MXPA06008374A - Peptides for inhibiting insects - Google Patents

Abstract

The subject invention pertains to the use of peptide fragments of cadhering (including cadherin-like proteins). The subject invention includes a cell (and use thereof) comprising a polynucleotide that expresses the peptide fragment. The subject invention includes methods of feeding the peptides to insects. In preferred embodiments, the peptides are fed to target insects together with one or more insecticidal proteins, preferably (but not limited to) B.t Cry proteins. When used in this manner, the peptide fragment can not only enhance the apparent toxin activity of the Cry protein against the insect species that was the source of the receptor but also against other insect species. Preferably, the cadherin is a Bacillus thuringiensis (B.t) insecticidal crystal protein (Cry) toxin receptor. Preferably, the peptide fragment is a binding domain of the receptor. In some preferred embodiments, the peptide is the binding domain nearest to the membrane proximal extodomain. Corresponding domains are identifiable in a variety of B.t. toxin receptors.

Description

PEPTIDES TO INHIBIT INSECTS CROSS REFERENCE TO A RELATED APPLICATION This application claims the priority of the provisional application of the United States with serial number 60 / 538,715, filed on January 22, 2004.

Government rights This invention was made in part with low government support Concession No. Al 29092 granted by the National Institutes of Health. The government may have certain rights in this invention.

BACKGROUND OF THE INVENTION Various receptors on insecticidal insect protein toxins of insect cells for Bacillus thuringiensis (B.t) are known in the art. See, for example, U.S. Patent Nos. 6,586,197; 6,429,360; 6,137,033; and 5,688,691. However, it was not taught, nor was it suggested in the prior art, the administration of cadherin-like protein fragments, especially fragments of B.t. toxin receptors, to insects. Bacillus thurinciiensis as an insecticide. Bacillus thuringiensis (B.t.) is an anaerobic facultative bacterium, Gram positive, mobile, spore-forming. The B.t. It is accepted as a biopesticide source friendly to the environment. The farmers have applied the B.t. as an insecticide spray for the control of lepidopteran and coleopteran type pests for more than 30 years. The United States Environmental Protection Agency has considered that aerosols of B.t. they are safe since they have been excluded from the tolerance requirement (a maximum permissible residue limit standard in foods). There are other alternatives for the application of B.t toxin to target insects. The genes of the B.t. toxin they are inserted into microorganisms that are associated with the habitat of the target insect so that the transformed organisms will colonize and continue to produce sufficient amounts of toxin to avoid damage to insects. Examples of these are the insertion of specific genes in bacteria that colonize the surface of the leaves of plants and roots externally, such as Pseudomonas cepacia, or internally, such as Clavibacter xyli. However, the release of living recombinant microorganisms causes some concern and regulatory restrictions. Alternative methods for introducing genes into microorganisms have been developed to minimize potential horizontal gene flow to other bacterial species. These include the use of transposase-negative derivatives of the Tn5 transposon, or suicide vectors that rely on homologous recombination for Integration to complete. In addition, there has been a development of non-viable recombinant organisms that could increase the persistence of toxins in the field, such as toxin-based products of B.t. in P. fluorescens. This method eliminates the concerns associated with the evaluation of live genetically engineered microorganisms. The proteins of B.t. they can be applied in transgenic plants. Examples of such plants, called B.t. plants, protected from attack by insects, include cotton and maize. The United States Environmental Protection Agency has approved the commercial plantation of cotton and corn B.t. since 1996. The mechanism of action of toxins B.t. it proceeds through several steps including the solubilization of ingested glass, the proteolytic activation of the protoxins, the adhesion of the toxin to receptors of the midgut and the insertion of the toxin into the apical membrane to form channels or ionic pores. It is assumed that the adhesion of the toxin to the vesicles of the rough-edged membrane (BBMV) is a two-step procedure that includes reversible and irreversible steps. Multiple receptors can be involved in the process of adhesion of the toxin and the insertion to the membrane. Tabashnik and others (Tabashnik 1992) described the phenomenon of synergy for toxins B.t. Cry and they developed a formula to calculate the synergy. Cry proteins are considered synergistic if the combined insecticidal power is greater than the sum of the individual components.

CrylAa and CylAc are synergistic in bioassays against the gypsy moth larvae (Lee and Dean, 1996). Other examples of synergy of B.t. for the Cry proteins of B.t. israelensis and combinations of spores and crystals against Plutella xylostella, the diamondback moth (Liu et al., 998). The molecules that are not of B.t. They are also known to synergize toxins. For example, ethylenediaminetetraacetic acid (EDTA) synergizes B.t. against P. xylostella. The synergy described here is new both in the nature of the synergistic molecule and in the effect detected in important Lepidoptera larvae. Receptors of Toxin B.t .. The characterization of receptors of the midgut of insects and the investigation of their interaction with Cry toxins provides a method to elucidate the mode of action of the toxins and design improved Cry toxins for the control of pests. Most of the midgut proteins that bind to the Cry toxin identified to date belong to two major families of proteins: the cadherin-like proteins and the aminopeptidases. There is in vitro and in vivo evidence that supports the relationship of aminopeptidases in the toxicity of Cry1 against the larva of lepidoptera. The aminopeptidases bind to the Cry toxins in a specific way allowing them to form pores in the membranes (Masson and others, 1995, Sangadala and others, 2001, Sangadala and others, 1994). Recent studies provide evidence that aminopeptidase can function as a receptor when expressed in cultured cells (Adang and Luo 2003) and insects (Gilí and Ellar 2002, Rajagopal et al., 2002). Aminopeptidases do not always confer susceptibility to Cry toxins when they are expressed in heterologous systems (Banks et al., 2003, Simpson and Newcomb 2000). Cadherin-like proteins are a class of Cryl receptor proteins in Lepidoptera larvae. Bombyx mori, the silk moth, has a 175 kDa cadherin-like protein called BtR175 that functions as a receptor for the Cryl Na and CrylAc toxins on the epithelial cells of the midgut (Hara et al., 2003; Nagamatsu et al. 1999). M. sixth has a 210 kDa cadherin-like protein, called Bt-R1, which serves as a receptor for CrylA toxins (Bulla 2002a, b, Vadlamudi et al., 1993, Vadlamudi et al., 1995). Bt-R-i binds to the toxins Cry ka, CrylAb, and CrylAc in ligand clots (Francis and Bulla 1997). The membranes purified from COS cells expressing Bt-R-i adhered to the three CrylA toxins in adhesion assays and ligand clots (Keeton and Bulla 1997). Additionally, the expression of Bt-Ri on the surface of COS7 cells led to toxin-induced cellular toxicity as monitored by microscopy with immunofluorescence with fixed cells (Dorsch and Oiros, 2002). The cadherin-like protein Bt-R-i has been suggested to induce a conformational change in CrylAb that allows the formation of a pre-pore toxin oligomer and increases the adhesion affinity for the aminopeptidase (Bravo and Oros 2004). In Bombyx mori, the cadherin-like protein BtR175 serves as a CrylAa receptor (Nagamatsu et al., 1998). Sf9 cells expressing BtR175 swell after exposure to the CrylAa toxin, presumably due to the formation of ion channels in cell membranes (Nagamatsu et al. 1999). When expressed in mammalian COS7 cells, BtR175 induces susceptibility to CrylAa (Tsuda et al., 2003). Hua et al. (Hua et al. 2004) developed a fluorescence-based assay using Drosophila S2 cells to analyze the function of the Manduca cadherin (Bt-R a) as a Cryl toxin receptor. The Bt-R1a cDNA that differs from Bt-R-i by 37 nucleotides and two amino acids and expresses it transiently in Drosophila melanogaster, Schneider 2 (S2) cells (Hua et al. 2004). Cells express Cry Aa toxins, CrylAb, and CrylAc bound to Bt-R? A on Bt-R-? To ligand clots, and in saturation adhesion assays. More CrylAb adhered in relation to Cry Aa and CrylAc, although each CrylA toxin adhered with high affinity (Kd values of 1.7 nM to 3.3 nM). The use of fluorescent microscopy and flow cytometry assays, (Hua et al. 2004) demonstrated that CrylAa, CrylAb and CrylAc, although not Cryl Ba, annihilated the S2 cells expressing the cadherin Bt-Rα. These results demonstrated that Bt-Rα to M. sexta cadherin functions as a receptor for Cryl A toxins in vivo and validates our cytotoxicity assay for future receptor studies. The participation of an interruption of the cadherin superfamily gene in resistance to CrylAc has been described for a resistant laboratory strain Heliothis virescens (Ganan et al., 2001). The encoded protein, called HevCaLP, has the binding properties expected for the CrylA receptor (Jurat-Fuentes et al. 2004). Similarly, larvae of Pectinophora gossypiella with alleles of resistance in the genes encoding a cadherin-like protein were resistant to the CrylA toxin (Morin et al., 2003). The toxins B.t. they bind to specific regions in the cadherin-like proteins. The domain II regions of CrylA toxins are involved in binding to Bt-R-i (Gómez et al., 2002; Gómez et al., 2001). The first toxin that binds to the region identified in Bt-R-i was a segment of seven amino acid residues located in repetition seven of the cadherin (CR7) (Gómez et al 2002; Gómez et al. 2001). (Dorsch et al. 2002) identified a second adhesion region of CrylAb within the repeat of cadherin 11 (CR11) in Bt-R1. Recombinant and synthetic peptides containing both amino acid sequences inhibited the toxicity of CrylAb in vivo when fed to larvae of M. sexta (Dorsch et al 2002; Gómez et al. 2001), demonstrating their relationship in the action of toxins. Previously, two Bt-Ri toxin binding regions were proposed in CR 7 (Gómez et al. 2001) and 11 (Dorsch et al. 2002) as functional receptor sites. U.S. Patent No. 60 / 538,753 entitled "Novel Binding Domain of Cadherin-like Toxin Receptor", from Adang et al., With Proxy Case No. UGR-104P, identifies an additional adhesion site recognized by the toxins Cry that works as a receiver. This additional binding site, which is also a functional receptor region, is included in the Proximal Extracellular Domain of the CR12 Membrane (MPED) of BtR1a (Hua et al. 2004). The protein HevCaLP of H. virescens has a CrylAc binding site in a comparable position (Xie et al. 2004), suggesting a conservation of the binding sites between cadherins of different insect species. There is no known report or suggestion that the ß.í. or a fragment thereof is fed, or otherwise administered, to an insect pest, with or without ß.í. protein, for the purpose of killing it or otherwise preventing the insect from feeding on a plant. Previous competitive binding studies suggest that there would be no change in toxicity (Gómez and others 2002) or a reduction in toxicity due to competitive union (Gómez et al 2001, Dorsch et al 2002, Gómez et al 2003, Xie et al. 2004).

