MXPA06009410A - Methods for enhancing insect resistance in plants - Google Patents

Abstract

Methods for creating and enhancing insect resistance in plants are provided. The methods comprise stably introducing into a plant a combination of polynucleotides comprising a sequence encoding a lipase polypeptide having insecticidal activity and a sequence encoding a Bt insecticidal protein, where each of these coding sequences is operably linked to a promoter that drives expression in a plant cell. Plants with enhanced insect resistance and seed thereof are also provided. The methods of the invention may be used in a variety of agricultural systems for controlling insect pests,including propagating lineages of insect-resistant crops and targeting coexpression of insecticidal lipase and Btinsecticidal protein to plant organs that are particularly susceptible to infestation.

Description

METHODS FOR INCREASING RESISTANCE TO INSECTS IN PLANTS FIELD OF THE INVENTION The present invention relates to the field of plant molecular biology and insect pest control, particularly methods for controlling insect species via the co-expression of insecticidal proteins that They have different modes of action. BACKGROUND OF THE INVENTION Insect pests are a serious problem in agriculture. They destroy millions of acres of fiber crops such as aiz, soy, pea and cotton. Annually, these pests cause up to $ 100 billion in crop damage in the US. only. In a current seasonal battle, farmers must apply billions of gallons of synthetic pesticides to combat these pests. However, synthetic pesticides present many problems. They are expensive, costing the US farmers. almost $ 8 trillion dollars per year. They encourage the emergence of insecticide-resistant pests, and can harm the environment. Other procedures for pest control have been tried. In some cases, crop farmers have introd "natural precleaners" of the species to be controlled, such as non-native insects, fungi, and bacteria similar to Bacillus thuringiensis.Alternatively, crop farmers have introd large colonies of insect pests sterile in the hope that in mating between sterilized insects and fecund wild insects the insect population decreases Unfortunately, the success has been uncertain and the cost considerable.For example, as a practical matter, introd species rarely remain in treated land - dispersing to other areas as an unintended consequence, predatory insects migrate, and fungi or bacteria wash out of plants in streams and rivers, and consequently, crop farmers need more practical and effective solutions. of microorganisms of the genus Bacillus are known to They possess pesticidal activity against a wide range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillus thuringiensis and Bacill us papilliae are among the most successful biocontrol agents discovered to date. The pathogenicity of the insect has been attributed to strains of: B. larvae, B. lentimorbus, B. papilliae, B. sphaericus, B. thuringiensis (Harwoo, ed. (1989) Bacillus (Plenum Press), page 306) and B. cereus (European Patent No. EP0792363). The pesticidal activity is shown to be concentrated in the inclusions of parasporal crystalline protein, although the pesticidal proteins have also been isolated from the stage of vegetative growth of Bacillus. Several genes encoding these pesticidal proteins have been isolated and characterized (see, for example, U.S. Patent Nos. 5,366,892 and 5,840,868). Microbial pesticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives for the chemical control of pests. Insecticidal proteins isolated from Bacillus thuringiensis strains, known as d-endotoxies or Cry toxins, are initially prod in an inactive protoxin form. These protoxins are proteolytically converted into an active toxin through the action of proteases in the insect's intestine. See, ~ nsr - cíotc-c _ c f, Y '', ce ™ !; -YY (• 'Y'- "' t? X ~ - y? Ce _" YYY X \ c, < M ", C'r; io L XYC! Y ') -' '" - • "-y , to proteolytic activation of the toxin can include the removal of the N- and C-terminal peptides of the protein, as well as the internal segmentation of the protein. Other proteases can degrade insecticidal proteins. See Oppert, ibid.; see also US Pat. Nos. 6,057,491 and 6,339,491. Once activated, the Cry toxin binds with high affinity to the receptors in the epithelial cells in the insect's intestine, thereby creating leak channels in the cell membrane, lysis of the insect's intestine, and death of the insect subsequent to through starvation and septicemia. See, for example, Li et al.,, I '«? <; -; - Y3: íM YY. Recently, agricultural scientists have developed crop plants with increased insect resistance by genetically engineering crop plants to produce insecticidal proteins from Bacillus. For example, the aiz and cotton plants genetically designed to produce Cry toxins (see, for example, Aronson (2002) '- «_«? Y s- Y, 59 (3): 417-425; i? -YYY , YY Y AND I .. O '.. Y-, 62 (3); 775-806) are now widely used in American agriculture and have provided the farmer with an environmentally friendly alternative to additional insect control methods. In addition, potatoes genetically engineered to contain Cry toxins pesticides have been sold to the American farmer. However, these insecticidal Bt proteins only protect plants from a relatively small range of pests. Thus, there is an immediate need for methods that increase the effects of Bt insecticidal proteins. BRIEF DESCRIPTION OF THE INVENTION Methods for creating or increasing resistance to insects in plants are provided. The compositions and methods of the invention can be used in a variety of systems to control plant and not plant pests, including the propagation of lineages of insect resistant crops and the direction of expression of pesticidal proteins to plant organs that are particularly susceptible to infestation, such as roots and leaves. These methods also find use in. the management of insect resistance. The methods of the invention comprise genetically modifying a plant to express at least one lipase polypeptide having insecticidal activity in combination with at least one Bacillus thuringiensis insecticidal protein. { Bt). The insecticidal properties of the lipase polypeptide coupled to the second mode of action of the Bt insecticidal protein provide synergistic control of insect pests. The compositions of the invention further comprise constructs that provide for the expression of insecticidal lipases, such as lipid acyl hydrolases, in combination with Bt insecticidal proteins in plants. DNA sequences encoding such Bt insecticidal lipases and proteins useful in the practice of the invention are also provided, including DNA sequences that are optimized for expression in plants. The DNA sequences encoding these insecticidal lipases can be used to transform plants and other organisms for pest control. Also provided are transformed plants, plant and cell tissues, and seeds thereof that have been genetically modified using the methods of the present invention to create or increase their resistance to insect pests. The compositions and methods of the invention can be used in a variety of agricultural systems to control plant and not plant pests, including the propagation of lineages of insect resistant crops and the direction of the coexpression of insecticidal insecticide and lipase proteins. from Bt to plant organs. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the results of the western corn rootworm bioassay (WCRW) of the pentin feed (lipase) and the Bt toxin to developing larvae. The diet causes a dose-dependent inhibition of larval growth as a percentage of wild-type controls. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods for creating and increasing insect resistance in plants by introducing polynucleotides encoding insecticidal lipases and insecticidal proteins of Bacillus thuringiensis (Bt). As will be described herein, these methods are useful for imparting insect resistance to a wide variety of plants, including crops and other domestic plant species. In particular modalities, the methods of the invention comprise stably introducing a combination of polynucleotides into plants, the combination comprising at least one polynucleotide comprising a sequence encoding an insecticidal lipase and at least one polynucleotide comprising a sequence encoding an insecticidal protein of Bt, each of which is operably linked to a promoter that induces expression in a plant cell. "Enter stably" is proposed to imply that the introduced nucleotide sequences are integrated into the plant genome. Once the combination of polynucleotides is introduced into the cells of the plant, the insecticide encoded lipase and the insecticidal Bt protein are transcribed and translated by the endogenous cellular machinery. When insects attempt to feed or lay eggs in the transgenic plant, the combined expression of the insecticidal lipase and the insecticidal protein of Bt either exterminates the insects or inhibits their growth. Thus, the cells of plants, organs, seeds and / or the whole plant in this way become resistant to infestation. Because the cells are stably transformed by these methods, the invention is useful in creating seed and filial lines that are also insect resistant. Additionally, it has been unexpectedly found that the coexpression of the Bt insecticidal protein and the lipase transgenes create a synergistic insecticidal effect. This effect is useful in decreasing the effective dose required. The synergy also decreases the effective amount of insecticidal protein that a plant must produce, thus decreasing the carbon / nitrogen load associated with plant defense and increasing the effective yield of the plant. Thus, many beneficial properties are conferred on a transgenic plant expressing a combination of these two classes of insecticidal proteins, i.e., a Bt insecticidal protein and a lipase polypeptide having insecticidal activity (hereinafter referred to as "lipases"). insecticides "). Insecticidal lipases, such as lipid acyl hydrolases, and Bt insecticidal proteins that can impact an insect pest find, use in practice of the methods of the invention. The term "impact an insect pest" or "impacting an insect pest" is proposed to imply the effect of employing any substance or organism to prevent, destroy, repel or mitigate an insect pest. Thus, many beneficial properties are conferred in a transgenic plant expressing insecticidal proteins, for example, lipase polypeptides and Bt polypeptides having pesticidal activity. As used herein, the term "pesticidal activity" is used to refer to the activity of an organism or a substance (such as, for example, a protein), whether toxic or inhibitory, that can be measured by, but is not limited to, plague mortality, plague weight loss, plague repellency, atrophy of plague growth and other behavioral and physical changes of a plague after feeding and exposure for a duration of time appropriate In this way, the pesticide activity impacts at least one measurable parameter of the plague condition. Similarly, "insecticide activity" can be used to refer to "pesticide activity" when the plague is an insect pest. "Atrophy" is proposed to imply greater than 50% inhibition of growth as determined by weight. General procedures for monitoring insecticidal activity include the addition of the experimental compound or organism to the diet source in an enclosed container. Assays for determining insecticidal activity are well known in the art. See, for example, US Pat. Nos. 6,570,005 and 6,339,144; incorporated herein by reference in its entirety. The optimal development stage for the test for insecticide activity is the larval or immature forms of an insect of interest. Insects can be created in total darkness at about 20 ° C to about 30 ° C and from about 30% to about 70% relative humidity. The bioassays can be performed as described in Czapla and Lang (1990) J. Econ. Entomol 83 (6): 2480-2485. Methods for creating insect larvae and performing bioassays are well known to one of ordinary skill in the art. The term "pesticidally effective amount" connotes an amount of a substance or organism having pesticidal activity when it is present in the environment of a pest. For each substance *? organism, the pesticidally effective amount is determined empirically for each affected pest in a specific environment. Similarly, an "insecticidally effective amount" can be used to refer to a "pesticidally effective amount" when the pest is an insect pest. "Creation or increase of insect resistance" is proposed to imply that the genetically modified plant according to the methods of the present invention has increased resistance to one or more insect pests relative to a plant having a similar genetic component. with the exception of the genetic modification described herein. The genetically modified plants of the present invention are capable of expression of at least one insecticidal lipase and, at least one Bt insecticidal protein, the combination of which protects a plant from an insect pest while impacting a pest. of insects of a plant. "Protects a plant from an insect pest" is proposed to imply limiting or eliminating damage related to insect pests to a plant by, for example, inhibiting the ability of an insect pest to grow, feed and / or reproduce or exterminate the plague of insects. As used herein, "impact of an insect pest of a plant" includes, but is not limited to, the deterrent factor of the insect pest, additional feeding on the plant, insect pests being damaged by, example, the inhibition of an insect's ability to grow, feed and / or reproduce, or the extermination of the insect pest. The toxic and inhibitory effects of insecticidal lipases and Bt proteins include, but are not limited to, atrophy of larval growth, extermination of eggs or larvae, reduction of either adult or juvenile feeding in transgenic plants relative to those observed. in the wild type, and the induction of avoidance of behavior in an insect with form is related to feeding, nesting or reproduction. The term "insecticidal lipase" is used in its broadest sense and includes, but is not limited to, any member of the lipid acyl hydrolases family that has toxic or inhibitory effects on insects. Also, the term "Bt insecticidal protein" is used in its broadest sense and includes, but is not limited to, any member of the Bt family of proteins that have toxic or inhibitory effects on insects, such as Bt toxins described. in the present and known in the art. Thus, as described herein, insect resistance can be conferred to an organism by introducing a nucleotide sequence encoding an insecticidal lipase with a sequence encoding a Bt insecticidal protein or by applying an insecticidal substance, including, but is not limited to, an insecticidal protein, an organism (for example, a plant or plant part thereof). Insecticidal lipases expressed in combination with Bt insecticidal proteins find use as an alternative to a previously implemented pesticide method such as pesticide application and / or modification. - previous genetics of a plant. Measures aimed at reducing the potential for insect pests to become resistant to a pesticide are called "insect resistance management". Insecticidal lipases, such as insecticidal lipid acyl hydrolases including but not limited to those disclosed herein, as well as Bt insecticidal proteins disclosed herein and known in the art are for use in such insect resistance management programs. because they can be used as an alternative to current pesticides. Therefore, insecticidal lipases and Bt insecticidal proteins that. They find use in the invention can also be further selected for use in insect resistance management programs. Those skilled in the art recognize that the selection of a particular insecticidal lipase, such as a lipid acyl hydrolase, and / or a particular Bt insecticidal protein will depend on the type of the resistant insect strain that emerges (or is likely to emerge). as well as the crops that are likely to suffer from the infestation. Any nucleotide sequence encoding a lipase polypeptide having insecticidal activity can be used to practice the methods of the invention. The term "insecticidal lipase" includes any member of the lipid acyl hydrolases family that has toxic or inhibitory effects on insects. Lipases are well known in the art. One class of lipases is the acyl hydrolase class of lipid, also known as triacylglycerol acylhydrolases or triacylglycerol lipases (called enzymes EC 3.1.1.3 under the naming system IUBMB). These enzymes catalyze the hydrolysis reaction: triacylglycerol + HO = diacylglycerol + a carboxylate. Lipid acyl hydrolases all share a conserved cleavable structural region, called the catalytic triad. The catalytic triad consists of a glycine-amino acid-X-serine-amino acid-X-glycine (GxSxG) moiety. It has been shown that the substitution of amino acids in this region abolishes the enzymatic activity. Notably, the enzymatic action of these lipid acyl hydrolases also correlates with significant insecticidal activity. See, for example, insecticide lipases disclosed in the co-pending North American Non-Probate Request filed on February 2005 (attorney's file No. 035718/286812) claiming the benefit of US Provisional Application Serial No. 60 / 546,605, entitled "Lipases and Methods of Use ", filed on February 20, 2004; incorporated herein by reference. * The combination of polynucleotides to be introduced into a plant may comprise a coding sequence for one or more insecticidal lipases. Insecticidal lipases can be derived from plants and not from plants. "No plants" is proposed to imply that it comprises all phylogenetic realms except Plant (ie, comprising the Eubacteria Kingdom, Euryarcheota Kingdom, Crenarcheota Kingdom, ,, - Protozoa Kingdom, Kingdom Mycota, Kingdom> And t; Y > - YÍ > '"' '' '' 'Examples of non-plant lipase sequences useful for the practice of the present invention include Candida lipase 1 (CLIP1) derived from the yeast Candida cylindracea (previously known as Candida rugosa) (NCBI Access No. XI6712) (see, for example, SEQ ID NO: 1. which codes SEQ ID NO: 2), lipase derived from Khizopus > - '> and ~ XX'B Access No. AF229435) (see, for example, SEQ AND NO: A encoding -PM ~ X: 6); lipase derived from Ni trosomonas europaea (NCBI Access No. NP842507 and deposited with the ATCC as Access No. 9 and, and see, for example, YO and YY 7, which codes for SEO "3 NO: 8), and lipase derived from porcine pancreas (see, for example, SEQ and NO: 4 as encoded by the optimized coding sequence of corn shown in SEQ "X NO: 3). Plant lipases for use in the practice of the methods of the invention include, but are not limited to, the pentin-1 lipase derived from the oil soybean tree (see, for example, SEQ. encodes SEO X NO: 10 and the pentin-1 nucleotide sequence optimized for increased expression, for example, SEQ _L NO: 11, which encodes SEO _J NO: 12); (see also, U.S. Patent Nos. 5,981,722 and 6, - ?, 144, incorporated herein by reference in their entirety); patatin lipase (see U.S. Patent 5,743,477, incorporated herein by reference in its entirety); (see also, for example, SEQ ID NO: 13, which encodes SEO l? NO; 14); and functional variants or fragments thereof. See also, Longhi et al., (1992) Bioc Xy Bi ophys. Acta 1131 (2): 227-232 and Lotti et al., (1993) Gene 124 íl): 45-55. In some embodiments, the methods of the present invention provide for the introduction of one or more nucleotide sequences encoding lipase comprising the sequences arranged in the SEO ID NOs: 1, 3, 5, 7, 9, 11 and 13. These sequences encode the insecticidal lipase polypeptides set forth in SEQ ID NOS: 2, 4, 6, 8, 10, 12 and 14, respectively. It is recognized that variants and fragments of these coding sequences can be used as long as they encode a lipase polypeptide having insecticidal activity. According to the methods of the present invention, at least one polynucleotide comprising a sequence encoding a Bt insecticidal protein is introduced into a plant in combination with the introduction of at least one polynucleotide comprising a coding sequence for a Lipase polypeptide having insecticidal activity. Any coding sequence for a Bt insecticidal protein can be used. "Bt insecticidal protein" is proposed to imply the broader class of toxins found in various strains of Bacillus thuringiensis, which includes such toxins as, for example, the vegetative insecticidal proteins and the 5-endotoxins or 'Cry toxins. Vegetative insecticidal proteins (eg, members of classes VIP1, VIP2 or VIP3) are secreted insecticidal proteins that undergo proteolytic processing by midgut insect fluids. They have pesticidal activity against a broad spectrum of Lepidoptera insects. See, for example, U.S. Patent No. 5,877,012, incorporated herein by reference in its entirety. Bt d-endotoxins are synthesized as protoxins and crystallize as parasporal inclusions. When swallowed by an insect pest, the microcrystal structure is dissolved by the alkaline pH of the insect's midgut, and the protoxin is cleaved by the insect intestine proteases to generate the active toxin. The activated Bt toxin binds to receptors in the intestinal epithelium of the insect, causing membrane damage and associated swelling and intestinal lysis. insect. The death of the insect results from starvation and septicemia. See, for example, Li et al., (1991) Nature 353: 315-821; Aronson (2002) Cell Mol. Life Sci. 59 (3): 417-425; Schnepf et al., (1998) Microbí l M¡.-1. Biol. Rev ,, 62 (3): 775-806. Bt d-endotoxins are toxic to larvae of a number of insect pests, including members of the orders Lepidoptera, Diptera and Coleoptera. The effectiveness of Bt insecticidal proteins as a toxin depends on the structure of the toxin, amount ingested, and the species of larvae that ingest the toxin. The d-endotoxins or Bt cry toxins are well known in the art (see, U.S. Patent Application Publication No. 2003/0177528, incorporated herein by reference in its entirety). There are currently over 250 known species of Bt d-endotoxins with a wide range of specificities and toxicities. See, for example, the toxic Bacillus thuringiensis proteins described in U.S. Patent Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al., Gene 48: 109 (1986). For an expansive list see Crickmore et al., (1998) Microbiol. Mol. Biol. Rev. 62: 807-813, and for regular updates see C-CIYC et al., (2003) "Y ~ e;, ':. -v ~ ¡- -i ^ -i- -cu} c "- .ir *",;? biols. susx.ac.V '' l íX: Y í, r? Y ~ YYÍCX, which can be accessed in the world network using the prefix "X. The criteria for inclusion in this list is that the proteins have significant sequence similarity to one or more toxins within the nomenclature or is a parasporal inclusion protein of Bacillus thuringiensis that exhibits pesticidal activity, or that has some experimentally verifiable toxic effect to an objective organism. The d-endotoxins are related to several degrees by similarities in amino acid sequences and tertiary structure and the means to obtain the structures of endotoxin chromosomes from B. thuringiensis are well known. The high resolution crystal structure solution exemplary of both of the Cry3A and Cry3B polypeptides are available in the literature. The resolved structure of the Cry3A gene (Li et al., (1991) Nature 353: 815-821) provides insight into the relationship between the structure and function of endotoxin. A combined consideration of the published structural analyzes of B. thuringiensis endotoxins and the reported function associated with particular structures, portions and the like indicate that specific regions of endotoxin are correlated with particular functions and discrete stages of the mode of action of the protein. For example, the d-endotoxins isolated from B. thuringiensis are generally described as comprising three domains: a bundle of seven helices that is involved in the formation of the pore, a three-lane domain that has been implicated in the binding of the receptor, and a beta-intercalation portion (Li et al., (1991) Nature 305: 815-821). Co-expression of any Bt d-endotoxin (either a holotoxin or insecticidal fragment) is useful for practicing this invention, including all Cry and Cyt species designated as Bt d-endotoxins. These toxins include Cry up to Cry 42, Cyt 1 and 2, Cyt-like toxin and binary Bt toxins. In the case of binary Bt toxins, those skilled in the art recognize that two Bt toxins must be co-expressed to induce the insecticidal activity of Bt. In some embodiments, the methods of the present invention provide for the introduction of a nucleotide sequence encoding a Bt d-endotoxin selected from the group consisting of Cry 1, Cry 3, Cry 5, Cry 8 and Cry 9. Of particular interest they are the Cry 8 d-endotoxins similar to Cry 8. "Similar to Cry8" is proposed to imply that the nucleotide or amino acid sequence shares a high degree of identity or sequence similarity to the previously described sequences classified as Cry 8, which includes such toxins as, for example, CrydBbl (see Genbank Access No. CAD57542) and Cry8Bcl (see Genbank Access No. CAD57543). See copending U.S. Patent Application Publication No. 2004/0210963, filed June 25, 2003, incorporated herein by reference in its entirety. "Insect Protoxin Similar to Cry 8" is proposed to imply the biologically inactive polypeptide that is converted to the insect toxin similar to Cry 8 activated in the cleavage at a proteolytic activation site by a protease. This is the insect toxin similar to activated Cry 8 that has pesticidal activity. As used herein, "insect toxin similar to Cry 8" refers to a biologically active pesticidal polypeptide that shares a high degree of sequence identity or similarity to the Cry 8 insect toxin sequences. In other embodiments, the methods of the present invention provide for the introduction of at least one Bt insecticidal protein, such as a nucleotide sequence encoding the Bt toxin selected from the group consisting of the sequences set forth in SEQ ID NO: 15 (Bt 1218K03), 17 (Bt 1218K04) and 19 (Bt 1218-1K054B). These sequences encode the Bt d-endotoxin polypeptides set out in SEQ ID NO: 16 (Bt 1218K03), 18 (Bt 1218K04) and 20 (Bt 1218-1 054B), respectively. It is recognized that variants and fragments of these coding sequences can be used while the encoded Bt toxin polypeptide has insecticidal activity. The Bt insecticidal proteins to be coexpressed with at least one insecticidal lipase may be naturally occurring proteins, or they may be genetically modified forms thereof which provide improved insecticidal properties. Examples of genetically modified or modified Bt insecticidal proteins include the mutant forms of insect protoxins similar to Cry 8 having a mutation comprising an additional, or alternative, protease sensitive site that is easily recognized and / or segmented by a category of proteases , such as mammalian proteases or insect proteases. The presence of an additional and / or alternative protease sensitive site in the amino acid sequence of the encoded polypeptide can improve the pesticidal activity and / or specificity of the polypeptide compared to that of the unmodified wild-type d-endotoxins. See, for example, the mutant forms of insect protoxins similar to Cry 8 disclosed in copending U.S. Patent Application Publication No. 2004/0210963, filed July 25, 2003, entitled "Genes Encoding Proteins with Pesticidal Activity"; incorporated herein by reference in its entirety. In other embodiments, the Bt insecticidal protein is a Bt d-endotoxin that has been genetically modified to protect the endotoxin from proteolytic inactivation by plant proteases. In this way, a proteolytic site within a Bt d-endotoxin that is susceptible to segmentation by a plant protease is mutated to comprise a site that is not sensitive to the plant protease, thereby protecting the protein from the plant. Proteolytic inactivation by a plant protease. Such proteolytic protection increases the stability of the active toxin in a transgenic plant and improves the resistance properties of the associated pest. See the copending US Patent Application Serial No. 10 / 746,914, filed on December 24, 2003, entitled "Genes Encoding Proteins with Pesticidal Activity"; incorporated herein by reference in its entirety. In alternative embodiments, the Bt insecticidal protein is a Bt d-endotoxin that has been genetically modified for enhanced proteolytic processing in the activated form of the protoxin. In this manner, the Bt d-endotoxins are modified to comprise at least one site of proteolytic activation that is not occurring naturally within the insect protoxin, and which has been designed to comprise a targeting site that is either sensitive to segmentation by a plant protease that resides within the cells of a plant, or is sensitive to segmentation by an insect-gut protease. "Sensitive to segmentation" is proposed to imply that the protease recognizes the cleavage site, and thus is capable of segregating the protoxin at that segmentation site. In both cases, the site of proteolytic activation that does not occur naturally is designed within a region of activation of the insect protoxin. "Activation region" is intended to mean a region within the insect protoxin wherein the proteolytic cleavage at the designed activation site results in the production of a biologically active insect toxin (i.e., the activated form of the insect protoxin). Proteolytic cleavage by plant protease or insect gut protease releases the activated insect toxin within a plant cell or within the insect gut, respectively. See copending United States Patent Application Serial No. 60 / 532,185, filed December 23, 2003, entitled "PlaY Activation of Insect Toxin" incorporated herein by reference in its entirety. Thus, the methods of the present invention comprise introducing a combination of polynucleotides into a plant, wherein the combination comprises at least one polynucleotide comprising a sequence encoding an insecticidal lipase, such as lipid acyl hydrolase, and at least one polynucleotide comprising a sequence encoding a Bt insecticidal protein, wherein each of these coding sequences is operably linked to at least one promoter that induces expression in a plant cell. Those skilled in the art recognize that the coexpression of transgenes can create subtractive, additive or synergistic phenotypic effects. Subtractive effects occur where a second event (for example, transformation with a second gene or cross-breeding with a second plant that expresses a gene of interest) decreases the insecticidal effectiveness in relation to the first event. Synergistic effects occur where the action of two or more agents working together produces a greater effect than the combined effect of the same agents used separately; see for example, McCutchen et al., (1997) J, F n. Ent üoX 90: 1170-1180; Preisler and collaborators, (1999) Y, cop. Exxioxaol .. 92: 598-603. Additive effects occur where two or more agents working together produce an effect at least equal to the combined effect of the same agents used separately. In some embodiments of the invention, coexpression of the combination of polynucleotides comprising sequences encoding the insecticidal lipase, such as a lipid acyl hydrolase, and the Bt insecticidal protein provides a synergistic effect, ie, synergistic increase in the resistance to one or more insect pests. The combination of polynucleotides for practicing the present invention may comprise full-length nucleotide sequences that encode the insecticidal lipase or the Bt insecticidal protein as well as fragments of the full-length coding sequences, wherein the polypeptide fragments are encoded. The term "fragment" is intended to mean a portion of the polynucleotide or a portion of the amino acid sequence and therefore the protein encoded in this manner. The fragments of a polynucleotide can encode protein fragments that retain the biological activity of the native protein and therefore retain the insecticidal activity. Where the combination of polynucleotides to be introduced into a plant comprises respective coding sequence fragments, the fragments in the same way • will encode protein fragments that retain the biological activity of the native protein and therefore retain the insecticidal activity. Thus, a fragment of a polynucleotide can encode a biologically active portion of an insecticidal lipase, such as a fragment of a lipid acyl hydrolase. A biologically active portion of a lipase, such as a biologically active portion of a lipid acyl hydrolase, can be prepared by isolating a portion of one of the polynucleotides encoding a lipase, which expresses the encoded region of the lipase protein ( example, by recombinant expression in vi tro), and by estimating the activity of the expressed portion of the lipase protein for lipid acyl hydrolase activity. For example, the lipid acyl hydrolases retain a conserved amino acid sequence called the catalytic triad (ie, GxSxG) as discussed supra. Thus, a fragment of a lipid acyl hydrolase having a catalytic triad finds use in the invention, since it retains the enzymatic activity. A fragment of a polynucleotide that encodes a biologically active portion of an insecticidal lipase useful in the invention, such as a lipid acyl hydrolase, will encode at least 15, 25, 30, 50, 100, 150, 200, 250 or 300 contiguous amino acids, or up to the total number of amino acids present in a complete lipase protein (eg, 549 amino acids for SEQ ID NO: 2, 450 amino acids for SEQ ID NO: 4, 392 amino acids for SEQ ID NO: 6 and 321 amino acids) for SEQ ID NO: 8. respectively). Polynucleotides that are fragments of nucleotide sequences encoding lipases, such as lipid acyl hydrolases, comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 940 contiguous nucleotides, or up to the number of nucleotides present in a full length polynucleotide disclosed herein (eg, 1650 nucleotides for SEQ ID NO: 1; 1,350 for SEQ? D NO: 3; 3120 nucleotides for SEQ ID NO: 5, of which 1178 contiguous nucleotides of 901-2079 are coding sequence; and 942 nucleotides for SEQ ID NO: 7). Additionally, a fragment of a polynucleotide can encode a biologically active portion of a Bt insecticidal protein. Structure-function relationships are well described in the art for Bt insecticidal proteins. The receptor binding domains and the crystal structure are known. See, for example, Carroll et al., (199.1 i Nature 353: 815-821; Hodgman and Ellar (1990) DNA Seq. 1: 97-106; Smedley and Ellar (1996) Microbiol. 142: 1617-1624; deMaagd et al., (1996) Microbiol. 62: 1537-1543; Knight et al., (1994) N.

