5).

[0334] Bioassay results from hundreds of randomly mutagenerated Txp40 mutants showed that the majority maintained different toxicity to NA-FAW and BR-FAW. However, we identified three mutants—Txp40-K119A / K213A / S11Y / M86I (SEQ ID NO: 130); Txp40-K119A / K213A / S11Y (SEQ ID NO: 131); and Txp40-K119A / K213A / F169V (SEQ ID NO: 132)—that maintained high activity against NA-FAW and, surprisingly, very high activity against BR-FAW compared to wild-type Txp40 (SEQ ID NO: 1) and the Txp40-K119A / K213A mutant (SEQ ID NO: 127) used as a template. One of these mutants, Txp40-K119A / K213A / S11Y (SEQ ID NO: 131), surprisingly exhibited significantly increased activity against soybean loopers (SBLs) compared to the mutant template Txp40-K119A / K213A. These results strongly suggest that amino acid positions 11, 119, and 213 of SEQ ID NO: 1 are crucial in determining the insecticidal efficacy of the Txp40 protein.

[0335] Further variant Txp40 proteins, which were prepared and tested against NA-FAW as described above, are shown in Table 6. All mutations in Table 6 refer to amino acid substitutions in SEQ ID NO: 1.

[0336] [Table 6]

[0337] The results shown in Table 6 support the finding that at least amino acid positions 119 and 213 of Sequence ID No. 1 are important for determining FAW activity.

[0338] Example 3. Testing of Txp40 activity against Vip3-resistant FAW To determine whether the toxicity of Txp40 and / or the mutant protein is due to a different MOA from the Vip3A protein, Txp40 wild-type and the above-described Txp40-K119A mutant protein were prepared as described in Example 2. The Txp40 proteins were mixed separately in Txp40 Buffer, and the purity of the lysed protein preparations was monitored using the Bio-Rad Experion system (BioRad, Hercules, CA).

[0339] The efficacy of purified Vip3A insecticidal protein against FAW strains resistant to it was tested. A feed overlay assay was performed essentially as described in Example 1. The Vip3A protein was dissolved in PBS. Two negative control treatments were PBS and Txp40 Buffer. Cry1Fa protein was used as a positive control for Vip3A-resistant FAW strains. Multiple concentrates of each protein were tested. Insecticidal activity was assessed on day 7 as effective mortality (larvae exhibiting growth inhibition and being in a mortal state were recorded as effectively dead).

[0340] The results showed that Vip3A-resistant FAW lines were not controlled by Vip3A, demonstrating that these lines are resistant to this protein. In contrast, Txp40 wild-type protein and the Txp40-K119A mutant resulted in a 60% mortality rate in Vip3-resistant insects, suggesting that the mechanism of action of Txp40 differs from that of the Vip3A protein, and therefore that stacking combinations with Vip3 are useful in mitigating the development of resistance in FAW populations.

[0341] Example 4. Simulated gastric juice test using E. coli lysate preparation. This example describes an assay for determining the SGF digestibility of Txp40 and / or Txp40 mutants. E. coli strain BL21 *Each Txp40 protein and / or mutant was produced in (DE3). The expression level of the mutants and the solubility of the mutants in the bacterial strains were determined. Bacterial lysates in buffer were diluted to 3 mg / mL (total protein concentration) for digestibility analysis. The digestion reaction was initiated at 37°C by adding 15 μL of lysate to 285 μL of simulated gastric juice [G-Con solution (2 mg / mL sodium chloride, pH 1.2) containing 10 units of pepsin / μg protein or approximately 1579 units of pepsin / mL]. After 5 minutes, 100 μL of the lysate-SGF reaction product was removed and the reaction was stopped by adding it to 100 μL of preheated (95°C) stop solution consisting of 65 parts tricine loading buffer (Bio-rad 2x tricine loading buffer w / 10β-mercaptoethanol) and 35 parts 500 mM sodium bicarbonate, pH 11.0. Time point zero (T0) was prepared by adding 5 μL of test lysate to 100 μL of preheated (95°C) stop solution and 95 μL of simulated gastric juice. All samples were heated to 95°C for 5 minutes and then stored on ice until SDS-PAGE analysis. Before standard protein gel electrophoresis, 30 microliters of each reaction were loaded onto 10-20 tris-tricine peptide gels. Immediately after electrophoresis, the tris-tricine gels were fixed with a 40 methanol:10 acetic acid mixture for 20 minutes. The gels were then stained with GelCode Blue protein dye at room temperature for 1 hour. After 1 hour, the polyacrylamide gels were destained with distilled water for at least 12 hours. The results consisted of "pass" and "fail" evaluations. A "fail" in the T5 test meant that intact or partially digested Txp40 protein and / or variants were detectable by GelCode Blue protein staining after gel electrophoresis, indicating that the protein was not completely digested in the SGF assay. A "pass" in the T5 test means that the intact Txp40 protein and / or variants were undetectable, indicating that the Txp40 protein and / or variants were digested in the SGF assay. The results of these experiments demonstrated that Txp40 and variant Txp40 proteins are completely digested in the SGF assay.