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the use of protein peptide fragments to control insects. In preferred embodiments, the source protein is a cadherin (including cadherin-like proteins) and / or crystal protein toxin receptors. { Cry) of Bacillus thuríngiensis. Preferably, the peptide fragment is a receptor binding domain. In some preferred embodiments, the peptide is the binding domain closest to the proximal ectodomain of the membrane. The corresponding domains can be identified in a variety of β-toxin receptors. Thus, one aspect of the invention relates to the use of an isolated polynucleotide that encodes a protein comprising (or consisting of) a fragment of a protein of cadherin type. In preferred embodiments, the peptides are fed to target insects together with one or more insecticidal proteins, preferably (but not limited to) the Cry proteins of ß.í .. When used in this way, the peptide fragment can not only potentiate the obvious activity of the Cry protein toxin against the insect species that were the origin of the receptor but also against other insect species. The present invention includes a cell (and the use thereof) that transports the polynucleotide and expresses the peptide fragment, including methods for feeding the peptide (preferably with Cry toxins of ß.i.) to the insects.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates truncated constructs of cadherin Bt-R1a expressed. on the surface of S2 cells using the vector plZT-V5-His (Invitrogen) and transfected in Drosphila S2 cells. Plasmids are designed for the encoded cadherin (CR) repeats. The numbers in parentheses indicate the amino acid residues of the start and end positions of CR. Units 7 and 11 of CR (in black) contain the Toxin Binding Regions 1 and 2, respectively. Figure 2 shows the results of toxin adhesion assays in original conditions (dot blotting and binding saturation assays). The designations are in accordance with the Bt-Rα constructions in Figure 1. Figure 2 shows the CrylAb that binds truncated and full-length Bt-R1a cadherin in S2 Drosophila cells under conditions of non-denaturing and competition by peptide CR12-MPED. A dot-blot was performed on the S2 cells (5 x 105 cells) in PVDF filters. After blocking, filters were placed with 125l-Cry1Ab or 125l-Cry1Ab plus purified CR12-MPED peptide in a 1000-fold excess (molar ratio). Figure 2 shows the binding of 125 I-Cry1Ab to the truncated fragment of expressed Cad12 containing CR12, but not to CR11 only. Figure 3 shows the amino acid sequence of cadherin Bt-R1a M. truncated sixth CR12-MPED in pET-30a (+) (Novagen). The letters in bold and underlined designate the amino acids Bt-R-? A. The truncated open reading frame of Bt-R? A is designated CR12-MPED. (264 residues in total - 206 residues of Bt-R1a (78%), 58 residues of pET-30a (+) (22%), PM = 28652 Dalton.). Figures 4A and 4B illustrate that CR12-MPED enhanced the potency of ß.í. CrylAb. Figures 4A and 4B show live and dead larvae, and illustrate the reduced size of larvae in all groups fed with combinations of Cryl Ab plus CR12-MPED. Figures 5A-5F show the effect of toxicity as body weight of Manduca sexta larvae, Heliothis virescens, Helicoverpa zea, Spodoptera frugiperda, and Plutella xylostella that survived fed with truncated cadherin peptide CR12-MPED with CrylA toxins. Figures 6A-6F show photographs of Manduca sexta larvae, Heliothis virescens, Helicoverpa zea, Spodoptera frugiperda, Plutella xylostella susceptible to ß.í., and Plutella xylostella resistant to ß.í. who survived fed a mixture of Cry toxins from ß.í.lA and truncated cadherin peptide CR12-MPED with CrylA toxins. Figure 7 shows that CR11-MPED enhances the toxicity of CrylAb for Manduca sexta (tobacco worm). Figure 8 shows the bioassay of CrylAc with cadherin fragments in the grasshopper of the soybeans (Pseudoplusia includens). Figure 9 shows the soybean grasshopper bioassay with Cry2Aa and different truncations of cadherin BtR? A. Ano-PCAP data are included. Figure 10A demonstrates that the CR12-MPED peptide was able to enhance the activity of the protoxin CrylAa, as well as truncated CrylAa digested with trypsin (Figure 10B) against P. includens. Figure 10B demonstrates that the peptide CR12-MPED was able to enhance the activity of truncated CrylAa digested with trypsin against P. includens. Figure 11 illustrates the results of the diet bioassay superimposed on a neonate of the grasshopper of the soybean (Pseudoplusia includens) a the mixture of CR12-MPED and 5 ng / cm2 of Cryl Ac with different ratios of toxin: peptide.

Figure 12 illustrates the results of the diet bioassay superimposed on the mortality of the neonate hatchlings of the cabbages (Trichoplusia ni) to the mixture of CR12-MPED and 8 ng / cm2 of CrylAc with different ratios of peptides: toxins.

Brief Description of the Sequences SEQ ID NO: 1 is a nucleotide sequence encoding the CR12-MPED peptide. SEQ ID NO: 2 is the amino acid sequence of the CR12-MPED peptide. This peptide can be referred to as "ß.í. Booster" or "BTB." SEQ ID NO: 3 shows the nucleotide sequence of truncated CR11-MPED of cadherin Bt-R1a from M. sexta, CR11-MPED can be mentioned as BtB2, which has 324 amino acid residues of Bacillus thuringiensis-R1a encoding a protein of approximately 35,447 Daltons (theoretical pl = 4.72). SEQ ID NO: 4 shows the amino acid sequence of truncated CR11-MPED of cadherin Bt-Ri from M. sexta. This peptide is as produced by the strain BL21 / DE3 / pRIL of E. co / 7 cloned with the vector pET-30a. SEQ ID NO: 5 shows the nucleotide sequence of CR1-3 of BtRia from M. sexta. SEQ ID NO: 6 shows the amino acid sequence of CR1-3 of BtRla from M. sexta. This peptide is as produced by the E. coli strain BL21 / DE3 / pRIL cloned with the pET-30a vector.