Molec. Microbiol. 11: 429-436; and 'Carroll and collaborators, (1997) J. Cell Sci. 110: 3099-3104. A fragment of a polynucleotide that encodes a biologically active portion of a Bt insecticidal protein useful in the invention will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350 or 380 contiguous amino acids, or to the total number of amino acids present in a full-length Bt insecticidal protein (eg, 408 amino acids for SEQ ID? O: 10, 408 amino acids for SEQ ID? O: 12, 386 amino acids for SEQ ID? O: 14, 673 amino acids for SEQ ID? O: 16, 673 amino acids for SEQ ID? O: 18 and 673 amino acids for SEQ ID? O: 20). Polynucleotides which are fragments of nucleotide sequences encoding Bt protein comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200 or 1220 contiguous nucleotides, or up to the number of nucleotides present in a full length polynucleotide disclosed herein (eg, 1307 nucleotides for SEQ ID NO: 9, of which 1226 contiguous nucleotides of 31-1257 are coding sequence, 1227 nucleotides for SEO ID NO: 11, 1404 nucleotides for SEQ ID NO: 13, 2022 nucleotides for SEQ ID NO: 15, 2022 nucleotides for SEQ ID NO : 17; and 2022 nucleotides for SEQ ID NO: 19). A biologically active portion of a Bt insecticidal protein can be prepared by isolating a portion of a full-length coding sequence for the Bt insecticidal protein of interest, which expresses the encoded region of the protein (e.g., by recombinant expression in vitro). vitro), and by estimating the activity of the encoded portion of the Bt insecticidal protein for insecticidal activity using assays well known in the art. The methods of the present invention can be practiced using biologically active variants of insecticidal lipases and / or Bt insecticidal proteins. "Variant" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having deletions (ie, truncations) at the 5 'and / or 3' end; deletion and / or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and / or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences which, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the lipase polypeptides useful in the invention, such as a lipid acyl hydrolase, or a Bt insecticidal protein. . Naturally occurring variants, such as allelic variants, can be identified with the use of well-known molecular biology techniques, such as, for example, with the polymerase chain reaction (PCR) and hybridization techniques as summarized below. Variable polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but still encoding an insecticidal lipase protein and / or Bt insecticidal protein useful in the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity than the particular polynucleotide as determined by the sequence alignment programs and parameters as described in another part in the present. The variants of a nucleotide sequence that encodes the particular insecticidal lipase or the Bt insecticidal protein (ie, the respective reference coding sequence) can also be evaluated by comparing the percent sequence identity between the lipase polypeptide or the Bt insecticidal protein encoded by a variant nucleotide sequence and the lipase or insecticide protein polypeptide of Bt encoded by the reference nucleotide sequence. Thus, for example, isolated nucleic acids encoding an insecticidal lipase polypeptide with a percent sequence identity given to the polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14 and / or a Bt insecticidal protein with a percent sequence identity given to the polypeptide of SEQ ID NO: 16, 18 or 20 can be used in the methods of the invention. The percent sequence identity between any two polypeptides can be calculated using the sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparing the shared identity percent shared by the two polypeptides they encode, the percent sequence identity of the encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or more sequence identity. "Variant protein" is intended to mean a protein derived from the native protein by suppression (called truncation) or addition of one or more amino acids to the N-terminal and / or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids in one or more sites in the native protein. The variant proteins comprised by the present invention are biologically active, that is they continue to exhibit the desired biological activity of the native protein, that is, retaining the insecticidal properties and / or lipase activity as described herein. Such variants may result from, for example, genetic polymorphism or human manipulation. Biologically active variants of a native insecticidal lipase or Bt insecticidal protein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native lipase or Bt insecticidal protein as determined by the sequence alignment programs described elsewhere in the present using the error parameters. A biologically active variant of a Bt insecticidal lipase or protein can differentiate from a native protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as little as 5, as little as as 4, 3, 2 or even 1 amino acid residue. The insecticidal lipases or Bt insecticidal proteins encoded by the invention can be altered in various ways including substitutions, deletions, truncations and amino acid insertions. The methods for such manipulations are generally known in the art. For example, amino acid sequence variants and lipase fragments, such as lipid acyl hydrolase fragments, and / or Bt insecticidal proteins can be prepared by mutations in the DNA. Methods for mutagenesis and alterations of nucleotide sequences are well known in the art. See, for example, Kunkel 1985) Proc. Nati Acad. Sci. EÜA 82: 488-492; Kunkel et al., (1987) Methods in Enzymol. 154: 367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., Atlas of Protein Sequence and Structure (Nat. Biomed. Res. Found., Washington, DC), incorporated herein by reference. Conservative substitutions, such as the exchange of one amino acid with another that has similar properties, may be optimal. Specifically, those of skill in the art will recognize that the regions of the nucleotide sequence and amino acid sequence that are not highly conserved in the lipid acyl hydrolases or Bt insecticidal proteins of the invention when compared to other regions within -the sequences, will generally be less tolerant to modification through amino acid substitutions. As such, the discussed catalytic triad previously found in lipid acyl hydrolases, which consist of a glycine-amino acid X-serine-amino acid X-glycine (GxSxG) portion, can be preserved in certain embodiments to retain enzymatic activity and / or biological Likewise, receptor binding domains and crystal structures are known for Bt proteins, and as such, conserved structures can also be preserved in certain embodiments to retain biological activity. Thus, the nucleotide sequences for use in the practice of the invention include both the naturally occurring sequences as well as the mutant forms. Similarly, the insecticidal proteins of the invention comprise both the naturally occurring proteins as well as the variations and modified forms thereof. Such variants will continue to exhibit the desired insecticidal activity. Obviously, the mutations that will be made in the DNA encoding the variant should not place the sequence outside the reading frame and optimally will not create complementary regions that could produce the secondary mRNA structure. See, Publication and Patent Application EP No. 75,444. The deletions, insertions and substitutions of the protein sequences comprised herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of substitution, deletion or insertion in advance, one skilled in the art will appreciate that the effect will be evaluated by routine classification tests. For example, the activity can be evaluated by a bioassay in which the lipase and / or Bt insecticidal protein is added to the diet of the corn rootworm larvae as described in Example 1. See, for example, Rose and McCabe (1973) J. Econ. Entomol 66: 393, incorporated herein by reference in its entirety. The variant nucleotide and protein sequences also comprise sequences and proteins derived from a mutagenic and recombinogenic process such as intermixing of DNA. With such a procedure, one or more different insecticidal lipase or Bt insecticidal protein encoding sequences can be engineered to create a new lipase or Bt insecticidal protein that possesses the desired insecticidal properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, the sequence portions that encode a domain of interest, such as the catalytic triad and variants thereof, can be inter-mixed between SEQ ID NO: 1 of the invention and other known lipase genes for get a new gene that. codes for a protein with an improved property of interest, such as an increased insecticidal activity. Alternatively, the domain of the St toxin and and one of the sequences as set forth in the SEQ ID NO: 15, SEQ ID NO: 17 and / or SEQ ID NO: 19, could be intermixed with another toxin protein domain of interest. Strategies for such between mixing of DNA are known in the art. See, for example, Stemmer (1994) Proc. Nati Acad. Sci. USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) Nature Biotech. 15: 436-438; Moore et al., (1997) J.