[0342] Example 5. Transformation of maize using the txp40-1 codon-optimized gene A binary vector construct suitable for Agrobacterium-mediated transformation of maize is prepared. The binary vector comprises a maize-optimized nucleic acid encoding txp40-K119A / K213A (SEQ ID NO: 127) and a vector encoding Txp40-K119A / K213A / S11Y (SEQ ID NO: 131), which is operably ligated at the 5-terminus to a promoter suitable for promoting expression in plants and operably ligated at the 3-terminus to a terminator sequence. Codon optimization of maize is performed, for example, using the method described in U.S. Patent No. 6,320,100 (incorporated herein by reference). The construct is transformed into Agrobacterium tumefaciens using standard molecular biology techniques known to those skilled in the art. To prepare Agrobacteria for transformation, cells are cultured overnight in liquid YPC medium at 28°C and 220 rpm. Agrobacterium transformation of immature maize embryos is carried out essentially as described in Negrotto et al., 2000 (Plant Cell Reports 19:798-803). In this example, all culture medium components are essentially as described in Negrotto et al. above. However, various culture medium components known in the art may be substituted.

[0343] After transformation, selection, and regeneration, plants are assayed using TaqMan® analysis for the presence of genes encoding selectable markers and the txp40 maize codon-optimized coding sequence. Plants are also tested for the presence of the vector skeleton. Plants that are negative for the vector skeleton and contain one copy of the transgene are moved to a greenhouse and assayed for resistance to FAW damage. The results will indicate that both proteins are expressed in the plants and are active against fall armyworm.

[0344] Example 6. Txp40-1 in combination with a second insecticide Txp40 and / or Txp40 variants are purified as bacterial lysates or as proteins, as in Example 1. A second insecticide, such as Cry protein and / or VIP and / or dsRNA, is prepared. In non-limiting examples, the Cry protein may be Cry1Ab, Cry1B, Cry1C, Cry1D, Cry1F, Cry1J and / or Cry2A proteins. In other non-limiting examples, the Vip3 protein may be Vip3A and / or Vip3B proteins. dsRNAs may target genes encoding vacuolar ATP synthase, beta-tubulin, 26S proteosome subunit p28 protein, EF1α48D, troponin I, tetraspanin, gamma-coatmer, beta-coatmer, and / or juvenile hormone epoxide hydrolase (International Publication Nos. 2018 / 026770, 2018 / 026773, and 2018 / 026774; U.S. Patent No. 7,812,219; as incorporated herein by reference). The purified Txp40 protein and the second insecticide are tested for insecticidal efficacy against FAW in a feed overlay assay carried out with the addition of the second insecticide, essentially as described in Example 1.

[0345] The examples and embodiments described herein are for illustrative purposes only, and in light of such description, various modifications or variations will be suggested to those skilled in the art and should be understood to fall within the spirit and scope of this application and claims.

[0346] All publications and patent applications referenced herein represent the level of skill of those skilled in the art in which the present invention relates. All publications and patent applications are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated by reference.

Claims

1. 1. A method for controlling Spodoptera pests, comprising contacting said Spodoptera pests with a Txp40 protein or a variant thereof comprising an amino acid sequence having at least 95% identity to SEQ ID NO:

1.

2. 10. The method of claim 1, wherein the Spodoptera pest is Spodoptera frugiperda pest.

3. 3. The method of claim 2, wherein the Spodoptera frugiperda pest is resistant to a Cry protein and / or a Vip3 protein.

4. The method of claim 3 , wherein the Vip3 protein is a Vip3A protein.

5. 4. The method of claim 3, wherein the Cry protein is a Cry1A, Cry1B, Cry1C, Cry1D, Cry1F, Cry1J, or Cry2A protein.