SEQ ID NO: 7 (Anof-PCAPseq.doc file) shows the nucleotide sequence of the putative cell adhesion protein of Anopheles gambiae (NCB1 LOCUS XM_321513). SEQ ID NO: 8 (Anof-PCAPseq.doc file) shows the amino acid sequence of the putative cell adhesion protein of Anopheles gambiae (NCBI LOCUS XMJ321513). SEQ ID NO: 9 shows the nucleotide sequence encoding "PCAP" - the truncation of the putative cell adhesion protein of Anopheles gambiae (PCAP). SEQ ID NO: 10 shows the truncated PCAP region (putative cell adhesion protein) of the Anopheles gatnbiae protein. This truncated peptide is referred to herein as PCAP or Ano-PCAP (213 amino acid residues - a 24416.56 Dalton protein, theoretical pl = 4.96). This peptide is how it is produced from the DNA cloned in the vector pET-30a and expressed in the strain of £. co / 7 BL21 / DE3 / pRIL. SEQ ID NO: 11 (Ano-Cad-Sequence (GH) doc) shows the cadherin sequence with full-length cDNA of Anopheles gambiae. The search in BLAST with the sequence corresponds to the DNA and predicted the protein sequence for a cDNA of partial Anopheles gambiae (NCBI Locus XM_312086). SEQ ID NO: 12 shows a partial nucleotide sequence of cadherin from Anopheles gambiae. SEQ ID NO: 13 shows the expressed peptide sequence ("Ano-CAD") of the fragment cloned in the pET-30a vector and expressed in the E. coli strain BL21 / DE3 / pRIL.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods and materials used to control insects and other pests, in particular pests in plants. More specifically, the present invention relates to the use of peptide fragments of a protein, preferably cadherin (including cadherin-type proteins), to control insects. Alternatively or in addition, the protein is preferably a toxin receptor of the crystal protein (Cry) of Bacillus thuringiensis (B.t.). These peptide fragments are provided, or made available, for target pests for ingestion. This can be achieved through a variety of means that are known in the art, some of which are explained in more detail below. Fragments of cadherin protein ectodomains (the portion of the protein that is left outside the cell when part of the cadherin protein is embedded in the cell membrane and part is exposed to the surface of the cell) are preferred. Preferably, the cadherins can be toxin receptors of the crystal protein (Cry) of Bacillus thuringiensis (B.t.). Preferably, the peptide fragment is a receptor binding domain. In some preferred embodiments, the peptide is the binding domain closest to the ectodomain proximal to the membrane. The corresponding domains are identifiable in a variety of B.t. toxin receptors. In preferred embodiments, the peptides are fed target insects together with one or more insecticidal proteins, preferably (but without limitation) the Cry proteins of ß.í .. When used in this way, the peptide fragment can, not only potentiate the activity Obvious to the Cry protein toxin against the insect species that was the origin of the receptor but also against other insect species. A related aspect of the invention relates to the use of an isolated polynucleotide that encodes a protein comprising (or consisting of) a fragment of a cadherin-like protein. The present invention includes a cell (and the use thereof) that transports the polynucleotide and expresses the peptide fragment, including methods for feeding the peptide (preferably with Cry toxins of ß.i.) to the insects. The nucleotide sequences can be used to transform hosts, such as plants, to express the fragments of the receptor (preferably cadherin fragments) of the present invention. The transformation of plants with the genetic constructions disclosed herein can be achieved using techniques known to those skilled in the art. Thus, in some embodiments, the present invention provides nucleotide sequences encoding the receptor fragments, preferably a cadherin-like Bt-R-i protein. The receptor used as the origin of this (these) domain (s) can be derived from several pests and insects, such as Manduca sexta larvae, Heliothis virescens, Helicoverpa zea Spodoptera frugiperda and Plutella xylostella. Many sequences of said receptors are available to the public. The peptide fragments of the invention can not only enhance the activity of a toxin against the insect species that was the origin of the receptor, but also against other insect species. Several pests can be sought, including but not limited to the larvae of Manduca sexta, Heliothis virescens, Helicoverpa zea Spodoptera frugiperda and Pluiella xylostella. Due to the unique and novel method of the present invention, pests can be sought which are not normally susceptible to Cry proteins of ß.í. For example, hemiptera represent a larger group of insects that normally have not been effectively controlled by the <; 5-endotoxins of ß.í .. Numerous species of pests of hemiptera, most notably the species Lygus, cause considerable damage to plants and economic losses every year. The digestive system of hemiptera (including aphids) is unusual among insects in several ways: certain hydrolytic digestive enzymes are absent, such as trypsin; the midgut lacks a peritrophic membrane, and there is no culture. These characteristics reflect the way of feeding to liquid diet and by suction, subject to evolutionary restrictions. Due to differences in diet, feeding mode, and digestive physiology and biochemistry, proteins that have insecticidal activity against leaf-chewing insects would not necessarily be expected to have activity against the saphat-feeding Hemiptera. However, "due to the new method of the invention, the present invention offers new alternatives for pest control." The present invention can be used to enhance and expand the spectrum (or range of insects) of toxicity of a toxic protein to insects. In some preferred embodiments, these peptide fragments can be used to enhance the potency of ß.í toxins to control insects.In some preferred embodiments, peptide fragments enhance the toxicity of Cryl toxins, but as shown herein The present invention is not limited to the use of said toxins Various types of plants and crops can be protected in a variety of ways when practicing the present invention Cotton and corn are the main crops that can be protected by the use of peptides (and proteins) of the present invention, as well as soybeans and rice. to protect these crops include producing transgenic crops that are designed to produce peptides (and proteins) according to the present invention. Preferred uses for spray applications include, but are not limited to, protecting vegetables and looking for forest pests (protecting planted trees and the like). Preferred pests to search in this manner include, without limitation, lepidoptera. Without the intention of limiting by a theory or specific theories of mechanisms of action, one possibility is that these fragments work in conjunction with the toxins of ß.í. and intensify the pesticide activity of the toxin. When the insects are fed with a Cry toxin, the peptide can change the effect of a toxin from a growth inhibitory effect to an insecticidal effect. In addition or alternatively, the fragments may exert, at least, a partial toxic effect by a separate mechanism of action. Even another possibility is that the fragments also, or alternatively, work indirectly to stabilize the βß toxin. Thus, said fragment can function independently of the Cry toxin (through another mechanism of action) and / or in conjunction with the Cry toxin to intensify the insecticidal potency of the Cry toxin. However, the mechanism (s) of action are not important to practice the present invention. Based on the disclosure of this text, one skilled in the art can practice various aspects of the present invention in a variety of ways. For example, the cadherin-like protein fragment can be expressed as a fusion protein with a Cry toxin of β.i. using techniques known to those skilled in the art. As described herein, preferred fusions would be chimeric toxins produced by combining a toxin (including a fragment of a protoxin, for example) and a fragment of a cadherin-like protein. In addition, mixtures and / or combinations of toxins and protein fragments of the cadherin type can be used in accordance with the present invention. These mixtures or chimeric proteins have the unexpected and remarkable properties of the intensified insecticidal potency. In the same way it would be observed in a similar manner as one skilled in the art., having the benefit of the present disclosure, will recognize that the peptides of the invention will potentially have a variety of functions, uses and activities. As indicated herein, the peptides of the invention can be administered together with a Cry protein. When used in this manner, the peptides of the present invention can effect a faster killing of the target insects, and / or may allow less Cry protein to be required to kill the insects. However, full lethality is not required. The ultimate goal is to prevent insects from damaging the plants. In this way, the prevention to feed oneself is sufficient. In this way "inhibiting" insects is all that is required. This can be achieved by making the insects "get sick" or by otherwise inhibiting them (including killing) from damaging the protected plants. The peptides of the present invention can be used alone or in combination with another toxin to achieve this inhibitory effect, which can be mentioned as "toxin activity". In this way, the inhibitory function of the peptides of the invention can be achieved by any mechanism of action, directly or indirectly related to the Cry protein, or completely independent of the Cry protein. In preferred embodiments, the present invention relates to the use of a repeat of the 12-MPED cadherin peptide of Manduca sexta Bt-R-ia cadherin-type protein to enhance the potency of β.i. toxins.