Mol. Biol. 272: 336-347; Zhang et al., (1997) Proc.

Nati Acad. Sci. USA 94: 4504-4509; Crameri and collaborators, (1998) Nature 391: 288-291; and US Patents Nos. 5,605,793 and 5,837,458. As an example, the specificity of altered substrate can be a parameter for the selection of gene intermingling products. The lipid acyl hydrolases comprise a diverse multigene family that is conserved across many species. The enzymes exhibit hydrolyzing activity for many glyco- and phospholipids. The substrates include monogalactosyldiacylglycerol, acyl steryl glucoside, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, as well as many other lipid substrates. Similarly, the membrane composition of various insects as well as plants can vary from species to species and can be affected by diet or growth conditions. Consequently, the activity of a given lipid acyl hydrolase for a given substrate could affect both specificity and potency. The solubility and stability of the protein could also be altered from the mixed gene products. Insecticidal proteins are active in the rigorous environment of the lumen of the insect's intestine. Its proteins are digested by proteases, and affected by reducing oxidizing conditions that vary according to the species of insect tested. Some forms of Bt insecticidal protein require proteolytic processing in the insect's midgut before they become active. Thus, the solubility and stability of insecticidal lipases or insecticidal proteins of Bt in the transgenic plant and in the lumen of the insect intestine could affect the biological activity and could be altered through strategies of gene mixing. In addition, conditions for enzyme reactions such as pH and optimum temperature can also affect the insecticidal activity of a lipase or Bt insecticidal protein. For example, the pH of the bowel of the rootworm of the aiz is 5.5-6.0. The selection of mixed gene products for enzymatic activity towards lipid substrates or Bt toxin receptors in this pH range is another parameter that could affect toxicity. The variants of an insecticidal lipase or Bt insecticidal protein must retain the desired biological activity of the native sequence, i.e., the pesticidal activity. Methods are available in the art to determine whether a variant polypeptide retains the desired biological activity of the native polypeptide. The biological activity can be measured using bioassays specifically designed to measure the activity of the native protein or polypeptide. See, for example, Czapla and Lang (1990) J. Econ. Environment 1. 83 (6): 2480-2485; Andrews et al., (1988) Bi ochem J 252: 199-206; and U.S. Patent No. 5,743, 477, all of which are incorporated herein by reference in their entirety. Additionally, antibodies raised against the native sequence polypeptide can be tested for their ability to bind the variant polypeptide, where the effective linkage is indicative of a polypeptide having a conformation similar to that of the native polypeptide. The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) " percentage of sequence identity ". . (a) As used herein, "reference sequence" is a defined sequence used as a basis for the sequence comparison. A reference sequence can be a subset in the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. (b) As used in the present "comparison window" it refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window can comprise additions and deletions (ie, spaces) compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences. Generally, the comparison window is at least 20 nucleotides contiguous in length, and optionally may be 30, 40, 50, 100 or longer. Those skilled in the art understand that to avoid high similarity of a reference sequence due to the inclusion of spaces in the polynucleotide sequence a space sanction is typically introduced and is subtracted from the number of matches. Methods of sequence alignment for comparison are well known in the art. Thus, the determination of the percent sequence identity between any of the two sequences can be performed using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453; the local search alignment method of Pearson and Lipman (1988) Proc. Nati Acad. 85: 2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Nati Acad. USA 872264, as modified by Karlin and Altschul (1993) Proc. Nati USA 90: 5873-5877. Computer implementations of these mathematical algorithms can be used for sequence comparison to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics GCG Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using the error parameters. The CLUSTAL program is well described by Higgins (1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nucleic Acids Res. 16-: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A weighted residue table PAM120, a space length penalty of 12 and a space penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST program of Altschul et al. (1990) J. Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, log = 100, word length = 12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. Searches of BLAST protein can be performed in the BLASTX program, log = 50, word length = 3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain spaced alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When using BLAST, Gapped BLAST and PSI-BLAST, the error parameters of the respective programs (for example, BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See www.ncbi.nlm.nih.gov. The alignment can also be done manually by inspection. Unless stated otherwise, the sequence identity / similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters:% identity and% similarity for a nucleotide sequence using the Weighting of GAP of 50 and Weight of length of 3 and the registration matrix nwsgapdna.cmp; % identity and% similarity for an amino acid sequence using the GAP Weighting of 8 and Weight of Length of 2 and the registration matrix BLOSUM62; or any equivalent program. By "equivalent program" any sequence comparison program is proposed which, for either of the two sequences in question, generates an alignment that has equalizations of nucleotide or identical amino acid residues and an identical sequence identity percent when compared to the corresponding alignment generated by GAP Version 10. ' GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48-443-453, to find the alignment of two complete sequences that maximizes the number of equalizations and minimizes the number of spaces. GAP considers all possible alignments and space positions and creates the alignment with the largest number of equalized bases and the smallest spaces. This allows the provision of a space creation sanction and a space extension penalty in units of equal bases. GAP must make use of the number of sanctions for the creation of space of equalizations for each space that it inserts. If a space extension penalty greater than zero is selected, GAP, must also make an advantage for each space inserted in the length of the space times of the space extension penalty. The error space creation penalty values and the version 10 space extension sanction values of the Wisconsin Software package. Genetics GCG for protein sequences are '8 and 2, respectively. For nucleotide sequences the error space creation penalty is 50 while the error space extension penalty is 3. The space creation and space extension sanctions can be expressed as a whole number selected from the group of Whole numbers consisting of 0 to 200, So, for example, the space creation space extension penalties can be 0, 1, 2, 3, '4, 5, 6, 7, 8, 9, 10, 15 , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater. GAP presents a member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP exhibits four figures of merit for the alignments: Quality, Relation, Identity and Similarity. Quality is the maximized metric in order to align the sequences. The Relationship is the quality divided by the number of bases in the shortest segment. Percent of Identity is the percent of the symbols that actually match. Percent of Similarity is the percent of symbols that are similar. The symbols that are through the spaces are ignored. A similarity is recorded when the register matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The registration matrix used in version 10 of the Genetics Wisconsin GCG software package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Nati, Acad. Sci. USA 89: 10915). (c) As used herein, "sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence on a comparison window specified. When the percentage of sequence identity is used in reference to proteins it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where the amino acid residues are replaced by other amino acid residues with similar chemical properties ( example, loading or hydrophobicity) and therefore do not change the functional properties of the molecule. When the sequences differ in conservative substitutions, the percent sequence identity can be adjusted upward to correct the conservative nature of the substitution. Sequences that differ with such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those skilled in the art. Typically, this involves registering a conservative substitution as a partial unequalization before it completes, in order to increase the percentage of sequence identity. Thus, for example, where an identical amino acid is given a record of 1 and a non-conservative substitution is given a record of zero, a conservative substitution is given a record between zero and 1. The record of conservative substitutions is calculated, for example, how it is implemented in the PC / GENE program (Intelligenetics, Mountain View, California). (d) As used herein, "percent sequence identity" means the value determined by comparing two sequences optimally aligned over a comparison window, wherein the portion of the polynucleotide sequence in. the comparison window may comprise additions or deletions (i.e., spaces) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to produce the number of equalized positions, by dividing the number of equalized positions by the total number of positions in the comparison window, and by multiplying the result by 100 to produce the percentage of sequence identity. The coding sequences for the insecticidal lipases and Bt insecticidal proteins encompassed by the invention can be provided in expression cassettes for co-expression in the plant or organism of interest. The cassette may include 5 'and 3' regulatory sequences operably linked to a polynucleotide encoding an insecticidal lipase, such as a lipid acyl hydrolase, and / or a Bt insecticidal protein. "Operably linked" is proposed to imply a functional link between two or more elements. For example, an operable link between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows expression of the polynucleotide of interest. Operably linked elements can be contiguous or non-contiguous. When used to refer to the binding of two protein coding regions, by operably linked it is proposed that the coding regions are in the same reading frame. The cassette may additionally contain additional genes to be cotransformed in the organism. Alternatively, the additional gene (s) may be provided in multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for the insertion of a polynucleotide encoding an insecticidal lipase, such as a lipid acyl hydrolase, and / or a Bt insecticidal protein, of So the gene is (or genes are) under the transcriptional regulation of regulatory regions. The expression cassette may additionally contain selectable marker genes. Such expression cassettes are provided with a plurality of restriction sites for the insertion of the lipase and Bt insecticidal protein sequence which is under the transcriptional regulation of the regulatory regions. The expression cassettes may additionally contain selectable marker genes. The expression cassette will include in the 5'-3 'transcription direction, a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence comprised by the invention, such as a DNA sequence that encodes the insecticidal lipase and / or a sequence encoding Bt insecticidal protein, and a functional transcriptional and translational (i.e., termination region) termination region in plants. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and / or the polynucleotides that encode lipase and / or polynucleotides that encode Bt insecticidal protein useful in the invention may be native / analogous to the host cell or each other. Alternatively, regulatory regions and / or polynucleotides that encode lipase and / or polynucleotides that encode Bt insecticidal protein useful in the invention can be heterologous to the host cell or to each other. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if it is from the same species, is substantially modified in its native form in composition and / or genomic site by the deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is a species different from the species from which the polynucleotide was derived, or, if it is from the same / analogous species, one or both are substantially modified from their original form and / or genomic site. or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. The termination region may be native with the transcription initiation region, can be native with the polynucleotides encoding operably linked lipase of interest and / or polynucleotides encoding Bt insecticidal protein of interest, can be native to the plant host, or can be derived from another source (ie, foreign or heterologous) to the promoter, the polynucleotide encoding insecticidal lipase and / or polynucleotides encoding Bt insecticidal protein of interest, the plant host, or any combination thereof). Suitable termination regions are available from the A. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al., (1991) Mol.