6. 2. The method of claim 1, wherein the contacting step is carried out using a microorganism transformed with at least one polynucleotide encoding the Txp40 protein or variant thereof.

7. The method of claim 1 , wherein the contacting step is carried out using a plant or part thereof comprising at least one polynucleotide encoding the Txp40 protein or variant thereof.

8. The method of claim 7 , wherein the plant is a corn plant or part thereof.

9. The method of claim 2, wherein the Spodoptera frugiperda is contacted with a Cry protein and / or a Vip3 protein in addition to the Txp40 protein or a mutant thereof.

10. 10. The method of claim 9, wherein the Cry protein and / or the Vip3 protein is selected from the group consisting of Cry1A, Cry1B, Cry1C, Cry1D, Cry1F, Cry2A and Vip3 proteins.

11. 2. The method of claim 1, wherein the Txp40 protein comprises SEQ ID NO:

1.

12. 1. A chimeric gene comprising a promoter operably linked to a heterologous nucleic acid molecule comprising a nucleotide sequence encoding a Txp40 protein having insecticidal activity against Spodoptera pests, said nucleotide sequence comprising: (a) any of SEQ ID NOs: 3-5 or 11-73; (b) has at least 95% identity to any of SEQ ID NOs: 3-5 or 11-73; (c) encoding a protein comprising the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 74-136; (d) encodes a protein comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1; or (e) A chimeric gene that is complementary to the nucleotide sequence of any one of (a) to (d).

13. A nucleic acid molecule encoding an insecticidal protein comprising the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:5 or any of SEQ ID NOs:11 to 73.

14. An insecticidal protein comprising the amino acid sequence of SEQ ID NO: 2 or any one of SEQ ID NOs: 74 to 136.

15. A recombinant vector comprising the chimeric gene of claim 12.

16. A transgenic host cell comprising the chimeric gene of claim 12.

17. The host cell of claim 16, which is a bacterial cell.

18. The host cell of claim 16, which is a plant cell.

19. 1. A transgenic plant that can be infested by the fall armyworm (Spodoptera frugiperda) pest, wherein the transgenic plant is protected from said pest by being stably transformed with at least one nucleic acid molecule encoding a Txp40 protein or a variant thereof comprising an amino acid sequence having at least 95% identity to SEQ ID NO:

1.

20. 20. The transgenic plant of claim 19, wherein the Txp40 insecticidal protein or variant thereof comprises the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, or any of SEQ ID NOs: 74 to 136.

21. 20. The transgenic plant of claim 19, wherein the nucleic acid molecule comprises the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or any of SEQ ID NOs: 11 to 73.

22. 20. The transgenic plant of claim 19, which is a corn plant.

23. 20. The transgenic plant of claim 19, wherein the armyworm pest is resistant to a Cry1F protein or a Vip3 protein.

24. A codon-optimized nucleotide sequence encoding a Txp40 protein or a variant thereof comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 1, wherein the codons are optimized for expression in corn plants.

25. 23. The codon-optimized polynucleotide of claim 22, wherein the nucleotide sequence comprises SEQ ID NO:

4.

26. 1. A method for reducing the occurrence of resistance to a Vip3 protein or a Cry1F protein in a population of fall armyworm (Spodoptera frugiperda), the method comprising the step of delivering to the fall armyworm population or its environment a transgenic plant comprising a polynucleotide encoding a Txp40 protein or a variant thereof comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1, and a polynucleotide comprising a nucleotide sequence encoding a Vip3A protein or a nucleotide sequence encoding a Cry1F protein, wherein the Txp40 protein or variant thereof and the Vip3 protein or the Cry1F protein are produced in the transgenic plant.

27. 1. A method for producing corn protected against fall armyworm (Spodoptera frugiperda), comprising: (a) introducing into a corn plant a polynucleotide encoding a Txp40 protein or a variant thereof comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1; and (b) planting the corn plant or progeny of the corn plant in an area that may be infested by fall armyworm pests, wherein the corn plant or progeny express the Txp40 protein.

28. 28. The method of claim 27, wherein the introducing step is accomplished by: a) transforming a corn cell with the polynucleotide and regenerating a corn plant that expresses the Txp40 protein or variant thereof; or b) crossing a first corn plant containing the polynucleotide with a second corn plant, resulting in a progeny corn plant that expresses the Txp40 protein or variant thereof.