A region (i.e., fragment) of a cadherin-like protein was identified that synergizes the insecticidal potency of a Cry toxin of ß.í. The receptor fragment binds to the toxin with high affinity, catalyzes cell death induced by toxins when expressed on the surface of cells of cultured insects, and potentiates (ie, synergizes) the insecticidal potency of a Cry toxin. However, in view of the present disclosure, it will be recognized that other peptides can be used in similar ways. For example, a new CrylAb binding site on Bt-R1a was identified as described in U.S. Patent No. of Series 60 / 538,753 entitled "Novel Binding Domain of Cadherin-like Toxin Receptor," by Adang and others, which carries Proxy Case No. UGR-104P, which identifies an additional binding site recognized by Cry toxins that functions as receiver. This additional binding site, which is also a functional receptor region, is included in the CR12 Membrane Proximal Extracellular Domain (MPED) of Bt-R1a (Hua et al. 2004). The HevCaLP protein of H. virescens has a CrylAc binding site in a compatible position (Xie et al. 2004), which suggests a conservation of the binding sites between the cadherins of different insect species. The full-length and truncated fragments of Bt-Rα were genetically engineered and expressed in Drosophila S2 cells to evaluate binding to CrylAb and receptor-mediated cytotoxicity. See, for example, Figure 1. In toxin adhesion assays under denaturing conditions (ligand staining), 1251-Cry1Ab bound full-length Bt-R-a, and truncated fragments of Cad7-12, Cad10 - 12, and Cad11-12. Binding assays in original conditions (dot blotting and binding saturation assays) revealed that the binding of 1251-Cry1Ab to the truncated fragment of expressed Cad12 contained CR12, but not to CR11 alone (See, eg, Figure 2) . In saturation binding assays, the 1251-Cry1Ab toxin bound with high affinity similar to Bt-R-? A, Cad7-12, Cad11-12, and full length Cad12, although the concentration of the receptors was higher for Cad 11-12. The fluorescence assisted classification assays (FAGS) showed that S2 cells expressing Bt-R1a, Cad7-12, Cad10-12, Cad11-12 or Cad12 were susceptible to CrylAb. The S2 cells expressing Cad7 or Cad11 were not annihilated by the toxin. Thus, a new receptor binding region in Bt-Rα is described here which is located at CR12. The binding to CR12 is necessary and sufficient to confer susceptibility to the CrylAb toxin for the cells of the insects. The present invention focuses in part on the unexpected finding that a peptide comprising CR12 and the membrane proximal ectodomain (MPED) (Dorsch et al. 2002) enhanced the toxicity of the Cr 1 toxins of β. when it was fed as a mixture to insect larvae. This peptide, called CR12MPED, is illustrated in Figure 3. The peptide not only functions as a Cry toxin enhancing agent against M. sexta, the original source of Bt-R-? A receptor, but also functions as an enhancing agent for Multiple toxins Cryl toxins against other Lepidoptera pests including H. virescens, H. zea and S. frugiperda. The use of a fragment of a B.t. in this way it has not been described or suggested previously. In preferred embodiments, the receptor fragment is a receptor binding domain. Without intending to link to any specific theory regarding the mechanism of action, the binding of this domain to a toxin of ß.í. could induce a conformational change in the ß.í. toxin, thereby making it more toxic, more able to bind to the toxin receptor, etc. In some preferred embodiments, the fragment comprises (or is formed of) the CR12-MPED domain. Peptides (such as CR12-MPED) and toxins may be fed or otherwise administered to the target pest (insect) in various ways, according to the present invention. In a preferred embodiment, a transgenic plant produces the peptide (such as CR12-MPED) and one or more ß.i. toxins. By consuming the peptide and the protein of ß.í. produced by said plant (for example, by eating the tissues of the plant and the cells containing the peptide and the protein), the insect will thereby contact the peptide and the protein. Together, they will exhibit enhanced toxic effects in the insect's intestine. Another preferred method of the present invention is to atomize the peptide (such as Cad12-MPED) onto transgenic plants of β.í. (such as corn, cotton, soybeans, and the like). The peptide may be in an appropriate vehicle, as is known in the art. By atomizing the peptide in this manner on the plant tissues consumed by the white pests, the pest will eat both the peptide (in the aerosol) and the protein ß.i. (produced by and present in the plant). Even another preferred method is to atomize both the peptide and the Cry protein of ß.í. about plants and the like. Such methods are known in the art (but until now lacked the synergizing peptides of the present invention). The ß.i toxins, and / or the peptide of the present invention, can be formulated with a vehicle acceptable for agricultural purposes, for example, which is suitable for spray application to plants and the like. In one embodiment, the present invention relates to the use of a polynucleotide encoding a CR12 binding domain of M. sexta. In a preferred embodiment, said polynucleotides comprise (or are formed of) a nucleotide sequence encoding the CR12-MPED peptide of SEQ ID NO: 2. (The "G" N-terminal - glycine residue - for example, can be deleted and the remaining fragment of the exemplified sequence can be used, according to the present invention.) Said nucleotide sequence is shown in SEQ ID NO: 1 In another embodiment, the present invention is directed to the use of a cell or cells transfected with a polynucleotide molecule comprising a nucleotide sequence encoding a CR12-MPED peptide, for example. In addition, the protein is preferably, but not necessarily, anchored to and localized to the cell membrane, and is capable of binding to a toxin. In a more preferred embodiment, said protein mediates an observable toxicity to said cell or cells, including death upon contact with a toxin. While CR12-MPED is an example mentioned above and elsewhere in this text, several other peptides are exemplified herein. Some other such peptides are described below in the Examples. Thus, it should be understood that these other peptides and their variants can be mentioned in the same way as CR12-MPED is. As described in the foundation section of the invention, many ß.i. toxins have been isolated and sequenced. Polynucleotides that encode any known toxin of ß.í or those that have not yet been discovered and their active fragments (see, for example, U.S. Patent No. 5,710,020) may be used in accordance with the teachings of this text. See Crickmore et al. (1998) for a description of other ß.i. toxins. A listing of Cry toxins from the Crickmore et al website is attached as Appendix A. These include, but are not limited to, polynucleotides that encode CrylA toxins such as Cryl Aa, Cryl Ab, Cryl Ac, preferably, as well as Cryl B, Cryl. C, Cryl F, D3 / 1 E, and Cry3A. Cry2 toxins are also preferred for concomitant administration with the peptides of the present invention. The toxin (s) can be selected from the Crickmore list, for example, based on the type of pests sought. For example, the rootworms were searched for in an example as explained below. Thus, anti-rootworm toxins (such as Cry34 / 35 toxins) can be used preferentially in such applications. The peptides of the invention can be used, in addition, to control mutant insects that are resistant to one or more toxins of B.t. In addition, Cry toxins (such as those described in U.S. Patent Nos. 6,825,006) can be modified.; 6,423,828; 5,914,318; and 5,942,664), in accordance with the present invention. In addition, they are contemplated here as well, to use ß.í toxins that are not Cry toxins (such as the "Vip" toxins as categorized in another section of the Críckmore website and others). Insecticidal proteins from organisms other than ß.r., such as Bacillus subtilis, are also contemplated for use. To provide an understanding of the number of terms used in the specification and the claims of this invention, the following definitions are provided. An "isolated" nucleic acid or polynucleotide (or protein) is a state or construct that can not be found in nature. In this way, it means the participation of "the hand of man". A polynucleotide encoding a peptide of the present invention, within the scope that the peptide does not exist in the state of nature, could be an isolated polynucleotide. This polynucleotide in a plant genome could also be "isolated" since it does not exist in its natural state. The term therefore covers, for example, (a) a DNA having the sequence of part of a naturally occurring genomic DNA molecule although it is not surrounded by the coding or non-coding sequences that border that part of the molecule in the genome of the organism further characterized because it occurs naturally; (b) a nucleic acid incorporated into a vector or genomic DNA of a prokaryotic or eukaryotic such that the resulting molecule is not identical to any naturally occurring genomic vector or DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by reaction of the polymerase chain (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. A nucleotide sequence is operatively linked when placed in a functional relationship with another nucleotide sequence. For example, a promoter is operably linked to a coding sequence if the promoter effects its transcription or expression. In a general sense, operatively linked means that the related sequences are contiguous and, when it is necessary to join two coding regions of proteins, contiguous and in reading frame. However, it is known that certain genomic elements, such as enhancers, can be operatively linked even remotely, that is, even if they are not contiguous. The polynucleotides of the present invention include an isolated polynucleotide "formed in essence by" a segment encoding a CR12MPED peptide, for example, attached to a marker or reporter molecule and can be used to identify and isolate ß.i toxins and the like. Probes comprising synthetic oligonucleotides or other polynucleotides can be derived from natural or recombinant nucleic acids of single or double chain structure or synthesized by chemical means. The polynucleotide probes can be labeled by any of the methods known in the art, for example, random labeling with hexamer, translation of the nick or the Klenow filling reaction. The polynucleotides can also be produced by chemical synthesis, for example, by the phosphoramidite method described by Beaucage and Caruthers (1981) Tetra. Letts., 22: 1859-1862 or the triester method according to Matteuci et al. (1981) J. Am. Chem. Soc, 103: 3185, and can be performed on commercial automatic oligonucleotide synthesizers. A double chain structure fragment of the single chain chemical synthesis product can be obtained by either synthesizing the complementary chain structure and tempering the chain structure together with appropriate conditions or by adding chain structure complementary using DNA polymerase with an appropriate starter sequence. DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system (i.e., vector) recognized by the host, including the desired DNA fragment encoding the desired polypeptide, and preferably further, will include transcriptional and translational initiation regulatory sequences operatively linked to the segment encoding a polypeptide. Expression systems (expression vectors) may include, for example, an origin of replication or an autonomous replication sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary information sites, such as sites of ribosome binding, RNA splice sites, polyadenylation sites, transcription terminator sequences and mRNA stabilizing sequences. In addition, signal peptides may be included where appropriate from the secreted peptides of the same or a related species, which allow the protein to cross and / or be included in the cell membranes or secreted from the cell. The cloning and expression vectors will similarly contain a selectable marker, i.e., a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. Although said marker gene can be transported over another polynucleotide sequence co-introduced into the host cell, it is more often included on said cloning vector. Only those host cells in which the marker gene has been introduced will survive and / or grow under selective conditions. Normally the selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, for example, ampicillin, neomycin, methotrexate, etc.; (b) complementary autotrophic deficiencies; or (c) supply of critical nutrients not available from the complex medium. The choice of the appropriate selectable marker will depend on the host cell; Appropriate markers for different hosts are known in the art.