• Gen. Genet. 262: 141-144; - Proudfoot (1991) Cell 64: 671-674; Sanfacon et al., (1991) Genes Dev. 5: 141-149; Mogen et al., (1990) Plant Cell 2: 1261-1272; Munroe et al., (1990) Gene 91: 151-158; Bailas et al., (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al., (1987) Nucleic Acids Res. 15: 9627-9639. Where appropriate, the polynucleotides can be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using preferred plant codons for enhanced expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of the preferred codon usage by the host. The methods are available in the art to synthesize preferred plant genes. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, and Murray et al., (1989) Nucleic Acids Res. 17: 477-498, incorporated herein by reference. Those skilled in the art recognize that the native DNA sequence encoding the Bt lipase sequences can not be expressed appropriately in plants. Therefore, certain modifications to the DNA sequence may be necessary to ensure proper protein expression and folding. For example, Candida cylindracea has unusual codon usage. This translates the CTG codon as a serine instead of the usual leucine as in other organisms, see Kwaguchi et al., (1989), Nature 6238: 164-166. As a consequence, if one tries to express the native DNA sequence in plants, the enzyme would contain leucines instead of serines. In some cases, this substitution could not affect the enzymatic activity. However, because the catalytic triad requires a serine at the active site, the substitution of serine to leucine transforms the native inactive encoded lipase into plants. Thus, by replacing the CTG codon with a codon that is read as a serine in plants the activity is restored. For example, replacing CTG with the codons TCT, TCC, TCA, TCG, AGT or AGC will cause the plant to translate the correct amino acid - serine - instead of leucine. The DNA sequence presented in the SEO ID NO: 1, which was derived from Candida cylindracea, includes these advantageous substitutions. Additional sequence modifications are known to increase the expression of the gene in a cell host. These include the removal of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence can be adjusted to average levels for a given cell host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is -modified to avoid predicted secondary hairpin mRNA structures. The expression cassettes may additionally contain 5 'leader sequences in the construction of the expression cassette. Such guide sequences can act to increase translation. Translation guides are known in the art and include: picornavirus guides, for example, guide EMCV (5 'non-coding region of Encephalomyocarditis) (Elroy-Stein et al. (1989) Proc. Nati. Acad. Sci.

USA 86: 6126-6130); Potivirus guides, for example, TEV guidance (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165 (2): 233-238), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated guide of the protein mRNA of the alfalfa mosaic virus coating (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); Guide to Tobacco Mosaic Virus (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and corn chlorotic mottle virus guide (MCMV) (Lo mel et al. (1991) Virology 81: 382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968. In the preparation of the expression cassette, the various DNA fragments can be manipulated to provide the DNA sequences in the proper orientation "and, as appropriate, in the appropriate reading structure. To this end, the adapters or linkers can be used to join DNA fragments or other manipulations can be involved to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like, for this purpose, in vitro mutagenesis, primer repair, restriction, Annealing, re-substitutions, eg, transitions and transversions, may be involved A number of promoters may be used in the practice of the invention Promoters may be selected based on the desired effect The nucleic acids encoding the insecticidal lipase and the Bt insecticidal protein can be combined with constitutive promoters, preferred d and tissue, or other promoters for expression in plants. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubicuitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last and collaborators (1991) Theor, Appl. Genet, 81: 581-588); MAS (Velten and Collaborators (1984) EMBO J. 3: 2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611. Generally, it will be beneficial to express the insecticidal protein sequences from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those of proteins related to pathogenesis (PR proteins), which are induced after infection by a pathogen; for example, PR proteins, SAR proteins, beta-1, 3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth J. Plant Pathol. 89: 245-254; Uknes et al. (1992) Plant Cell 4: 645-656; and Van Loon (1985) Plant Mol. Virol. 4: 111-116. See also WO 99/43819 incorporated herein by reference. Of interest are the promoters that are expressed locally at or near the site of the pathogen's infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9: 335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2: 325-331; Somsisch et al. (1986) Proc. Nati Acad. Sci. USA 83: 2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2: 93-98; and Yang (1996) Proc. Nati Acad. Sci. USA 93: 14972-14977. See also, Chen et al. (1996) Plant J. 10: 955-966; Zhang et al. (1994) Proc. Nati Acad. Sci. USA 91: 2507-2511; Warner et al. (1993) Plant J. 3: 191-201; Siebertz et al. (1989) Plant Cell 1: 961-968; U.S. Patent No. 5,750,386 (inducible by nematode); and the references cited therein. Of particular interest is the inducible promoter for the PRms gene of maize, whose expression is induced by the pathogen Fusarium moniliforme (see, for example, Cordero et al. (1992) Physiol, Mol Plant Plant, 41: 189-200).

Additionally, as pathogens find entry into plants through injury or. insect damage, a promoter inducible by injury can be used to induce the expression of insecticidal proteins. Such injury inducible promoters include the potato proteinase inhibitor gene (pin II) (Ryan (1990) Ann. Rev. Plzytopath. 28: 425-449; Duan and collaborators, (1996) Nature Biotechnology 14: 494-498); win 1 and win 2, the U.S. Patent No. 5,428,148; win 1 and win 2 (Stanford et al., (1989) Mol. Gen. Genet 215: 200-208); systemin (McGurl et al., (1992) Science 225: 1570-1573); WIP 1 (Rohmeier et al., (1993) Plant Mol. Biol. 22: 783-792; Eckelkamp et al., (1993) FEBS Letters 323: 73-76); MPI gene (Corderok et al., (1994) Plant J. 6 (2): 141-150); and the like, incorporated herein by reference. Chemically regulated promoters can be used to modulate the expression of an insecticidal insect protein sequence in a plant through the application of an exogenous chemical regulator. Depending on the objective, the promoter can be a chemically inducible promoter, where the application of the chemical induces the expression of the gene, or a chemically repressible promoter, where the application of the chemical represses the expression of the gene. Chemically inducible promoters are known in the art and include, but are not limited to, the corn In2-2 promoter, is activated by moderators of benzenesulfonamide herbicide, the corn GST promoter, which is activated by the hydrophobic electrophilic compounds that they are used as pre-emergent herbicides, and the PR-la tobacco promoter, which is activated by salicylic acid. Other chemically regulated promoters of interest include promoters responsive to steroids (see, for example, the glucocorticoid-inducible promoter in Schena et al., (1991) Proc. Nati.

Acad. Sci. USA 88: 10421-10425 and McNellis et al., (1998) Plant J. 14 (2): 247-257) and the tetracycline-inducible and repressible promoters by tetracycline (see, for example, Gatz et al., (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Patent Nos. 5,814,618 and 5,789,156), incorporated herein by reference. Preferred tissue promoters can be used to direct the increased expression of insecticidal lipase and Bt insecticidal protein within a particular plant tissue. Preferred tissue promoters include those discussed in Yamamoto et al., (1997) Planl J. 12 (2): 255-265; Kawamata et al., (1997) Plant Cell Physiol. 38 (7): 792-803; Hansen et al., (1997) Mol. Gen Genet. 254 (3); 337-343; Russell et al., (1997) Transgenic Res. 6 (2): 157-168; Rinehart et al., (1996) Plant Physiol. 112 (3): 1331-1341; Van Camp et al., (1996) Plant Physiol. 112 (2): 525-535; Canevascini et al., (1996) Plant Physiol. .112 (2): 513-524; Yamamoto et al., (1994) Plant Cell Physiol. 35 (5): 773-778; Lam (1994) Resul ts Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23 (6) .1129-1138; Matsuoka et al. (1993) Proc Nati. Acad. Scí. USA 90 (20): 9586-9590; and Guevara-Garcia et al., (1993) Plant J. 4 (3): 495-505. Such promoters can be modified, if necessary, for weak expression. Preferred leaf promoters are known in the art. See, for example, Yamamoto and collaborators, (1997) Plant J. 12 (2): 255-265; Kwon et al., (1994) Plant Physiol. 105: 357-67; Yamamoto et al., (1994) Plant Cell Physi ol. 35 (5): 773-778; Gotor et al., (1993) Plant 3: 509-18; Orozco et al., (1993) Plant Mol. Biol. 23 (6): 1129-1138; and Matsuoka et al., (1993) Proc. Nati Acad. Sci. USA 90 (20): 9586-9590. Preferred root promoters are known and can be selected from the many available from the literature or de novo isolates of several compatible species. See, for example, Hirey collaborators, (1992) Plant Mol. Biol. 20 (2): 207-218 (specific soybean glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3 (10): 1051-1061 (Root specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol. Biol. 14 (3): 433-443 (root specific promoter of the manpina synthase (MAS) gene from Agrobacterium tumefaciens; and Miao et al., (1991) Plant Cell 3 (l): ll-22 (full-length cDNA clone which encodes cytosolic glutamine synthetase (GS), which is expressed in roots and nodules of soybean root.) See also Bogusz et al., (1990) Plant Cell 2 (7): 633-641, where two root-specific promoters isolated from genes of hemoglobin from the nitrogen-fixing non-legume Parasponia andersonii and the non-nitrogen-fixing non-ligneous related Trema tomentosa are described.The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the non-legume Nicotiana tabacum and the legume Lotus corniculatus, and in both cases the activity of root specific promoter was preserved, Leach and Aoyagi (1991) describe in their analysis the promoters of the highly expressed Agrobacteri rolC and rolD root induction genes. um rhizogenes (see Plant Science (Limerick) 79: 69-76). They concluded that the preferred DNA determinants of tissue and of dissociated enhancers in these promoters. Teeri et al. (1989) used the lacZ gene fusion to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the root tip epidermis and that the TR2 'gene is root specific in the plant intact and stimulated by leaf injury and tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8 (2): 343-350). The TRl 'gene, fused to. nptll (neomycin phosphotransferase II) showed similar characteristics. Additional preferred root promoters include the VÍENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol. Biol, (1995) .29 (4): 759-772); and the rolB promoter (Capana et al., (1994) Plant Mol. Biol. 25 (4): 681-691 See also U.S. Patents Nos. 5,837,876, 5,750,386, 5,633,363, 5,459,252, 5,401,836, 5,110,732, and 5,023,179. "seed preferred" includes both of the "seed-specific" promoters (those promoters active during seed development such as "seed storage protein" promoters as well as the "seed germination" promoters. (those active promoters during germination of the seeds). See Thompson et al. (1989) BioEssays : 108, incorporated herein by reference. Such preferred seed promoters include, but are not limited to, Ciml (message induced by cytokinin); cC19Bl (zein of 19 kDa of corn); and milps (myo-inositol 1-phosphate synthase); (See WO 00/11177 and U.S. Patent No. 6,225,529, incorporated herein by reference). Gamma-zein is an endosperm-specific promoter. Globulin 1 (Glb-1) is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, β-petrol phaseolin, napin, β-conglycinin, soybean lectin, cruciferin and the like. For monocotyledons, seed-specific promoters include, but are not limited to, 15 kDa zein from aiz, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where the seed preferred promoters of the endl and end2 genes are disclosed; incorporated herein by reference. Where low level expression is desired, weak promoters will be used. Generally, a "weak promoter" is proposed to imply a promoter that drives the expression of a coding sequence at a low level. By the term "low level", levels of approximately 1/1000 transcripts are proposed to approximately 1 / 100,000 transcripts to approximately 1 / 500,000 transcripts. Alternatively, it is recognized that weak promoters also comprise promoters that are expressed in only a few cells and not in others to give a total of low level of expression. Where a promoter is expressed at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels. Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99/43838 and US Patent No. 6,072,050), the core 35M CaMV promoter and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also, US Patent No. 6,177,611, incorporated herein by reference. The expression cassette may also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are used for the selection of transformed cells or tissues. Marker genes include genes that encode antibiotic resistance, such as those that encode neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes that confer resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones and 2, 4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al., (2004) Biotechnol, Bioeng 85: 610-9 and Fetter et al., (2004) Plant Cell 16: 215-28), cyano fluorescent protein (CYP) (Bolte et al., (2004) J. Cell Science 117: 943-54 and Kato et al., (2002) Plant Physiol. 129: 913-42), and fluorescent protein Yellow (PhiYFP ™ from Evrogen; see, Bolte et al., (2004) J. Cell Science 117: 943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech 3: 506-511; Christopherson et al., (1992) Proc. Nati Acad. Sci. USA 89: 6314-6318; Yao et al., (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6: 2419-2422; Barkley et al., (1980) in The Operon, pages 177-220; Hu et al., (1987) Cell 48: 555-566; Brown and collaborators, (1987) Cell 49: 603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et al., (1989) Proc. Nati Acad. Aci USA 86: 5400-5404; Fuerst and collaborators, (1989) Proc. Nati Acad. Sci. USA 86: 2549-2553; Deuschle et al., (1990) Science 248: 480-483; Gossen (1993) Ph. D. Thesis, University of Heidelberg; Reines and collaborators, (1993) Proc. Nati Acad. Sci. USA 90: 1917-1921; Labow et al., (1990) Mol. Cell. Biol. 10: 3343-3356; Zambretti et al., (1992) Proc. Nati Acad. Sci. USA 89: 3952-3956; Baim et al., (1991) Proc. Nati Acad. Sci. USA 88: 5072-5076; Wyborski et al., (1991) Nucleic Acids Res. 19: 4647-4653; Hillenand-Wiss an (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb et al., (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph. D. Thesis, University of Heidelberg; Gossen et al., (1992) Proc. Nati Acad. Sci. USA 89: 5547-5551; Oliva et al. "(1992) Antimicrob Agents Chemother 36: 913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Volume 78 (Springer-Verlag, Berlin); Gili et al. (1988) Nature 334 : 721-724 Such descriptions are incorporated herein by reference The above list of selectable marker genes is not intended to be limiting Any selectable marker gene may be used in the present invention The methods of the invention involve the introduction of A "polypeptide" or "polynucleotide" in a plant. "Introduction" is intended to mean the presentation of the polynucleotide or polypeptide plant in such a way that the sequence gains access to the interior of a plant cell.The methods of the invention do not depend of a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptide gains access to the interior of at least one cell of the The methods for introducing polynucleotides or polypeptides into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods and virus mediated methods. "Stable transformation" is proposed to imply that the nucleotide construction introduced into the plant is integrated into the genome of the plant and is capable of being inherited by the plant's progeny. "Transient transformation" is intended to mean that a polynucleotide is introduced into a plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant. Transformation protocols as well as protocols for introducing polypeptide or polynucleotide sequences into plants can vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods for introducing polypeptides and polynucleotides into plant cells and subsequent insertion into the genome of the plant include microinjection (Crossway et al. (1986) Biotechniques 4: 320-334), electroporation (Riggs et al. (1986) Proc. Nati, Acad. Sci. USA 83: 5602-5606), Agrobacterium-mediated transformation (U.S. Patent Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and ballistic particle acceleration (see, for example, U.S. Patent Nos. 4,945,050; 5,879,918; 5,88; 6,244; 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment", in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin), and McCabe and collaborators (1988) Biotechnology 6: 923-926), and the Lecl transformation (WO 00/28058). Weissinger and collaborators (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 81: 611-614 (soy); McCabe et al. (1988) Bio / Technology 6: 923-926 (soybean); Finer and McMullen (1991) In Vi tro Cell Dev.

Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet 96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al (1988) Proc. Nati Acad. Scí. USA 85: 4305-4309 (corn); Klein et al (1988) Biotechnology 6: 559-563 (corn); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91: 440-444 (corn); Fromm et al (1990) Biotechnology 8: 833-839 (corn); Hooykaas-Van Slogteren et al. (1984) Nature (London) -311: 763-764; U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Nati Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman and collaborators (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet 84: 560-566 (transformation mediated by Whisker); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize via Agrobacterium tumefaciens); all of which are incorporated herein by reference. The polynucleotide constructs of the invention can be introduced into the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest into a viral DNA or RNA molecule. It is recognized that a insecticidal lipase and / or Bt insecticidal protein useful in the invention can be initially synthesized as part of a viral polyprotein, the latter can be processed by proteolysis in vivo or in vi tro to produce the desired recombinant protein. In addition, it is recognized that the promoters of the invention also comprise promoters used for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and the expression of a protein encoded therein, which involve viral DNA or RNA molecules are known in the art. See, for example, U.S. Patent Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367 and 5,316,931; and Porta et al. (1996) Molecular Biotechnology 5: 209-221; incorporated herein by reference. In specific embodiments, the insecticidal lipase sequences and the Bt insecticidal proteins useful in the invention can be provided to a plant using a variety of transient transformation methods. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al., (1986) Mol. Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44: 53-58; Hepler et al., (1994) Proc. Nati Acad. Sci. 91: 2176-2180 and Hush et al., (1994) J. Cell Sci. 107: 775-784, all of which are incorporated in the present by reference. Alternatively, a polynucleotide encoding insecticidal lipase and / or polynucleotide encoding Bt insecticidal protein can be transiently transformed in the plant using techniques known in the art. Such techniques include the vector system. and the precipitation of the polynucleotide in a manner that prevents subsequent release of the DNA. Thus, the transcription of the DNA bound to a particle can occur, but the frequency with which it is released will become responsible for the genome is greatly reduced. Such methods include the use of particles coated with polyethylimine (PEI; Sigma # P3143). Methods are known in the art for the digested insertion of a polynucleotide to a specific location in the genome of the plant. In a modality, the insertion of the polynucleotide into a desired genomic location is achieved using a site-specific recombination system. See, for example, WO 99/25821, WO 99/25854, WO 99/25840, WO 99/25855 and WO 99/25853, all of which are incorporated herein by reference. Briefly, the polynucleotide of the invention can be contained in a transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated in its genome an objective site that is flanked by two non-recombinogenic recombination sites corresponding to the transfer cassette sites. An appropriate recoinase is provided and the transfer cassette is integrated into the target site. The polynucleotide of interest is thus integrated into a specific chromosomal position in the genome of the plant. The cells that have been transformed can be grown in plants according to conventional manners. See, for example, McCormick et al., (1986) Plant Cell Reports 5: 81-84. These plants can then be cultured, either pollinated with the same transformed strain or different strains, and the resulting progeny that has expression of the desired, identified phenotypic characteristic. Two or more generations can be cultured to ensure that the expression of the desired phenotypic characteristic is stably maintained and inherited and then the seeds harvested to ensure the expression of the desired phenotypic characteristic that has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into its genome. In some embodiments, the combination of nucleotide sequences to be introduced into a plant comprises a single nucleotide sequence encoding an insecticidal lipase and a single nucleotide sequence encoding a Bt insecticidal protein, each with its own expression cassette. Where two cassettes are jointly expressed in the same plant, the cassettes are referred to herein as "stacked" constructions. These expression cassettes may be in different constructions or in the same construction. Where the expression cassettes are both present in the same construction, the construction is also referred to herein as a "molecular stacking" construct. In still other embodiments, the nucleotide sequence encoding an insecticidal lipase and a nucleotide sequence encoding a Bt insecticidal protein are fused and thus expressed as a fusion polynucleotide in a single expression cassette. Such construction is referred to herein as a "fusion" construct. In some other embodiments, this combination of nucleotide sequences can be stacked with some third (or more) polynucleotide sequence of interest in order to create plants with a desired attribute. An attribute, as used herein, refers to the phenotype derived from a particular expressed nucleotide sequence or groups of sequences. For example, the combination of polynucleotides encoding a Bt insecticidal protein and an insecticidal lipase of the present invention can be stacked with any other of the polynucleotides encoding polypeptides having pesticidal and / or insecticidal activ such as lectins (Van Damme et al., (1994) Plant Mol. Biol. 24: 825 and the like The combinations generated can also include multiple copies of any of the polynucleotides of interest.

For example, in one embodiment, at least one copy of the nucleotide sequence encoding the Bt insecticidal protein is expressed in combination with a plurality of copies of the nucleotide sequence encoding the insecticidal lipase. In another embodiment, at least one nucleotide sequence encoding an insecticidal lipase is expressed in combination with a plurality of nucleotide sequences encoding Bt insecticidal proteins. The combination of polynucleotides encoding the insecticidal proteins can also be stacked with any other gene or combination of genes to produce plants with a variety of desired attribute combinations including, but not limited to, desirable attributes for feeding animals such as genes from high oil (e.g., U.S. Patent No. 6,232,529); balanced amino acids (eg, hordothionines (U.S. Patent No. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); high-licina barley (Williamson et al., (1987) Eur. J.

Biochem. 165: 99-106; and WO 98/20122) and high-methionine proteins (Pedersen et al., (1986) J.

Biol. Chem. 261: 6279; Kirihara et al., Gene 71: 359 (1988): and Musumura et al., (1989) Plant Mol. Biol. 12: 123)); increased digestibility (eg, modified storage proteins (North American Application Serial No. 10 / 053,410, filed November 7, 2001), and thioredoxins (North American Application Serial No. 10 / 005,429, filed December 3, 2001) )); the descriptions of which are incorporated herein by reference. The combination of polynucleotides encoding the lipase and Bt insecticidal proteins can also be stacked with desirable attributes for resistance or disease to herbicides (eg, fumonisin detoxification genes (US Patent No. ,792,931); avirulence genes and disease resistance (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089); acetolactate synthase (ALS) mutants that lead to herbicidal resistance such as S4 and / or Hra mutations; genes that code for resistance to herbicides that act for the inhibition of the action of glutamine synthase, such as phosphinothricin or coarse (eg, gene bar); genes encoding glyphosate resistance (eg, the EPSPS gene and the GAT gene; see, for example, U.S. Publication No. 20040082770 and WO 03/092360)); and desirable attributes for processing or process products such as high in oil (eg, US Patent No. 6,232,529); modified oil (for example, fatty acid desaturase genes) (U.S. Patent No. 5,952,544; WO 94/11516)); modified starch (eg, ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Patent No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase and acetoacetyl-CoA reductase (Schubert et al., (1988) J. Bacteriol. 170: 5837-5847) facilitate the expression of polyhydroxyalkanoates (PHAs) )); the descriptions of which are incorporated herein by reference. The polynucleotides of the present invention could also be combined with polynucleotides that provide agronomic attributes such as male sterility (e.g., see U.S. Patent No. 5,583,210), stem strength, flowering time or transformational technology attributes such as regulation of the cell cycle or the direction of the gene (for example / WO 99/61619, WO 00/17364 and WO 99/25821); descriptions of which are incorporated herein by reference. These stacked combinations, including the combination of polynucleotides comprising sequences encoding insecticidal lipase and Bt insecticidal protein., can be created by any method including, but not limited to, cross-breeding by any conventional methodology • or TopCross, or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired attributes can be used as the objective to introduce additional attributes by subsequent transformation. The attributes can be introduced simultaneously into a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained in the same transformation cassette (cis). The expression of the sequences can be conducted by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette which will suppress the expression of the polynucleotide of interest. This can be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of attributes in the plant. It is further recognized that the polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination site. See, for example, WO 99/25821, WO 99/25854, WO 99/25840, WO 99/25855 and WO 99/25853, all of which are incorporated herein by reference. As used herein, the term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calluses, plant clusters and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, • flowers, branches, fruits, grains, ears, corn, farfollas, stems, roots, root points, anthers and the like. The grain is proposed to imply mature seed produced by commercial farmers for purposes other than the cultivation or reproduction of the species. Progeny, variants and mutants of regenerated plants are also included within the scope of the invention, as long as these parts comprise the introduced polynucleotides. The nucleotide sequences encoding insecticidal lipases and Bt insecticidal protein can be engineered and used to express the proteins in a variety of hosts including, but not limited to, microorganisms and plants. In addition, the present invention can be used for transformation of any plant species, including, but not limited to, monocots and dicotyledons. Examples of plant species of interest include, but are not limited to, maize (Tea mays), Brassica sp. (for example, B. napus, B. rapa, B. júncea), particularly those Brassica species useful as a source of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Sécale cereale,) sorghum (Sorghum bicolor, Sorghum vulgare), millet (for example, pearl millet) (Pennisetum glaucum), millet proso (Panicum miliaceum), millet of foxtail (Setaria italica), millet spread (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean ( Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hipogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), casava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucífera), pineapple (Ananas comosus),. citrus trees (Citrus spp.), cacao (Theobroma cacaco), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica) , olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almonds (Prunus amygdal us), beets (Beta vulgaris), sugar cane (Saccharum spp.), Oats, barley, vegetables, ornamental plants and conifers. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (for example, Lactuca sativa), green beans (Phaseolus vulgaris), beans (Phaseolus limensis), peas (Lathyrus spp.), And members of the genus Cucumis such as cucumber (C. sativus ), cantaloupe (C. cantalupensis), and melon (C. meló). Ornamental plants include azalea (Rhododendron spp.), Hydrangea (Macrophylia hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), Tulips (Tulipa spp.), Daffodils (Narcissus spp.), Petunias (Petunia hybrida), carnation (Dianthus caryophyllus), red shepherdess (Euphorbia pulcherrima), and chrysanthemum. Conifers that can be employed in the practice of the present invention include, for example, pine trees such as incense pine (Pinus taeda), pine tree (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta) ), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesil); western pinabete (Tsuga canadensis); Sitka fir (Picea glauca); red wood (Sequoia sempervirens); typical firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western red cedar (Thuja plicata) and yellow Alaskan cedar (Chamaecyparis nootkatensis). In specific embodiments, the plants of the present invention are crop plants (e.g., corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other modalities, the corn and soybean plants are optimal, and in other modalities the maize plants are optimal. Plants of particular interest include grain plants that provide seeds of interest, oilseed plants and leguminous plants. The seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil seed plants include cotton, soybean, safflower, sunflower, Brassica, corn, alfalfa, palm, coconut, etc. Legume plants include beans and peas. The beans include guar, carob, fenegreco, soybeans, garden beans, cowpea, ungo, bean, fava bean, lentils, chickpea, etc. The methods of the invention can be used to protect plants from pests, especially insect pests. In particular, proteins and nucleotide sequences that are inhibitory or toxic to insects of the coleoptera order can be obtained and used in the methods of the invention. The embodiments of the present invention can be effective against a variety of pests. For purposes of the present invention, pests include, but are not limited to, insects, fungi, bacteria, nematodes, acarids, protozoan pathogens, liver flukes for animal sites and the like. In particular, proteins and nucleotide sequences that are inhibitory or toxic to insect pests are encompassed by the present invention. Insect pests include insects selected from the Coleoptera orders, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera. Insect pests of particular relevance include those that infest major crops. For example: Corn: Ostrinia nubilalls, European corn borer; Agrotis Ípsilon, black cutworm; Helicoverpa zea, maize maso worm; Spodoptera frugiperda, devastating autumn worm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, stem borer of lesser maize; Diatraea saccharalis, sugarcane borer; Diabrotica virgifera, western corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotus spp., Centipede; Cyclocephala borealis, northern masked bumblebee (white larva); Cyclocephala immaculata, southern masked bumblebee (white larva); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn beetle; Splzenoplzorus maidis, weevil insect of corn; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, bug bugs; Melanoplus femurrubrum, red-legged lobster; Melanoplus sanguinipes, migratory lobster; Hylemya platura, corn worm; Agromyza parvicornis, spotted maize miner; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, ant stealing; Tetranychus urticae, two-spotted red mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, devastating autumn worm; Helicoverpa zea, corn cob worm; Elasmopalpus lignosellus, minor corn stem borer; Underground felting, granulated cutworm; Phyllophaga crinita, white larva; Eleodes, Conoderus and Aeolus spp., Centipedes; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn beetle; Sphenophorus maidis, weevil of corn; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugar cane aphid; Blissus leucopterus leucopterus, bug bugs; Contarinia sorghicola, jej6n de sorgo; Tetranychus cinnabarinus, carmine red mite; Tetranychus urticae, two-spotted red mite; Wheat: Pseudaletia unipunctata, worm devastating; Spodoptera frugiperda, devastating autumn worm; Elasmopalpus lignosellus, stem borer of minor maize; Agrotis orthogonia, worm cutter of the west; Elasmopalpus lignosellus, minor corn stem borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, cloverleaf weevil; Diabrotica undecimpuctata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis graminum, green bug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, red-legged lobster; Melanoplus differentialis, differential locust; Melanoplus sanguinipes, migratory lobster; Mayetiola destructor, Hesse fly; If todiplosis mosellana, wheat midge; Meromyza americana, wheat stem worm; Hylemya coarctata, wheat bulb fly; Frankliella fusca, tobacco trips; Cephus cinctus, wheat stem sawfly; Tulipae, billowed mire of wheat; Sunflower: Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; zygogra ma exclamatíonis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtíana, sunflower seed midge; Cotton: Heliothis virescens, cotton worm; Helicovelpa zea, cotton weevil; Spodoptera exigua, beet devastating worm; Pectinophora gossypiella, pink weevil; Anthonomus grandis, cotton weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, small cotton lobster; Trialeurodes abutilonea, white fly with stripes; Lygus lineolaris, bleached plant bug; Melalloplus femurrubrum, red-legged lobster; Melanoplus diflerentialis, differential lobster; Thrips tabaci, onion thrips; Franklinkiella filsca, tobacco trips; Tetranychus cinnabarinus, carmine red mite; Tetranychus urticae, two-spotted red mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, devastating autumn worm; Helicoverpa zea, corn cob worm; Colaseis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Si tophilus oryzae, rice weevil; Nephotettix nigropictus, rice grasshopper; Blissus leucopterus leucopterus, bug bugs; Acrosternum hilare, green bug; Soy: Pseudoplusia incl udens, soy measuring caterpillar; Anticarsia gemmatalis, hay caterpillar; Plathypena scabra, green clover worm; Ostrinia nubilalis, European corn barrel; Agrotis Ípsilon, black cutworm; Spodoptera exigua, beet devastating worm; Heliothis virescens, cotton weevil; Helicoverpa zea, cotton weevil; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato grasshopper; Acrosternum hilare, green forest bug; Melanoplus femurrubrum, red-footed grasshopper; Alelanoplus differentialis, differential grasshopper; Hylemya platura, corn seed larva; Sericothrips variabilis, soybean thrips; Thrips tabad, onion thrips; Tetranychus turkestani, red strawberry mite; Tetranychus urticae, two-spotted red mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, green bug; bug bugs, for example, Blissus leucopterus leucopterus; Acrosternum hilare, green forest bug; Euschistus servus, coffee bug; Jylemya platura, corn seed larva; Mayetiola destructor, Fly of Hese; Petrobia latens, acaro de trgo coffee; Oilseed rape: Brevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, cruciferous beetle; Mamestra configurata, devastating worm Berta; Plutella xylostella, diamond back moth; Delia ssp. Root maggots. It is recognized that having discovered the beneficial effects of the coexpression of these two classes of insecticidal proteins, similar protection of insect pests could be realized by their co-application to the environment of the target species (s). Thus, at least one insecticidal lipase and at least one Bt insecticidal protein can be used together to protect plants, seeds and plant products of insect pests in a variety of ways. When used for coaplication, the two kinds of insecticidal proteins can be applied as a single pesticidal composition; alternatively, they can be co-applied as two separate pesticidal compositions, one comprising an effective amount of the insecticidal lipase, the other comprising an effective amount of the Bt insecticidal protein. Where more than one member of any class of insecticidal proteins is used to practice the invention, the additional member (s) may be applied as a separate pesticidal composition, or as part of the pesticidal composition comprising either the other insecticidal lipase (s), the other insecticidal protein (s) of Bt, or the other insecticidal lipase (s) (s) ) • the other insecticide protein (s) of Bt (ie, all members of these two classes of insecticidal proteins that are co-applied as a single pesticide composition). For example, the insecticidal lipase and the Bt insecticidal protein can be used in a method that involves placing an effective amount of one or more pesticidal compositions comprising both the lipase and the Bt insecticidal protein in the plague environment by a process selected from the group consisting of spraying, spraying, dispersion or seed coating. Before the propagation of the plant the material (fruit, tuber, bulb, bulbous stem, grains, seeds), but especially seeds, is sold as a commercial product, this is usually treated with a protective coating that includes herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with additional carriers, surfactants or application promotion adjuncts usually employed in the formulation art to provide protection against damage caused by bacterial pests , fungal or animal. In order to treat the seed, the protective coating can be applied to the seeds either by impregnating the tubers or granules with a liquid formulation or by coating them with a combined wet or dry formulation. In addition, in special cases, other methods of application to plants are possible, for example, treatment directed to the buds or to the fruit. The plant seed of the invention comprising a combination of polynucleotides encoding an insecticidal lipase and Bt insecticidal protein can be treated with a seed protective coating comprising a seed treatment compound, such as, for example, captan, carboxy , thiram, metalaxyl, pirimiphos-methyl and others that are commonly used in the treatment of seeds. In one embodiment within the scope of the invention, a protective seed coat comprising a pesticidal composition comprising both an insecticidal lipase and the Bt insecticidal protein is used alone or in combination with one of the seed coatings usually used. in seed treatment. It is recognized that polynucleotides comprising sequences encoding insecticidal lipases and Bt insecticidal proteins can be used to transform pathogenic insect organisms to provide host organism production of these insecticidal proteins, and subsequent application of the host organism to the environment of the host. (s) target plague (s). Such host organisms include baculoviruses, fungi, protozoa, bacteria and nematodes. Optimally, the host organism is co-transformed with polynucleotides comprising the coding sequences for both the insecticidal lipase and the Bt insecticidal protein to ensure co-expression of these proteins and maximum exposure to the combination of their pesticidal activities. Alternatively, the individual classes of insecticidal proteins can be expressed in different classes of the same host organism, or in different host organisms, with the subsequent co-application of the different classes or different host organisms to the environment of the plague (s). target, while the expression of these two classes of insecticidal proteins within different classes or different host organisms provides the combined presentation of both classes of insecticidal proteins to the environment of the target pest (s). In this manner, the combination of polynucleotides encoding the insecticidal lipase and the insecticidal Bt protein can be introduced via a suitable vector into a microbial host and the host applied to the environment, or to plants or animals. The term "enter" in the context of inserting a nucleic acid into a cell, means "transfection" or "transformation" or "transduction" including the reference, to the incorporation of a nucleic acid into a 'eukaryotic or prokaryotic cell where the nucleic acid can be stably incorporated into the genome of the cell (eg, chromosome, plasmid, plastidase or mitochondrial DNA), converted into an autonomous, or transiently expressed replicon (eg, transfected mRNA). Host microorganisms that are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere and / or rhizoplane) of one or more crops of interest can be selected. These microorganisms are selected to be able to compete successfully in the particular environment with the wild-type microorganisms, provide stable maintenance and expression of the sequences encoding the lipase and Bt insecticidal proteins and desirably, provide improved protection of these insecticidal proteins from degradation and environmental inactivation. Such microorganisms include bacteria, algae and fungi. Of particular interest are microorganisms such as bacteria, for example, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, _ Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc and Alcaligenes, fungi, particularly yeast, for example, Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula and Aureobasidium. Of particular interest are such bacterial species of the phytosphere such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacterxylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinlandir and yeast species of the phytosphere. as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurefatii, Saccharofnyces rosei, S. pretoriensis, S. cerevisiae, Spor obo fnyces rosues, S odous, Kluyveromyces ve ona and Aureobasidium pollulans. Of particular interest are pigmented microorganisms. A number of ways are available to introduce the combination of polynucleotides comprising sequences encoding the lipase and Bt insecticidal proteins into the microorganism host under conditions that permit stable maintenance and, the expression of these nucleotide coding sequences. For example, the expression cassettes can be constructed to include the nucleotide constructs of interest operably linked to the transcriptional and translational regulatory signals for the expression of the nucleotide constructs, and a nucleotide sequence homologous to a sequence in the host organism, through which the integration will occur, and / or a replication system that is functional in the host, through which integration or stable maintenance will occur. Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcription initiation starters, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals and the similar ones. See, for example, U.S. Patent Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al., (2000); Molecular Cloning: A Laboratory Manual (3- edition, Cold Spring Harbor Laboratory Press, Plainview, NY); Davis et al., (1980) Advanced -Bacterial Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and references cited therein.) Suitable host cells, where cells containing pesticidal proteins will be treated to prolong the activity of the insecticidal proteins in the cell when the treated cell is applied to the environment of the target plague (s), can include either prokaryotes or eukaryotes, usually being limited to those cells that do not produce substances toxic to higher organisms, such as Nevertheless, organisms that produce toxic substances to higher organisms could be used, where the toxin is unstable or application level sufficiently low to avoid any possibility of toxicity to a mammalian host. lower eukaryotes, such as fungi, illustrative prokaryotes, both g ram-negative as gram-positive, include Enterobacteriaceae, such as Escherichia, Erwifaia, Shigella, Salmonella and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacteria, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among the eukaryotes are fungi such as Phycomycetes and Ascomycetes, which includes yeast, such as Schizosaccharomyces; and yeast of Basidiomycetes, such as Ythodotorula, Aureobasidium, Sporobolomyces and the like. Characteristics of particular interest in the selection of a host cell for purposes of the production of pesticidal protein include the ease of introduction of the <; sequence that encodes the pesticide protein in the host, availability of the expression systems, efficiency of expression, stability of the protein in the host, and the presence of auxiliary genetic capabilities. Characteristics of interest for use as a pesticide icrocapsule include protective qualities for the pesticide, such as the walls of the thick cell, pigmentation and packaging or intracellular formation of inclusion bodies; affinity to the leaf; lack of toxicity to the mammal, attraction to pests for ingestion; ease of extermination and fixation without damage to the toxin; and the similar ones. Other considerations include ease of formulation and handling, economy, storage stability, and the like. Host organisms of particular interest include yeast, such as Rhodotorula spp. , Aureobasidium spp. , Saccharomyces spp. , and Sporoboloinyces spp. , phylloplane organisms such as Pseudomonas spp. , Erwinia spp. , and Flavobacterium spp. , and other such organisms, including, Pseudomonas aerugxnosa, Pseudomonas fluoescens, Saccharomyces cerevisiae, Bacxillus thuringxensxs, Escherichxa coli, Bacillus subtilis and the like. The combination of polynucleotides comprising sequences encoding the lipase and Bt insecticidal proteins comprised by the invention can be introduced into microorganisms that multiply in plants (epiphytes) to deliver these two classes of insecticidal proteins to potential target pests. Epiphytes, for example, can be gram-positive or gram-negative bacteria. Bacteria that colonize the root, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a strain of Bacillus cereus colonizing the roots can be isolated from roots of a plant (see, for example, Handelsman et al. (1991) Appl. Envxron Microbiol. 56: 713-718). The combination of polynucleotides comprising sequences encoding the lipase and Bt insecticidal proteins can be introduced into a Bacillus cereus colonizing the root by standard methods known in the art. For example, the sequences encoding these insecticidal proteins can be introduced, for example, into the Bacillus colonizing the root by means of electrotransformation. Specifically, the nucleotide sequences encoding the lipase and Bt insecticidal proteins can be cloned into a shuttle vector, eg, pHT3101, and the shuttle vector can be transformed into the Bacillus colonizing the root by electroporation ( Lerecius et al. (1989) FEMS Microbiol. Letts, 60: 211-218).