Those skilled in the art will recognize that DNA sequences may vary due to the degeneracy of the genetic code and the use of codons. All DNA sequences encoding exemplified and / or suggested peptides (and proteins) are included. For example, the CR12 peptides thereof are included in this invention, including the DNA of SEQ ID NO: 1 (plus an ATG prior to the coding region), which encodes SEQ ID NO: 2. Additionally, those skilled in the art will recognize that allelic variations may occur in the DNA sequences that will not significantly change the activity of the amino acid sequences of the peptides encoding the DNA sequences. All said equivalent DNA sequences are included within the scope of this invention and the definition of the regulated promoter region. The skilled artisan will understand that the exemplified sequences (such as the sequence CR12MPED of SEQ ID NO: 1) can be used to identify and isolate non-exemplified nucleotide sequences encoding functional equivalents of the sequences given in, or an amino acid sequence. of more than 90% identity with it and that has an equivalent biological activity. DNA sequences that have at least 90%, or at least 95% identity with a DNA sequence mentioned and that encode functional peptides (such as CR12-MPED) are considered equivalent sequences and are included in the present invention. Next, other numerical scales are provided for variant polynucleotides and amino acid sequences (eg, 50-99%). Following the teachings of this invention and using the knowledge and techniques known in the art, the skilled worker will be able to perform the extensive number of operational modes having the DNA sequences equivalent to those mentioned herein without the expense of undue experimentation. As used herein, the percent sequence identity of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990) Proc. Nati Acad. Sci. USA 87: 2264-2268, modified as in Karlin and Altschul (1993) Proc. Nati Acad. Sci. USA 90: 5873-5877. Said algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol .. 215: 402-410. Searches of nucleotides by BLAST are performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences with the desired percentage of sequence identity. To obtain alignments with gaps for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucí. Acids Res. 25: 3389-3402. When the BLAST and Gapped BLAST programs are used, the default parameters of the respective programs (NBLAST and XBLAST) are used. See the website ncbi.nih.gov. The polynucleotides (and the peptides and proteins they encode) can also be defined by their hybridization characteristics (their ability to hybridize to a given probe, such as the complement of a DNA sequence exemplified herein). Several degrees of stringency hybridization can be employed. The more stringent the conditions, the greater the complementarity that is required for the formation of duplexes. The stringency can be controlled by temperature, probe concentration, probe length, phonic resistance, time, and the like. Preferably, hybridization is conducted under conditions of moderate to high stringency by techniques known in the art, as described, for example, in Keller, GH, MM Manak (1987) DNA Probes, Stockton Press, New York, NY, pp. . 169-170. As used herein "moderate to high stringency" conditions for hybridization refers to conditions that achieve the same, or nearly the same, degree of hybridization specificity as conditions "such as those described herein." Examples of moderate to high stringency conditions are provided here. Specifically, the hybridization of DNA immobilized in Southern blots with gene-specific probes labeled with 32P was transformed using standard methods (Maniatis and oiros.) In general, hybridization and subsequent washes were performed under conditions of moderate to high stringency that allowed the detection of target sequences with homology to the sequences exemplified here. of double-stranded structure, hybridization was carried out overnight at 20-25 ° C below the melting temperature (Tf) of the DNA hybrid in 6XSSPE, dx Denhardt's solution, 0.1% SDS, 0.1 mg / ml of denatured DNA The melting temperature is described by the following formula by Beltz et al. (1983). Tf = 81.5 ° C + 16.6 Log [Na +) + 0.41 (% G + C) -0.61 (% formamida) 600 / length of the duplex in base pairs. The washes are usually carried out in the following manner: (1) Twice at room temperature for 15 minutes in 1XSSPE, 0.1% SDS (low stringency wash). (2) Once at Tf-20 ° C for 15 minutes in 0.2xSSPE, 0.1% SDS (moderate stringency wash). For the nucleotide probes, the hybridization was carried out overnight at 10-20 ° C below the melting temperature (Tf) of the hybrid in dxSSPE, 5x Denhardt's solution, 0.1% SDS, 0.1 mg / ml from Denatured DNA The Tf for the nucleotide probes was determined through the following formula of Suggs et al. (1981): Tf (° C) = 2 (number T / A base pairs) +4 (number G / C base pairs) Normally the washings were carried out in the following manner: (1) Twice at room temperature for 15 minutes IxSSPE, 0.1% SDS (low stringency wash). (2) Once at the annealing temperature for 15 minutes in IxSSPE, 0.1% SDS (moderate stringency wash) In general, the salt and / or temperature can be altered to change the stringency. With a labeled DNA fragment of more than about 70 or more bases in length, the following may be used: Low: 1 or 2xSSPE, room temperature Low: 1 or 2xSSPE, 42 ° C.

Moderate: 0.2X or IxSSPE, 65 ° C. Height: 0.1 x SSPE, 65 ° C. Duplex formation and stability depend on substantial complementarity between the two chain structures of a hybrid and, as noted above, a certain degree of non-equivalence can be tolerated. Therefore, the polynucleotide sequences of the present invention include mutations (both single and multiple), deletions, and insertions in the described sequences, and combinations thereof, in which said mutations, insertions, and deletions allow the formation of stable hybrids with a polynucleotide of interest sought. Mutations, insertions, and deletions can occur in a determined polynucleotide sequence using standard methods known in the art. In the future other methods may become known. The mutation, insertion, and deletion variants of the polynucleotide and the amino acid sequences of the invention can be used in the same way as the sequences exemplified as long as the variants are substantially similar to the original sequence. As used herein, "substantial similarity" of the sequence refers to the extension of nucleotide similarity that is sufficient to allow the variant polynucleotide to function in the same capacity as the original sequence. Preferably, this similarity is greater than 50%; more preferably, this similarity is greater than 75%; and even more preferably, this similarity is greater than 90%. The degree of similarity necessary for the variant to work in its intended capacity will depend on the intended use of the sequence. The mutation, insertion, and deletion mutation modality that is designed to improve the function of the sequence or otherwise provide a methodological advantage is known to a person skilled in the art. The identity and / or similarity can also be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% compared to the sequence exemplified here. The similarity / identity and / or homology of the amino acid will be the lowest in the critical regions of the protein that explain the biological activity and / or are involved in determining the three-dimensional configuration that is ultimately responsible for the biological activity. Regarding this point, certain amino acid substitutions are acceptable and can be expected if these substitutions are found in regions that are not critical for activity or are conservative amino acid substitutions that do not affect the three-dimensional configuration of the molecule. For example, amino acids can be placed in the following classes: non-polar, polar without charge, alkaline and acid. Conservative substitutions by which an amino acid of one kind is replaced with another amino acid of the same type fall within the scope of the present invention so long as the substitution does not materially alter the biological activity of the compound. Table 1 provides a listing of examples of amino acids that belong to this class.

TABLE 1 Amino Acid Examples Examples of Amino Acids Non-polar Ala, Val, Leu, Lie, Pro, Met, Phe, Polar without charge Trp Gly, Ser, Thr, Cys, Tyr, Asn, Gin Asp Acid, Glu Basic Lys, Arg, His In some cases, substitutions can also be made conservative The critical factor is that these substitutions should not move away significantly of the biological activity of the toxin. The practice of some embodiments of the present invention could require the use of expression vectors comprising one or more polynucleotides comprising a nucleic acid sequence exemplified, and are capable of expressing the peptides of the invention, in an appropriate host cell. In the vectors of the present invention, the polynucleotide encoding the peptide is operably linked to transcription and / or translation elements to effect expression of the peptide in an appropriate host cell. Regulatory elements can be derived from genes of mammals, microbes, viruses or insects and include, example, promoters, enhancers, initiation sequences of transcription and translation, termination sequences, origins of replication, and leader and transport sequences. Appropriate regulatory elements are selected from optimal expression in a desired host cell. Possible regulatory sequences may include, but are not limited to, any promoter that has already been shown to be constitutive of expression, such as those of viral origin (eg, the Baculovirus promoter) or so-called "housekeeping" genes (ubiquitin, actin, tubulin ) with its corresponding termination sequences / poly A +. In addition, the gene can be placed under the regulation of inducible promoters and their termination sequences such that gene expression is induced by light (rbcS-3A, cab-1), heat (promoters of hsp genes) or wounds (mannopin, HGPGs). Other suitable promoters include the metallothionein promoter, dexamethasone promoter, alcohol dehydrogenase promoter, and the baculovirus promoters, i.e., the initial promoter (eg, IE-1 and et1), the late promoters (eg, vp39). and p6.9), the very late promoters (e.g., polh and p10) and the hybrid promoter (e.g., vp39 / po1 h). It is clear to a person skilled in the art that a promoter can be used in both original and truncated form, and can be equated with its own sequence or heterologous termination sequence / polyA +. In a preferred embodiment, the vectors of the invention are regulated by the promoter D. melanogasfer HSP70. Expression vectors can be constructed by molecular biology methods known as described for example in Sambrook et al. (1989), or any of a number of laboratory manuals on recombinant DNA technology that are widely available. Expression vectors in which the polynucleotides of the present invention can be cloned under the control of an appropriate promoter are also available commercially. Recombinant viral vectors, including retroviral, baculoviral, parvoviral and densoviral vectors may be used although they are not particularly preferred. In host cells containing vectors having an inducible promoter that controls the expression of the nucleic acid encoding CR12-MPED, for example, expression is induced by methods known in the art and is appropriate for the selected promoter. For example, expression of the nucleic acids under the control of the metallothionein promoter is induced by the addition of cadmium chloride or copper sulfate to the host cell culture medium. In a specific embodiment, the present invention includes the use for pest control of a host cell containing a vector comprising nucleotide sequences encoding CR12-MPED under the control of a promoter. The host cell can be prokaryotic or eukaryotic, including bacterial, yeast, insect and mammalian cells. Insect and mammalian cells are preferred. Preferred host cells in particular include cell lines, such as, for example, Spodoptera frugiperda (Sf9 and Sf21) and Trichoplusia ni (Tn cells), Stigma acrae (Ea4 cells), Drosophila melanogaster (Dm cells), Choristoneura fumiferama (Cf cells). -1), Mamestra brassicae (MaBr-3 cells), Bombyx morí (MnN-4 cells), Helicoverpa zea (Hzlb3 cells), and Lymantria dispar (Ld652Y cells), among others. The host cells can be transformed, transfected or infected with the expression vectors of the present invention through methods known to those of ordinary skill in the art. Transfection can be accomplished by known methods, such as liposome-mediated transfection, calcium phosphate-mediated transfection, microinjection and electroporation. The transgenic cells of the present invention can be obtained by transfection with a polynucleotide comprising an exemplified (or suggested) nucleic acid sequence. Equipped with the teachings of this text, those with experience would be able to transfect cells with the future polynucleotides encoding exemplified isolated peptides, to produce cells that produce the peptides of the present invention. Progeny cells that maintain the polypeptide encoding a peptide are, of course, within the scope of the present invention, as are the transgenic plants. The term "transfection" as used herein refers to an introduction of a foreign DNA or RNA into a cell by mechanical inoculation, electroporation, agroinfection, particle bombardment, microinjection or other known methods. The term "transformation" as used herein refers to a stable incorporation of an RNA or foreign DNA into the cell that produces a permanent, inheritable alteration in the cell. Consequently, the expert will understand that the transfection of a cell can produce the transformation of that cell. In preferred embodiments, expression of the peptide and / or the gene encoding the toxin directly or indirectly results in the intracellular production (and maintenance) of the peptide / protein. When the transgenic / recombinant / transformed / transfected cells (or their contents) are ingested by the pests, the pests will ingest the toxic peptides / proteins. This is a preferred way in which contact of the pest with the toxin is caused. The result is the control of the plague. Suctioning pests can also be controlled in a similar way. Alternatively, said microbial hosts, for example Pseudomonas such as P. fluorescens, may be applied where the pests are present.; the microbes can proliferate there, and be ingested through the objective pests. When the gene (s) are introduced through a suitable vector into the microbial host, and said host is applied to the environment in a living state, certain host microbes should be used. The microorganism hosts that are selected are known to occupy the "phytosphere" (phytoplank, phytosphere, rhizosphere, and / or risoplane) of one or more crops of interest. These microorganisms are selected as they are successfully able to compete in a particular environment (culture and other insect habitats) with the wild type microorganisms, provided for the maintenance and stable expression of the polypeptide pesticide gene expression, and, desirably provide a improved pesticide protection from degradation and environmental inactivation. It is known that a large number of microorganisms inhabit the phylloplane (the surface of the leaves of plants) and / or the rhizospheres (the earth that surrounds the roots of the plant) of a wide variety of important crops. These microorganisms include bacteria, algae and fungi. Of particular interest are microorganisms, such as, for example, the genus Pseudomonas. Erwinina, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobiüm, Rhodopseudomonas, Metlrylphilíus, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi particularly yeast, for example, the genus Sccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are the bacterial species of phytosphere such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia acescens, Acetobacter xylium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorulla rubra, R. glutinis, R. marina, R. aurantiaca, Cruptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. prestoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae and Aureobasidium pollulans. Pigmented microorganisms are also of interest. One aspect of the present invention is the transformation / transfection of plants, plant cells, and other host cells with polynucleotides of the present invention expressing proteins of the present invention. Plants transformed in this way can become resistant to attack through the target pest (s). A wide variety of methods are available for introducing a gene encoding a pesticidal protein in a target host under conditions that allow stable maintenance and expression of the gene.

These methods are well known to those skilled in the art and are described, for example, in U.S. Patent No. 5,135,867. For example, a large number of cloning vectors are available that comprise a replication system in E. coli and a marker that allows the selection of transformed cells for preparation for the insertion of foreign genes into higher plants. The vectors include, for example, pBR322, the pUC series, the M13mp series, pACYC184, etc. Accordingly, the sequence encoding the toxin can be inserted into the vector at a suitable restriction site. The resulting plasmids are used for transformation into E. coli. E. coli cells are grown in an appropriate client environment, after they are harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biological biochemical-molecular methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence can be divided and ligated with the following DNA sequence. Each sequence of plasmids can be cloned into the same or other plasmids. Depending on the method of inserting the desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, the at least one right edge, usually the right border and the left border of T-DNA of the Ti or Ri plasmid, is they have to unite the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively investigated and described in EP 120 156; Hoekerna (1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B.V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4: 1-46; and An et al. (1985) EMBO J. 4: 277-287. A large number of techniques are available for inserting DNA into a plant host cell. These techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes, people transforming, melting, injecting, biolistic (bombardment of micro particles), or electroporation as well as other possible methods. If Abrobacteria is used for transformation, the DNA to be inserted has to be cloned into special plasmids, mainly either in an intermediate vector or in a binary vector. The intermediary vectors they can be integrated into the Ti or Ri plasmid through homologous recombination due to sequences that are homologous to the sequence in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary to transfer ai T-DNA. Intermediary vectors can not replicate themselves in Agrobacterium. The intermediate vector can be transferred into Agrobacteria tumefaciens by means of an auxiliary plasmid (conjugation). Binary vectors can replicate themselves in E coli and in Agrobacteria. These may comprise a selection marker gene and a linker or polylinker which are constructed through the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978]) Mol. Gen. Genet. 163: 181-187). The Agrobacterium used as a host cell is to comprise a plasmid carrying a vir region. The vir region is necessary to transfer the T-DNA into the cell of the plant. Additional T-DNA may be contained. The battery thus transformed is used for the transformation of plant cells. Plant explants can advantageously be cultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of DNA into the plant cell. The whole plants can then be regenerated from the infected plant material (for example, pieces of leaves, segments of stem, roots, but also protoplasts or cells grown in suspension) in a medium or suitable, which may contain antibiotics or biocides for selection. The plants thus obtained can then be tested for the presence of inserted DNA. No special demands of plasmids have been made in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives. Transformed cells grow inside plants in the usual way. These can form germ cells and transmit the trait (s) to the progeny plants. Said plants can grow in a normal way and cross with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties. In some preferred embodiments of the invention, the genes encoding the bacterial toxin are expressed from transcription units inserted into the genome of the plant. Preferably, said transcriptional units are recombinant vectors capable of stable integration into the genome of the plant and enable the selection of transformed plant lines expressing mRNA encoding the proteins. Once the inserted DNA has been integrated into the genome, it is relatively stable there (and does not come out again). It normally contains a selection marker which confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually used marker should therefore allow the selection of transformed cells to place the cells that do not contain the inserted DNA. The gene (s) of interest preferably are expressed either through constitutive or induced promoters in the plant cell. Once expressed, the mRNA is translated into proteins, therefore incorporating amino acids of interest within the protein. The genes encoding a toxin expressed in the plant cells may be under the control of a constitutive promoter, a tissue-specific promoter, or an inducible promoter. There are several techniques for introducing foreign recombinant vectors into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include the introduction of coated genetic material onto micro particles directly into the cells (U.S. Patent Nos. 4,945,050 to Cornell and 5,141, 131 to DowEIanco, now Dow AgroSciences, LLC). In addition, until they can be transformed using Agrobacterium technology, see United States Patent No. 5,177,010 for the University of Toledo; 5,104,310 from Texas A &M; European Patent Application 0131624B1; European Patent Application 120516, 159418B1 and 176, 112 of Schilperoot; U.S. Patent Nos. 5,196,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot; European Patent Applications 116718, 290799, 320500 all by Max Planck; European Patent Applications 604662 and 627752, and Patent of E.U.A. No. 5,591, 616, of Japan Tobacco; European Patent Applications 0267159 and 0292435, and Patent of E.U.A. No. 5,231, 019, all of Ciba Geigy, now Novartis; Patents of E.U.A. Nos. 5,463,174 and 4,762,785, both from Calgene; and the Patents of E.U.A. Nos. 5,004,863 and 5,159 135, both from Agracetus. Another transformation technology includes beard technology. See Patents of E.U.A. Nos. 5,302,523 and 5,464,765, both from Zeneca. Electroporation technology has also been used to transform plants. See WO 87/066614 of Boyce Thompson Institute; the Patents of E.U.A. Nos. 5,472,869 and 5,384,253, both from Dekalb; and WO 92/09696 and WO 93/21335, both from Plant Genetic Systems. In addition, viral vectors can also be used to produce transgenic plants that express the protein of interest. For example, monocotyledonous plants can be transformed with a viral vector using the methods described in U.S. Patents. Nos. 5,569,597 of Mycogen Plant Science and Ciba-Giegy, now Novartis, as well as the Patents of E.U.A. Nos. 5,589,367 and 5,316,931 both from Biosource. As previously mentioned, the form in which the DNA construct is introduced into the plant host is not critical to this invention. Any method that provides efficient transformation can be used. For example, various methods for plant cell transformation are described herein and include the use of Ti or Ri plasmids and the like to carry out the Agrobacterium-mediated transformation. In many cases, it will be desirable to have the construction used for the transformation bordered on one or both of them through the T-DNA edges, more specifically the right edge. This is particularly useful when the construction uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as a mode for transformation, although the T-DNA borders may find use with other transformation modes. When Agrobacterium is used for the transformation of the plant cell, a vector can be used which can be introduced into the host for homologous recombination with T-DNA or the Ti or Ri plasmid present in the host. The introduction into the vector can be carried out through electroporation, parental pairing and other techniques for transforming gram-negative bacteria, which are known to those skilled in the art. The form of vector transformation within the Agrobacterium host is not critical to this invention. The Ti or Ri plasmid containing the T-DNA for recombination may be capable or incapable of causing gall formation and is not critical to said invention as long as the vir genes are present in said host. In some cases where Agrobacterium is used for transformation, the construction of the expression is within the T-DNA borders it is inserted into a broad spectrum vector such as pRK2 or derivatives thereof as described in Ditta et al., ( PNAS USA (1980) 77: 7347-7351 and EPO 0 120 515, which is incorporated herein by reference.Where or more markers are included within the expression construct and the T-DNA as described herein which allows selection of the Transformed Agrobacterium and Transformed Plant Cells The particular marker used is not essential for this invention, with the preferred marker depending on the host and the construct used.For the transformation of plant cells using, the explants can be combined to incubate with the Agrobacterium for a sufficient time to allow the transformation of the same., the Agrobacterium is annihilated through selection with the appropriate antibiotic and the plant cells are cultured with the appropriate selective medium. Once the callus is formed, shoot formation can be stimulated through the use of appropriate plant hormones according to methods well known in the art of plant tissue culture and plant regeneration. However, an intermediate stage of callus is not always necessary. These from the formation of the shoots, said cell plants can be transferred to a medium that stimulates root formation thereby completing the regeneration of the plant. Plants after being able to grow to plant them, and such planting can be used to establish future generations. With respect to the transformation technique, the gene encoding the bacterial toxin is preferably incorporated within the gene transfer vector adapted to express said gene in a plant cell through the inclusion in the vector of a regulatory element of the promoter of plant, as well as 3 'untranslated transcription termination regions such as Nos and the like. In addition to the numerous technologies for the transformation of plants, the type of tissue to be contacted with foreign genes can also vary. Such tissue could include but not be limited to embryogenic tissue, types I, II and III of callus tissue, hypocotyl, meristem, root tissue, tissues for expression in phloem, and the like. Almost all plant tissues can be transformed during differentiation using appropriate techniques described herein. A variety of selectable markers can be used, if desired. The preference of a particular marker is at the discretion of the technician, but any of the following selectable markers can be used along with any other gene not listed here that could function as a selectable marker. In addition to selectable marker, it can be appreciated to use a reporter gene. In some cases a reporter gene can be used with or without a selectable marker. The reporter genes are genes that are typically not present in the recipient organism or tissue and typically encode proteins as a result of a phenotypic change or enzymatic property. An assay for detecting reporter gene expression can then be carried out in a suitable time after said gene has been introduced into the recipient cells. Accordingly, the skilled artisan will note that the preferred polynucleotides for practicing the present invention encode proteins (or peptides) capable of expression in cells, localization in the cell membrane and binding to the toxin. Accordingly, fragments of exemplified sequences, as well as functional mutants, can be used in the same way in the practice of the present invention. Said fragments and mutants may be easily obtained by following the teachings of this text coupled with the prior art. For example, by using the polynucleotides specifically exemplified as probes, useful polynucleotides can be obtained under conditions of appropriate stringency. Standard hybridization conditions include hybridization with nonspecific DNA, such as salmon DNA, at 50 ° C and washing at 45 ° C. To obtain polynucleotides having the lowest detectable homology with the exemplified CR12-MPED (for example), hybridization is conducted under conditions of low standard stringency (30-37 ° C and 4-6xSSC). The polynucleotides most closely related to CR12-MPED (for example) can be obtained under standard conditions of moderate stringency (40-50 ° C in 1 x SSC). Having thus described in detail the preferred embodiments of the present invention, it should be understood that the various embodiments described are merely illustrative of the present invention and that many obvious variations thereof are possible without departing from the spirit or scope of the invention. invention. In consecuense, one skilled in the art will amply recognize that the present invention is not limited to the embodiments described herein. The description provided herein in the following examples refers to the preferred method using the available strategy of the published protocols to construct DNA vectors and to apply the target tissue of the present invention successfully towards the goal of achieving the transformation of corn plastics. cereal. Any molecular cloning and necessary recombinant DNA technique would be performed by standard methods (Sambrook et al., 1995). All patents, patent applications, provisional applications and publications mentioned or cited herein are incorporated by reference in their entirety to the extent that they are not inconsistent with the explicit teachings of this specification. Below are examples illustrating procedures for practicing the invention. These examples should not be constructed as a limit. All percentages are by weight, and all proportions of solvent mixtures are by volume unless otherwise specified.