Expression systems can be designed so that "these insecticidal proteins are secreted out of the cytoplasm of gram-negative bacteria, E. coli, for example." The advantage of having the lipases and insecticidal proteins of Bt secreted are: (1) avoidance of potential cytotoxic effects of the expressed pesticidal protein and (2) improve the efficiency of the purification of these insecticidal proteins, including, but not limited to, increased efficiency in the recovery and purification of the protein by volume of cellular broth and decreased time and / or costs of recovery and purification by unit protein Insecticidal proteins can be made to be secreted in E. coli, for example, by fusing an appropriate E. coli signal peptide to the amino-terminal end of the insecticidal protein. The signal peptide recognized by E. coli can be found in already known proteins that are secreted in E. coli, for example the protein to OmpA (Ghrayeb et al. (1984) EMBO J. 3: 2437-2442). O pA is a major protein of the outer membrane of E. coli, and thus its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide does not need to be modified prior to processing as could be the case for other signal peptides, for example, the lipoprotein signal peptide (Duffaud et al. (1987.) Meth.

Enzymol. 153: 492). Lipase and Bt insecticidal proteins can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same way that Bacillus thuringiensis strains have been used as insecticidal sprays. In the case of an insecticidal protein (s) that is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and / or deletions prevent the secretion of the insecticidal protein (s) in the growth medium during the fermentation process. The insecticidal proteins are retained within the cells, and the cells are then processed to produce the encapsulated insecticidal proteins. Any suitable microorganism can be used for this purpose. Pseudomonas have been used to express Bacxillus thuringiensxs endotoxins as encapsulated proteins. and the resulting cells processed and sprayed as an insecticide. Gaertner et al. (1993), in Advanced Engineered Pesticides, ed. L. Ki (Marcel Decker, Inc.). Alternatively, insecticidal proteins are produced by introducing heterologous genes into a cellular host. The expression of heterologous genes results directly or indirectly in the intracellular production and maintenance of these insecticidal proteins. These cells are then treated under conditions that prolong the activity of the Bt lipase and insecticide gene produced in the cell when the cell is applied to the environment of the target pest (s). The resulting product retains the pesticidal activity of these insecticidal proteins. These naturally-encapsulated pesticide proteins can then be formulated according to conventional techniques for application to the environment harboring a target pest, eg, soil, water, and foliage of the plants. See, for example, EPA 0192319 and the references cited therein. In the present invention, a transformed microorganism (which includes, whole organisms, cells, spore (s), insecticidal protein (s), pesticide component (s), component (s) that impacts pests, mutant (s)) s) optimally dead or alive cells and cellular components, including mixtures of living and dead cells and cellular components, and including broken cells and cell components) or an isolated insecticidal protein can be formulated with an acceptable carrier in separate or combined pesticidal compositions which are, for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder and an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, and also encapsulations in, for example, polymeric substances. Such compositions disclosed in the above can be obtained by the addition of an active agent on the surface, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye , a UV protector, a regulatory solution, a flow agent or fertilizers, donors of icronutrients, or other preparations that influence the growth of the plant. One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, crop assistants, and fertilizers, may be combined with carriers, surfactants or adjuvants usually employed in the formulation technique or other components to facilitate the handling of the product and the application for the particular target pests. Suitable adjuvant carriers can be solid or liquid and correspond to the substances ordinarily employed in the formulation technology, for example, natural or regenerated mineral substances, solvents, dispersants, wetting agents, glidants, binders or fertilizers. the present invention (ie, the combination of at least one lipase and at least one Bt insecticidal protein) are normally applied in the form of compositions and can be applied to the crop, plant or seed area to be treated. For example, the compositions, pesticides can be applied to grain in the preparation or during storage in a grain bin or silo, etc. The pesticidal compositions can be applied simultaneously or in succession with other compounds. to a pesticidal composition containing at least one lipase and / or insecticidal protein of Bt in cluye, but not limited to, foliar application, seed coating and application to the soil. The number of applications and the proportion of application depend on the intensity of infestation by the corresponding plague. Suitable surface active agents include, but are not limited to, anionic compounds such as carboxylate, for example a metal; carboxylate of a long chain fatty acid; an N-acyl sarcosinate; mono- or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfate; alkyl phenol sulphates ethoxylated; lignin sulfonates; petroleum sulfonates; alkyl aryl sulfonates such as alkyl benzene sulfonates or lower alkyl naphthalene sulfonates, for example, butyl naphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates, such as amide sulfonates, for example, the sulphonated condensation product of oleic acid and N-methyl taurine; or dialkyl sulfosuccinates, for example, sodium sulfonate or dioctyl succinate. Nonionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or phenols substituted with fatty alkyl or alkenyl with ethylene oxide, fatty esters of polyhydric alcohol esters, for example, fatty acid esters of sorbitan, condensation products of such esters with ethylene oxide, for example, polyoxyethylene sorbitan fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols. such as 2, 4, 7, 9-tetraethyl-5-decin-4,7-diol, or ethoxylated acetylenic glycols. Examples of an active agent on the cationic surface include, for example, an aliphatic mono-, di- or polyamine such as an acetate, mathenate or oleate; or oxygen containing amine such as polyoxyethylene alkylamine amine oxide; an amine linked amide prepared by condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt. Examples of inert materials include, but are not limited to, inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates or botanical materials such as cork, powdered corn cobs, peanut pods, rice pods and walnut shells. The pesticidal compositions comprising the lipase and / or Bt insecticidal proteins may be in a form suitable for direct application or as a concentrate of primary composition which requires dilution with a suitable amount of water or other diluent before application. The concentration of pesticide will vary depending on the nature of the particular formulation, specifically, whether it is a concentrate or is used directly. The composition contains from 1 to 98% of a liquid solid inert carrier and 0 to 50%, optimally 0.1 to 50% of a surfactant. These compositions will be administered in the proportion labeled for the commercial product, optimally about 0.01 lb. - 5.0 Ib. per acre when in dry form and approximately 0.01 pts. - 10 pts. per acre when it is in liquid form. In a further embodiment, the pesticidal compositions as well as the transformed microorganisms capable of expressing the lipase in the Bt insecticidal proteins, can be treated prior to formulation to prolong the pesticidal activity when applied to the environment of a target pest while the pretreatment is not harmful to the activity. Such treatment may be by chemical and / or physical means as long as the treatment does not adversely affect the -properties of the composition (s). Examples of chemical reagents include, but are not limited to, allogeting agents; aldehydes such as formaldehyde and glutaraldehyde; antiinfectives, such as zephyrin chloride; alcohols, such as isopropanol and ethanol; and histological fixatives, such as Bouin's fixative and Helly's fixative (see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freeman and Co.)). In other embodiments of the invention, it may be advantageous to treat Bt insecticidal proteins with a protease, for example trypsin, to activate the protein prior to the application of a pesticidal protein composition comprising this class of insecticidal proteins to the environment of the target pest. Methods for the activation of protoxin by a serine protease are well known in the art. See, for example, Cooksey (1968) Biochem. J. 6: 445-454 and Carroll and Ellar (1989) Biochem. J. 261: 99-105, the teachings of which are incorporated herein by reference. For example, a suitable activation protocol includes, but is not limited to, the combination of a polypeptide to be activated, and trypsin in a weight ratio of 1/100 Bt / trypsin protein in 20 nM NaHCO 3, pH 8 and the digestion of the sample at 36 ° C for 3 hours. Pesticidal compositions (including transformed microorganisms) can be applied to. environment of a plague. insect by, for example, spraying, atomizing, sprinkling, dispersing, coating or casting, introduction into or on the ground, introduction into irrigation water, by treating seed in general application or sprinkling in time when the plague has begun to appear or before the appearance of pests as a protective measure. For example, the pesticidal composition (s) and / or so-formed microorganism (s) can be mixed with grain to protect the grain during storage. It is generally important to obtain good pest control in the early stages of plant growth, as this is the time when the plant may be most severely damaged. The pesticidal compositions may conveniently contain another insecticide if this is thought necessary. In one embodiment of the invention, the pesticidal composition (s) is applied directly to the soil, at the time of planting, in a granular form of a carrier composition and dead cells of a Bacillus strain. or transformed microorganisms of the invention. Another embodiment is a granular form of a composition comprising an agrochemical such as, for example, a herbicide, an insecticide, a fertilizer, in an inert carrier, and killed cells such as a Bacillus strain or transformed microorganism of the invention. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL Example 1: Effect of the Combination of an Insecticide Lipase and Bt Insecticide Protein on Diabrotica Larvae Insect diets for larvae of southern corn rootworms and western corn rootworm are known in the art, see , for example, Rose and McCabe (1973 J. Econ. Entomology 66: 393, incorporated herein by reference.) The insect diet was prepared and emptied onto a tray, 1.5 ml of diet was supplied in each cell with an additional 50 μl of sample preparation. contains the insecticidal lipase and the Bt insecticidal protein of interest applied to the diet surface.Alternatively, 50 μl of PBS buffer adjusted for the concentration of ammonium sulfate was applied to the -diet of the control group. Western corn rootworm classification, 50μl 'of a 0.8 egg agar solution was applied to the layers.The trays were allowed to dry under a hood.After drying, the layers were placed on trays and stored 3-5 days at a temperature of 26 ° C. The trays were then recorded counting the "live" versus "dead" larvae and tabulating the results.The results were expressed as a percentage of mortality. of the feeding of the combination to the Diabrotica larvae are shown in Figure 1 and Table 1. Figure 1 shows the results of western corn rootworm bioassay of the pentin lipase feed as set forth in SEQ ID NO: 12 and the Bt insecticidal protein as set forth in SEQ ID NO: 18 of the developing larvae. The diet causes a dependent mortality, from the dose as a percentage of the wild-type controls. Table 1 shows -the results of the bioassay of the feeding in tabular form. The mortality records are shown in relation to the controls. The combination causes a dose-dependent increase in larval mortality.