EXAMPLE 1 Summary of the Antagonistic Binding and Blocking of Toxicity Assays Previously, it was shown that the toxin-binding regions on Bt-R1 acted as antagonists for CrylAb by blocking toxicity (Dorsch et al, 2002, Gomez et al. 2001, Gomez et al. 2003). The CR12-MPED region encoded by the Bt-R1 region including cad 12 and the MPED was evaluated using similar experiments. The CR12-MPED region (SEQ ID NO: 2, encoded by SEQ ID NO: 1) was over-expressed in E. coli and purified. Peptide was mixed with an LC50 dosage of CrylAb and fed to M. sexta larvae. CrylAb toxin was obtained by activation of the trypsin of the protoxin (Accession Number AAA22330). It was expected that CR12-MPED would block toxicity when mixed with CrylAb toxin and fed to the larvae. The results of the bioassay were quite surprising. The peptide CR12-MPED did not suppress the toxicity of CrylAb but it increased in a very surprising way the mortality of Manduca larvae that received CrylAb as food. Higher concentrations of CR12-MPED mixed with a constant amount of CrylAb given as food to the larvae killed more larvae. The peptide CR12-MPED potentiated the toxicity of CrylAb. This initial result was formed after further experimentation. CR12-MPED increases the potency of a Cry toxin that is already highly active against a susceptible insect.

EXAMPLE 2 Test Bioassay 1 with M. Sexta "group 1" - 9ng / cm2 of CrylAb (toxin ratio: peptide 1: 0), "group 2" - 9ng / cm2 of CrylAb plus 9ng / cm2 of CR12-MPED (ratio 1: 1), "group 3" - 9ng / cm2 of CrylAb plus 90ng / cm2 of CR12-MPED (ratio 1: 10), "group 4" - 9ng / cm2 of Cry Ab plus 450ng / cm2 of CR12- MPED (ratio 1: 50), "group 5" - 9ng / cm2 of Cy1Ab plus 900ng / cm2 of CR12-MPED (ratio 1: 100), "group 6" - 9ng / cm2 of CrylAb plus 4500ng / cm2 of CR12-MPED (ratio 1: 500) "group 7" - water only as control. Each group had 16 replications. After 7 days, many larvae in groups 4 and 5 were dead. This was attributed to the possibility that the Tris-HCl pH regulator, such as the CR12MPED peptide, was in 10mM Tris-HCl (pH 8.0). Alternatively, the CR12-MPED peptide could have enhanced the toxicity of CrylAb. To determine if the high toxicity of the Cry1Ab / CR12-MPED mixture was reproducible, the bioassay experiment was repeated with additional controls.

EXAMPLE 3 Test bioassay 2 with m. Sixth A 1: 500 ratio group (ie, group 6 above) was not included because the 1: 50 and 1: 100 ratios gave an enhanced effect. Four additional controls were included: 10mM Tris-HCl (pH 8.0), 9ng / cm2 of CR12-MPED, 9Ong / cm2 of CR12-MPED, and 900ng / cm2 of CR12-MPED. In two days, almost all the larvae in the highest concentration of CR12-MPED / CrylAb were dead, but the larvae that received the toxin were not dead. The treatment groups with CR12-MPED / C 1Ab showed the same trend as that obtained in the first study. Table 2 shows the percentage of mortality for the treatments in both studies performed with M. sexta. CR12-MPED increased the power of ß.í. CrylAb in both studies. Figures 4A and 4B show live and dead larvae from Study 2. Note the reduced size of larvae in all groups that received food with combinations of CrylAb plus CR12-MPED.

TABLE 2 Results of the bioassay for CrylAb with CR12-MPED for M. sexta larvae. 9ng / cm2 9ng1Ab / cm2 + 9ng1Ab / cm2 + 9ngl Ab / cm2 + 9nglAb / cm2 + 9ngAb / cm2 1Ab 9ngCR12- 90ngCR12- 450ngCR12- 900ng CR-12 + 4500ng PED MPED MPED CR12- + MPED Study of 31.3% 18.8% 56.3% 62.3% 62.5% 100% mortality 1 Study of 31.3% 45.5% 100% 100% mortality 2 mortality 1 Study of 16-7% 0% 0% 0% mortality 2 EXAMPLE 4 Additional Bioassays Preliminary data suggested that CR12-MPED synergizes the CrylAb toxicity of ß.í. for H. virescens. This is an important point since H. virescens is the largest cotton white of S.L. The ability of the CR12-MPED peptide to function synergistically with other combinations of insect pests and toxins can now be evaluated in view of the present disclosure.

EXAMPLE 5 Synergistic effect of CR12-MPED peptide on larval mortality Heliothis virescens, Helicoverpa zea, Spodoptera frugiperda, and Plutella xylostella that received CR12-MPED peptide food plus Cry A toxins Eggs were incubated and maintained on an artificial diet in which toxin or / and CR12-MPED peptide were added or not added. Bacillus thuringiensis toxins (CrylAa, lAb and lAc) were used in LC50 dosing according to the Toxin Specificity database for Bacillus thuringiensis (see web page at glfc.forestry.ca/bacillus). The three toxins as used were obtained by trypsin activation of the protoxins (CrylAa: DH37 (Accession Number AAA22353), CrylAb: NRD12 (Accession Number AAA22330), and CrylAc DH73 (Access AAA22331)). The concentration of each toxin is listed in the following tables. Neonates of H. virescens and H. zea were transferred to receptacles in a microassay tray containing the diet with or without toxin and / or CR12-MPED peptide. Seven days later, the mortality and the body weight of the larvae were measured. Mortality and body weight were recorded after seven days of feeding with toxins or / and CR12-MPED peptide. Each group has seventeen larvae per treatment. The CR12-MPED peptide concentration was in several mass ratios relative to CrylA toxin as shown in Tables 3-7 and in Figures 5A-5E. Figures 6A-6F are photographs showing the survival of larvae of Manduca sexta, Heliothis virescens, Helicoverpa zea, Spodoptera frugiperda, Susceptible to B.t. Plutella xylostella, and resistant to ß.í. Plutella xylostella fed a mixture of Cry toxins from ß.í.lA and truncated cadherin peptide CR12-MPED.