TABLE 1 K04 (μg / cm2) 0 10 25 50 100 0 * 8 14 12 22 44 G - -H Ü 10 16 42 75 53 69? O-i '25 25 53 59 67 76 50 35 46 62 58 74 100 32 52 58 61 73 * Example 2: Construction of the Plasmid A plasmid vector comprising the sequence set forth in SEQ ID NO: 11 operably linked to a ubiquitin promoter (RB-ubi-pentPinlI), and another operably linked to an actin promoter. rice (rice-pentin-PinlI RB-Actin) and a plasmid vector comprising the sequence set forth in SEQ ID NO: 19 operably linked to a ubiquitin promoter (Ubi-1218K054B-PinII:: 35s-pat-35s -LB) were made. Example 3 Transformation and Regeneration of Transgenic Plants Immature maize embryos from greenhouse donor plants are bombarded with a plasmid comprising the sequence set forth in SEQ ID NO: 11 operably linked to a ubicuitin promoter or rice actin promoter in combination with a plasmid comprising. the sequence set forth in SEQ ID NO: 19 operably linked to a ubiquitin promoter. A selectable marker gene such as PAT (Wohleben et al. (1988) Gene 70: 25-37), which confers resistance to the herbicide Bialaphos, is used. Alternatively, the selectable marker gene is provided in a separate plasmid. The transformation is done as follows: The media recipes are shown below. Before the transformation, the ears are disinfected and sterilized on the surface in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed twice with sterile water. The immature embryos are removed and the embryo-side axis is placed downward (the side of the embryo upwards), 25 embryos per plate, in the 560Y medium for 4 hours and then aligned within the 2.5 cm target area , in the preparation of the bombing. These plasmids plus the plasmid DNA containing a PAT selectable marker are classified into 1.1 μm tungsten pellets (average diameter) using a CaCl 2 precipitation procedure as follows: 100 μl of tungsten particles prepared in water. 10 μl (1 μg) of DNA in EDTA buffer (1 μg of total DNA) 100 μl of 2.5 M CaCl2 10 μl of spermidine 0.1 Each reagent is sequentially added to the suspension of tungsten particles, while remaining in the vortex forming apparatus of the multitube. The final sonic mixture briefly and allowed to tube under constant vortex formation for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, the liquid is removed, washed with 500 ml of 100% ethanol and centrifuged for 30 seconds. Again the liquid is removed and 105% of 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten / DNA particles are briefly sonicated and 10 μl is stained on the center of each macrocarrier and allowed to dry for approximately 2 minutes before bombardment. The sample plates are bombarded at level # 4 in the particle gun # HE34-1 or # HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles / DNA. After bombardment, the embryos are stored in the medium at 560Y for 2 days, then transferred into the 560R selection medium containing 3 mg / liter of Bialafos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred into the medium at 288J to initiate regeneration of the plant. After the maturation of the somatic embryo (2-4 weeks), well-developed somatic embryos are transferred to the medium for germination and transferred to the lighted culture room.Approximately 7-10 weeks later, the developing seedlings are transferred to the 272V hormone-free medium for 7-10 days until the seedlings are well established. The plants are then transferred to inserts in seed plugs (equivalent to 2.5"pot) containing potting soil and are grown for 1 week in a growth chamber, subsequently being grown in 1-2 additional weeks to the greenhouse, then transferred to 600 classic pots (1.6 gallons) and grown to maturity Plants are monitored and recorded for insecticidal activity Bombardment medium (560) comprises 4.0 g / 1 of base salts N6 (SIGMA C-1416), 1.0 ml / 1 Eriksson Vitamin Mix (1000X SIGMA-1511), 0.5 mg / 1 thiamine HCl, 120.0 g / 1 sucrose, 1.0 mg / 1 2,4-D, and 2.88 g / 1 L- proline (brought to volume with H20 DI after adjustment to pH 5.8 with KOH), 2.0 g / 1 of Gelrite (added after bringing to volume with H20 DI), and 8.5 mg / 1 of silver nitrate (added after sterilization the medium at room temperature.) The selection medium (560R) comprises 4.0 g / 1 of base salts N6 (SIGMA C-1416), 1.0 ml / 1 Eriksson Vitamin Mix (1000X SIGMA-1511), 0.5 mg / 1 thiamine HCl, 30.0 g / 1 sucrose, and 2.0 mg / 1, 2,4-D (brought to volume with H20 DI after adjustment to pH 5.8 with KOH); 3.0 g / 1 Gelrite (was added after carrying volume with H20 D-I); and 0.85 mg / 1 silver nitrate and 3.0 mg / 1 bialaphos (ambQS added after sterilizing the medium and cooling to room temperature) The plant regeneration medium (288J) comprises 4.3 g / 1 MS salts (GIBCO 11117 -074), 5.0 ml / 1 of MS vitamins extract solution (0.100 g of nicotinic acid, 0. 02 g / 1 of thiamine HCL, 0.10 g / 1 of pyridoxine HCL and 0.40 g / 1 of glycine brought to volume with H20. Purified D-I) (Murashige and Skoog (1962) Physiol Plant 15: 473), '100 mg / 1 myo-inositol, 0.5 mg / 1 zeatin, 60 g / 1 sucrose, and 1.0 ml / 1"of 0.1 mM abscisic acid (brought to volume with H20 DI purified after adjusting to pH 5.6); 3.0 g / 1 Gelrite (added after carrying volume with H20 D-I); and 1.0 mg / 1 indoleacetic acid and 3.0 mg / 1 bialaphos (added after sterilizing the cooled medium at 60 ° C). The hormone-free medium (272V) comprises 4.3 g / 1 of MS salts (GIBCO 11117-074), 5.0 ml / 1 of vitamin MS extract solution (0.100 g / 1 of nicotinic acid, 0.02 g / 1 of thiamine HCL, 0. 10 g / 1 of pyridoxine HCL and 0.40 g / 1 of glycine brought to volume with purified H20 DI), 0.1 g / 1 of myo-inositol, and 40.0 g / 1 of sucrose (brought to volume with purified H20 DI after adjust the pH to 5.6); and 6 g / 1 of bacto-agar (added after bringing to volume with purified H20 D-I) sterilized and cooled to 60 ° C. Example 4: Ag? Obacteritan-mediated transformation for Agrobacterium-mediated transformation of. corn with a combination of an expression cassette comprising SEQ ID NO: 11 operably linked to a ubicuitin promoter or a rice actin promoter and an expression cassette comprising SEQ ID NO: 19 operably linked to a promoter of ubiquuitin, the Zhao method may be employed (U.S. Patent No. 5,981,840 and its International Patent Publication No. WO 98/32326, the contents of which are incorporated herein by reference). Briefly, the immature embryos are isolated from corn and the embryos are contacted with an Agrobacterium suspension, where the bacteria are capable of transferring the combination of a lipase expression cassette and a Bt insecticide protein expression cassette to at least one cell of the claim of at least one of the immature embryos (stage 1: infection tea). At this stage, immature embryos usually submerge in a suspension of Agrobacterium for the initiation of the inoculation. The embryos are co-cultivated for a time 'with the Agrobacterium (stage 2: stage of co-cultures) -. Usually, immature embryos are grown in solid medium after the infection stage. After this period of coculture an optional "spare" stage is contemplated. In this resting stage, the embryos are incubated in the presence of at least one known antibiotic to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). Generally, the immature embryos were cultured on a solid medium with antibiotic, but without a selection agent, for the elimination of Agrobacterium and for a resting phase for the infected cells. Right away, the. Inoculated embryos are cultured in the medium containing a selective agent and the transformed callus in growth is recovered (step 4: the selection step). Generally, immature embryos are cultured on a solid medium with a selective agent that results in the selective growth of transformed cells. The calluses are then regenerated in the plants (step 5: the regeneration stage), and, generally, the calluses grown in the selective medium are grown in the solid medium to regenerate the plants. The transformed plants are then cultured and selected according to the methods in Example 3. Example 5: Transformation of the Soybean Embryo Soybean embryos are bombarded with a combination of plasmids, one having the expression cassette comprising SEQ ID NO: 11 operably linked to a ubicuitin promoter or a rice actin promoter and the other having the expression cassette comprising SEQ ID NO: 19 operably linked to a ubicuitin promoter as follows. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from sterilized immature seeds • On the surface of the variety of 'culture of A2872, it is grown in light and dark at 26 ° C on an appropriate agar medium for six to ten weeks. The somatic embryos they produce, secondary embryos that are then agitated and placed in an adequate liquid medium. After repeated selection for stacking of somatic embryos that multiplied as embryos of early globular stage, the suspensions are maintained as described below. Soybean embryogenic suspension cultures can be maintained in 35 ml of liquid medium on a rotary shaker, 150 rpm, at 26 ° C fluorescent lights in a day / night program at 16: 8 hours. The cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue in 35 ml of liquid medium. The culture embryogenic suspension cultures can then be transformed by the particle gun bombardment method (Klein et al. (1987) Nature (London) 327: 70-73, North American patent No, 4,945, 0509. ün DuPont Biolistic PDS1000 instrument / HE (helium retro-fit) can be used for transformations A selectable marker gene that can be used to facilitate the transformation of soy from a transgene composed of the 35S promoter of the Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313: 810-812), the hygromycin phosphotransferase gene of plasmid pJR225 (from E. coli, Gritz et al. (1983) Gene-25: 179-188) and the 3 'region of the nopaline ribza gene of the T-DNA of the Ti plasmid of Agrobacterium tumefaciens The exsion cassette comprises SEQ ID NO: 11 operably linked to the ubiquitin promoter and the exsion cassette comprising SEQ ID NO: 19 operably linked to the ubiquitin promoter. they can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector bearing the marker gene. To 50 μl of the suspension of gold particles of 1 μm of 60 mg / ml is added (in order): 5 μl of DNA (1 μg / μl), 20 μl of esopermidine (0.1 M = and 50 μl of CaCl 2 ( 2.5 M) The particle aration is then stirred for three minutes, rotated in a micro-centrifuge for 10 seconds and the supernatant is removed.The DNA coated particles are then layered once in 400 μl of 70% ethanol. and resuspend in 40 μl of anhydrous ethanol.The DNA / particle suspension can be sonicated three times for one second each.Five microliters of the DNA coated gold particles are then loaded onto each acro-carrier disk.Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid is removed from the tissue with a pipette For each transformation experiment, approximately 5-10 tissue plates are bombarded normally. The membrane rupture sure is adjusted to 1 100 psi, and the chamber is evacuated to a vacuum of 28 inches of mercury. The tissue is placed approximately 3.5 inches away from the retention screens and bombarded three times. After bombardment, the tissue can be divided in half and placed back in liquid and cultivated as described above. Five to seven days after the bombardment, the liquid medium can be exchanged with fresh medium, and eleven- to twelve days after bombardment with fresh medium containing 50 mg / ml hygromycin. This selective medium can be renewed every week. Seven to eight weeks after the bombardment, the transformed tissue, green, can be observed growing from necrotic embryogenic piles. Not transformed. The isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line can be treated as an independent transformation event. These suspensions can then be subcultured and maintained as stacking of immature or regenerated embryos in whole plants by maturation and germination of individual somatic embryos. Example 6: Sunflower Meristem Tissue Transformation Sunflower meristem tissues are transformed with an expression cassette comprising SEQ ID NO: 11 openably linked to a ubicuitin promoter or a rice actin promoter and the expression cassette which comprises SEQ ID NO: 19 operably linked to the ubicuitin promoter as follows (see also, European Patent No. EP 0 486233, incorporated herein by reference, and Malone-Schneberg et al. (1994) Plant Science 103: 199-207). Mature sunflower seeds (Helianthus annuus L.) are shelled using an individual wheat head crusher. The seeds are sterilized on the surface for 30 minutes in a 20% Clorox bleaching solution with the addition of two drops of Tween 20 per 50 ml of solution. The seeds are rinsed twice with sterile distilled water. Split embryonic axis explants are prepared by a modification of procedures described by Schrammeijer et al. (Schrammeijer et al. (1990) Plant Cell Rep. 9: 55-60). The seeds are imbibed in distilled water for 60 minutes after the surface sterilization process. The cotyledons of each seed are then broken, producing a clean fracture in the plane of the embryonic axis. After the excision of the tip of the root, the explants are bisected longitudinally between the primordial leaves. The two sheets are placed, the surface is cut upwards, in the GBA medium consisting of mineral elements of Murashige and Skoog (Murashige et al. (1962) Physiol. Plant 15: 473-497), Shepard vitamin additions (Shepard (1980) in.

Emergent Techniques for the Genetic Improve ent of Crops (University of Minnesota Press, St. Paul, Minnesota), 40 mg / 1 adenine sulfate, 30 g / 1 sucrose, 0.5 mg / 1 6-benzyl aminopurine (BAP), 0.25 mg / 1 acid indole-3-acetic acid (IAA), 0.1 mg / 1 gibberellic acid (GA3), pH 5.6, and 8 g / 1 Phytagar. The explants are subjected to the bombardment of micropoyectiles before the treatment with Agrobacterium (Bidney and collaborators (1992) Plant Mol. Biol. 18: 301-313).

Thirty to forty explantas. They are placed in a circle in the center of a plate of 60 X 20 mm for this treatment.

Approximately 4.7 mg of 1.8-mm tungsten microprojectiles are resuspended in 25 ml of sterile TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are used by bombardment. Each plate is bombarded twice through a 150 mm nytex screen placed 2 cm above the samples in a PDS 1000® particle acceleration device.

The EHA105 strain of Agrobacterium tumefaciens pacified is used in all transformation experiments, a binary plasmid vector comprising the expression cassette containing the lipase expression cassette and a cassette of Bt toxin expression is introduced into the strain EHA105 Agrobacterium by the freezing route as described by Holsters et al. '(1978) Mol. Gen Genet 163: 181-187. This plasmid also comprises a selectable marker gene of kanamycin (ie, nptll). Bacteria for plant transformation experiments are grown overnight (28 ° C and 100 RPM continuous stirring) in liquid YEP medium (10 gm / 1 yeast extract, 10 gm / 1 Bactopeptone and 5 gm / 1 of NaCl, pH 7.0) with the appropriate antibiotics required for the bacterial strain and the maintenance of the binary plasmid. The suspension is used when it reaches an OD600 of approximately 0.4 to 0.8. The Agrobacterium cells are pelletized and resuspended to a final OD600 of 0.5 in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm / 1 of NH4C1 and 0.3 gm / 1 of MgSO4. Freshly bombarded shells are placed in an Agrobacterium suspension, mixed and left undisturbed for 30 minutes. The explants are then transferred to the GBA medium and co-cultivated, the surface is cut down, at 26 ° C and 18-hour days. After three days of co-cultivation, the explants are transferred to 374B (medium GBA lacking growth regulators and a reduced sucrose level of 1%) supplemented with 250 mg / 1 cefotaxime and 50 mg / 1 sulfate of kanamycin. The explants are grown for two to five weeks and are then transferred to the fresh 374B medium lacking kanamycin for one to two weeks of continuous development. The explants with existing areas of growth-different antibiotics "that have not produced adequate reactions for excision are transferred to the GBA medium containing 250 mg / 1 cefotaxime dunte a second 3-day phytohormone treatment. kanamycin, green, are analyzed for the presence of NPTII by ELISA and for the presence of transgene expression by the ELISA assay and the Bt. insecticide protein bioassay. See, for example, U.S. Patent No. 5,743,477, incorporated in the present by reference in its entirety, and Hosteller et al. (1991) 'Methods Enzymol., 197: 125-134 and Rose and McCabe (1973) J. Econ. Entomology 66: 393. NPTII positive clumps are grafted to the rhizomes. of Sunflower Particle Cultivated in vitro Pioneer® 6440. The seeds, sterilized on the surface are germinated in medium 48-0 (Murashige and Skoog salts of medium concentration, sucrose at 0.5 %, 0.3% gelrite, pH 5.6) and are cultured under the conditions described for the culture of explants. The upper portion of the seedling is removed, a 1 cm vertical slot is made in the hypocotyl, and the transformed sprout is inserted in the cut. The entire area is wrapped with parafilm to secure the shoot. Infected plants can be transferred to the soil after a week of in vitro culture. The grafts in the earth are maintained under high humidity conditions followed by a slow acclimatization to the greenhouse environment. The transformed sectors of To plants (parental generation) are matured in the greenhouse and are identified by NPTII ELISA and / or by the activity of lipase and Bt toxin from the analyzes -of leaf extracts while the transgenic seed collected from the T0 positive plants of NPTII are identified by the analysis of lipase activity and the Bt toxin analysis of small portions of the dry seed cotyledon. An alternative sunflower transformation protocol allows the recovery of transgenic progeny without the use of chemical selection pressure. The seeds are shelled and sterilized under the surface for 20 minutes in a 20% chlorine bleach solution with the addition of two to three drops of Tween 20 per 100 ml of solution, then rinsed three times with distilled water. The sterilized seeds are embedded in the dark at 26 ° C for 20 hours on filter paper moistened with water. The cotyledons and the root radical are removed, and meristem explants are cultured in 374E (GBA medium consisting of MS salts, Shepard vitamins, 40 mg / 1 adenine sulfate, 3% sucrose, 0.5 mg / 1 6-BAP, 0.25 mg / 1 of IaA, 0.1 mf / 1 of GA and 0.8% of Phytagar at pH 5.6) for 24 hours in the dark. The primary leaves are removed to expose the apical meristem, about 40 explant are placed with the apical dome facing upwards in a circle of 2 cm in the center of 374M (medium GBA with 1.2% Phytagar) and then cultivated in the medium for 24 hours in the dark. Approximately 18.8 mg of 1.8 μm tungsten particles are resuspended in ethanol in 150 μl of pure ethanol. After sonication, 8 μl of at the center of the macrocarrier surface. Each plate is bombarded twice with 650 psi with 250 psi rupture discs in the first shelf at 26 mm Hg vacuum from the helium gun. The plasmid of interest is introduced into the strain EHA105 of Agrobacterium tumefaciens by thawing as previously described. The pellets of bacteria grown overnight at 28 ° C in a liquid YEP medium (10 g / 1 yeast extract, 10 g / 1 Bactopeptone and 5 g / 1 NaCl, pH 7.0) in the presence of '50 μg / 1 kanamycin is resuspended in an inoculation medium (12.5 mM 2 mM 2- (N-morpholino) ethanesulfonic acid, MES, 1 g / 1 NH4-C1 and 0.3 g / 1 MgSO4 at pH 5.7) to treat a final concentration of 4.0 in OD600. Explants bombarded with particles that profile GBA medium (374E) and a droplet of bacteria suspension are placed directly on top of the meristem. The explants are co-cultivated in the medium for 4 days, after which the explants are transferred to medium 374C (GBA with 1% sucrose and not BAP, IAA, GA3 and supplemented with 250 μg / ml cefotaxime). The seedlings are grown in the medium for approximately two weeks under day conditions of 16 hours and incubation of 26 ° C. The explants (about 2 cm long) of two weeks of culture in the 374C medium are classified by the activity of lipase and Bt toxin using assays known in the art and disclosed herein. After the positive explants are identified, those shoots that fail to exhibit lipase activity and Bt toxins are discarded, and each positive explant is subdivided into nodal explants, a nodal explant contains at least one potential node. The nodal segments are grown in the GBA medium for three or four days to promote the formation of auxiliary shoots of each node. They are then transferred to the 374C medium and allowed to develop for an additional four weeks. Developing shoots are separated and grown for an additional four weeks in the 374C medium. Samples of . The cumulative counts of each recently recovered ratoon are again classified by appropriate protein activity assays. During this time, positive sprouts recovered from an individual node will generally be enriched in the transgenic sector detected in the initial trial before the nodal culture. The recovered suckers positive for the expression of lipase and Bt toxin are grafted to the Pioneer Hybrid 6440 in vitro cultivated sunflower seedling system. The rhizomes are prepared as follows. The seeds are shelled and sterilized on the surface for 20 minutes in a 20% chlorine bleach solution with the addition of two to three drops of Tween 20 per 100 ml of solution, and rinsed three times with distilled water. The sterilized seeds are germinated in the filter moistened with water for three days, then transferred in medium 48 (medium concentration MS salt, 0.5% sucrose, 0.3% gelrite, pH 5.0) and cultivated at 26 ° C under dark for three days, then incubate under 16-hour daytime culture conditions. The upper portion of the selected seedling is removed, a vertical groove is made in each hypocoty, and the transformed seedling is inserted in a V cut. The cut area is wrapped with parafilm. After a week of cultivation in the middle, the grafted plants are transferred to the soil. In the first two weeks, they are kept under high humidity conditions to acclimatize to an environment in the greenhouse. Throughout the specification the word "comprising" or variations such as "is understood" or "comprising" shall be understood to imply the inclusion of the element, integer or established stage, or group of elements, integers or steps, but not the exclusion of any element, whole number or stage, or group of elements, integers or stages The article "a" and "an" is used in the present to refer to more than one (ie, at least one ) of the grammatical object of the article. By way of example, "an element" means one or more elements. All publications and patent applications mentioned in the specification are inductive to the level of those skilled in the art to which this invention pertains. All publications or patent applications are hereby incorporated by reference to the same extent as if each publication or individual patent application was specifically and individually indicated to be incorporated by reference. Although the above invention has been described in some detail by way of illustration in the example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (20)

1. CLAIMS 1. A method for creating or increasing insect resistance in a plant, the method characterized in that it comprises stably introducing into the plant a combination of polynucleotides, the combination comprising: a) at least one polynucleotide comprising a sequence encoding a lipase polypeptide having insecticidal activity operably linked to a promoter that induces expression in a plant cell, and b) at least one polynucleotide comprising a sequence encoding an insecticidal protein of Bácillus thuringiensis (Bt) operably linked to a promoter which induces expression in a plant cell.
2. 2. The method according to claim 1, characterized in that at least one polynucleotide of the combination is introduced into the plant through reproduction.
3. The method according to claim 1, characterized in that at least one polynucleotide sequence of the combination is introduced into the plant through the transformation.
4. 4. The method according to claim 1, characterized in that the insect resistance is created or increased against any species of the selected orders of the group consisting of Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera and Trichoptera.
5. The method according to claim 1, characterized in that insect resistance is usually created against one or more pests selected from the group consisting of western corn rootworms, northern corn rootworm, rootworm of southern corn, Mexican corn rootworm, weevils and centipedes.
6. 6. The method of compliance with the claim 1, characterized in that the plant- is a dicotyledonous one.
7. 7. The method according to claim 1, characterized in that the plant is a monocot.
8. 8. The method according to claim 7, characterized in that the monocotyledon is corn.
9. The method according to claim 1, characterized in that at least one of the promoters is selected from the group consisting of a constitutive promoter, an inducible promoter, and a preferred tissue promoter.
10. The method according to claim 9, characterized in that the preferred tissue promoter is selected from the group consisting of a preferred root promoter, a preferred leaf promoter and a preferred seed promoter.
11. 11. The method according to claim 1, characterized in that the expression of the combination of polynucleotides synergistically increases the insect resistance of the plant.
12. 12. The method in accordance with the claim 1, characterized in that the combination of polynucleotides comprises at least one nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 OR SEQ ID NO: 13; b) a nucleotide sequence comprising at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13; c) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO: l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: ll or SEQ ID NO: 13;; wherein the sequence encodes a polypeptide having insecticidal activity; d) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14; e) a nucleotide sequence encoding a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, wherein the polypeptide has insecticidal activity; and f) the nucleotide sequence of any of the preceding paragraphs (a) through (e), wherein the codon usage is optimized for the expression of a plant. The method according to claim 1, characterized in that the combination of polynucleotides comprises at least one nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19; b) a nucleotide sequence comprising at least 80% sequence identity to the sequence set forth in SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19; c) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, wherein the sequence encodes a polypeptide having insecticidal activity; d) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20; e) a nucleotide sequence encoding a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, wherein the polypeptide has insecticidal activity; and f) the nucleotide sequence of any of the preceding paragraphs (a) to (e), wherein the codon usage is optimized for expression in a plant. The method according to claim 13, characterized in that the combination of polynucleotides comprises at least one nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 0 SEQ ID NO: 13; b) a nucleotide sequence comprising at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13; c) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO: l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13; wherein the sequence encodes a polypeptide having insecticidal activity; d) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14; e) a nucleotide sequence encoding a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, wherein the polypeptide has insecticidal activity; and f) the nucleotide sequence of any of the preceding paragraphs (a) to- (e), wherein the codon usage is optimized for expression in a plant. 15. A plant comprising a combination of polynucleotides stably integrated into its genome, the combination characterized in that it comprises: a) at least one polynucleotide comprising a sequence encoding a lipase polypeptide having insecticidal activity operably linked to a promoter ' induces expression in a plant cell; and b) at least one polynucleotide comprising a sequence encoding an insecticidal protein of Bacillus thuringiensis (Bt) operably linked to a promoter that induces expression in a plant cell. The plant according to claim 15, characterized in that the combination of polynucleotides comprises at least one nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID N0: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 OR SEQ ID NO: 13; b) a nucleotide sequence comprising at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13; c) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO: l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13; wherein the sequence encodes a polypeptide having insecticidal activity; d) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14; e) a nucleotide sequence encoding a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, wherein the polypeptide has insecticidal activity; and f) the nucleotide sequence of any of the preceding paragraphs (a) to (e), wherein the codon usage is optimized for expression in a plant. The plant according to claim 15, characterized in that the combination of polynucleotides comprises at least one nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID N0: 15, SEQ ID N0: 17, SEQ ID N0: 19; b) a nucleotide sequence comprising at least 80% sequence identity to the sequence set forth in SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19; c) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, wherein the sequence encodes a polypeptide having insecticidal activity; d) a nucleotide sequence encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20; e) a nucleotide sequence encoding a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16,. SEQ ID NO: 18, SEQ ID NO: 20, wherein the polypeptide has insecticidal activity; and f) the nucleotide sequence of any of the preceding paragraphs (a) to (e), wherein the codon usage is optimized for expression in a plant. The plant according to claim 17, characterized in that the combination of polynucleotides comprises at least one nucleotide sequence selected from the group 'consisting of: a) the nucleotide sequence set forth in SEQ ID N0: 1, SEQ ID NO : 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:, SEQ ID NO: 11 0 SEQ ID NO: 13; b) a nucleotide sequence comprising at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13; c) a nucleotide sequence comprising at least 15 contiguous nucleotides of the sequence set forth in SEQ ID NO: l, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13; wherein the sequence encodes a polypeptide having insecticidal activity; d). a sequence of nucleotides encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14; e) a nucleotide sequence encoding a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, wherein the polypeptide has insecticidal activity; Y . f) the nucleotide sequence of any of the preceding paragraphs (a) through (e), wherein codon usage is optimized for expression in a plant. 19. The plant according to claim 15, characterized in that the plant is a monocot. 20. Transformed seed of the plant of claim 15.