TABLE 3 Sixth Manduca CR12- CR12- CR12- CR12- MPED (0 *) MPED (1) MPED (10) MPED (100) H20 0% 0% 0% 0% Cry1Aa (5.2nglcm2) 6.25% 6.25% 43.75% 100% Cryl Ab (9ng / cm2) 31.3% 45.5% 100% 100% CrylAc (5.3ng / cm2) 37.5% 43.75% 93.75% 100% * The values in parentheses designate the mass ratio of CR12-MPED: Cry protein TABLE 4 Heliothis virescens CR12- CR12- CR12- CR12- MPED (0 *) MPED (1) MPED (10) MPED (100) H20 0% 0% 0% 0% Cry1 Aa (5.2nglcm2) 6.25% 37.5% 50% 56.25% Cryl Ab (0.16ng / cm2) 0% 12.5% 50% 75% CrylAc (4ng / cm2) 0% 56.25% 75% 100% The values in parentheses designate the mass ratio of CR12-MPED: Cry protein TABLE 5 Helicoverpa zea CR12- CR12- CR12- CR12- MPED (0 *) MPED (1) MPED (10) MPED (100) H20 0% 0% Cry1Aa (2.07uglcm2) 68.75% 70.83% Cr / 1 Ab (1.6ug / cm2) 0% 33.33% CryiAc (0.12ug / cm2) 50% 62.5% 100% 100% * The values in parentheses designate the mass ratio of CR12-MPED: Cry protein TABLE 6 Spodoptera fruc / iperda CR12- CR12- CR12- CR12- MPED (0 *) MPED (1) PED (10) MPED (100) H20 6.25% 12.5% 0% Cry1Aa (50 nglcm2) 18.75% 0% 18.75% 6.25% Cryl Ab (50 ng / cm2) 0% 25% 37.5% 50% CrylAc (50 ng / cm2) 6.25% 6.25% 50% 62.5% * The values in parentheses designate the mass ratio of CR12-MPED: Cry protein TABLE 7 Plutella xylostella (not resistant) CR12- MPED (0 *) CR12- MPED (100 *) Mortality Degree of Mortality Degree of pupa formation * The values in parentheses designate the mass ratio of CR12-MPED: Cry protein EXAMPLE 6 Theories Regarding the mechanism (s) of action Without intending to be limited to any specific theory regarding mechanisms of action, possible explanations are provided below for the "synergistic" or enhancing effects that the peptides of the present invention possess on the insecticidal activity of the ß.i proteins. The peptide it can bind to the protein (such as a Cry protein) causing a change in the conformation of the toxin, thereby allowing the dissociation produced by the midgut proteins and facilitating the subsequent binding and insertion events to the membrane. A protein / peptide complex could increase binding to receptor molecules such as aminopeptidase or other proteins associated with malt that are classified in the cell membrane. There is evidence to support this hypothesis additionally, since cadherin increases the affinity of CrylAb for the aminopeptidase of M. Sexta (Bravo et al. 2004). With the help of a peptide of the present invention, the toxin could be pooled or collected on the surface of BBMV and form pores on the surface of the cell. The peptides can function as an adapter or bridge to connect the toxin to the cell membrane. Alternatively or additionally, the peptide can function independently of the Cry toxin, for example. The peptides could have a partial or complete toxic effect elsewhere, separately, or the peptides could function indirectly to intensify the Cry toxin. For example, the peptide could contribute to the stability of the Cry toxin in the insect's intestine. The exact mechanism (s) of action, however, are relatively unimportant, since one skilled in the art can now make and use a wide variety of embodiments of the present invention as explained herein.

EXAMPLE 7 Additional Studies 7. A Expression and purification of the CR12-MPED peptide in E. coli. Two primers with restriction sites of Neo I and Xho I were designed according to the sequence of BtR1aEC12-MPED. CR12-MPED encoding Bt-R1a (136211 e-Pro1567) was cloned into pET-30a (+) vector (Novagen). The vector pET-30a (+) / CR12MPED was transferred in E. coli strain BL21 / pRIL. The white protein fused with 6x His-tag in both N- and C- terms was over-expressed by induction with 1mM IPTG when the culture reached OD600 0.5-0.6. The culture was harvested 4 hours after induction. The purification of CR12-MPED was in accordance with "Protocol 7" in The QIAexpressionist (2nd edition, summer 1992, QIAGEN) with minor modifications. The resulting peptide was dialyzed against 10mM Tris-HCl (pH 8.0) and confirmed by 15% SDS-PAGE and western blot with anti-BtRi serum (1: 5000). The peptide EC12-MPED was used in binding competition assays. The PVDF membrane dotted with S2 cells expressing truncated cadherin was incubated with EC12-MPED peptide and 1251-Cry1Ab toxin in a mass ratio of 500: 1, respectively. In addition, CR12-MPED was evaluated with Cryl toxins in insect bioassays (described below). 7B. Insect tests. The LC50 for CrylAb against neonatal larvae of M. sexta is from 5 to 10 ng / cm2 (see the web page: glfc.cfs.nrcan.gc.ca/bacillus 10/1/03). In a bioassay we set this LC50 value for CrylAb and we selected 9 ng / cm2 of CrylAb to evaluate the effect of CR12-MPED peptide. Toxin preparations were diluted with demineralized water, mixed with varying concentrations of CR12-MPED and then 50 ml was applied to the surface of the insect diet (Southland Products, Lake Village, Arkansas). Eggs were obtained from M. sexta Carolina Biologicals. Mortality was determined after 7 days. Eggs were obtained from H. virescens and Helicoverpa zea from Benzon Research and bioassays were conducted as for M. sexta. 7C. Results We expressed full-length and truncated peptides of BtR1a in S2 cells to investigate their relationship in toxicity and binding of CrylAb. All truncated cadherin constructs are contained in the leader signal peptide as well as the transmembrane and cytoplasmic domain for expression on the cell membrane.

The truncated cadherin fragment designations included the number of ectodomain cadherin (CR) repeats they contain and the region included. For example, Cad7-12 encodes CR7 and the rest of Bt-R1a codes for the carboxy terminus. The transfected S2 cells expressed full-length and truncated cadherin fragments, which were recognized to be against Bt-Ri in immunoblots. As reported subsequently (Hua et al. 2003), the full-length Bt-R-i cadherin expressed in S2 cells had a slightly smaller molecular size than the Bt-R-i of M. sexta BBMV. In contrast, the truncated Bt-R-i fragments of Cad7 and Cad7-12 expressed in S2 cells had a molecular size slightly higher than predicted. Clots of protein ligands from transfected S2 cells were probed with 1251-labeled CrylAb toxin. 1251-Cry1Ab toxin bound to truncated Bt-R13 containing Cad7-12, 10-12, and 11-12. The expressed truncated proteins that did not contain both CR11 and 12 (ie Cad7, Cad 11, Cad12 and Cad-MPED) did not bind 125I-Cry1Ab in the spots. These results were in agreement with the previous data of truncated fragments of Bt-Ri blots (Dorsch and others 2002), which showed that CrylAb binds to a region that includes both CR11 and 12. To verify if the space between the region of binding to the toxin and cell membrane was important for binding to the toxin, CR11 was commuted with CR12 and cloned into pIZT vector. Both spot spots and ligand spots showed that Cad 12/11 did not bind to the CrylAb toxin.

It has been reported that ligand blotting, which includes denaturation conditions, produces Cry toxin binding results that are sometimes inconsistent with the toxin adhesion assays performed in original conditions (Daniel et al., 2002).; Lee and Dean 1996). To investigate the possibility of alteration by spotting with ligand binding epitopes that are functional in original conditions, we perform dot spotting. S2 cells expressing truncated cadherin fragments were dotted on PVDF filters and binding to the 125I-Cry1Ab toxin was evaluated. Consistent with the ligand spotting experiments, the proteins containing the Bt-Ri CR11 and 12 ectodomains (full-length cadherin, Cad7-12, Cad10-12, Cad11-12) specifically bound 125l-Cry1Ab . The peptide expressed from Cad12 also bound to the toxins, which came as a surprise since it did not bind to the toxin in the spots. Cad7 and Cad11 did not join CrylAb. Although Cad12-11 contained both CR11 and CR12 domains, it did not bind to the labeled toxin CrylAb after interchanging with each other. These results suggest that the arrangement between CR11, CR12, and MPED is important for binding to toxins. It is interesting to clarify that the expressed Cad12 peptide, which contained only CR12 and MPED ectodomain, bound CrylAb specifically. This result was not observed in ligand staining and it is evidence that the original conditions are necessary for the binding of CrylAb to the CR12 ectodomain, and that the CR12 ectodomain is sufficient for the binding of the CrylAb toxin. MPED may also be important in maintaining the secondary structure of CR12 (a.k.a. EC12) or possibly collaborating with CR12 in the binding of the toxin. These results identify the CR12 ectodomain as a critical CrylAb binding epitope on Bt-R-ia. Interestingly, when counting the radioactivity of the individual spots, the truncated Cad11-12 peptide containing both CR11 and 12 ectodomain bound to more CrylAb toxin than any other expressed truncated peptide, including full length cadherin (the data is not shown). To quantify the binding of CrylAb to expressed Bt-R-? A fragments, saturation assays of CrylAb binding with cell suspensions were performed as previously reported (Hua et al. 2004). According to the spot spotting results, cells expressing full-length cadherin, Cad7-12, Cad11-12 and Cad12 were bound to CrylAb. The binding of the toxin was specific and saturable in all cases, and the cells expressing cad11-12 bound to more toxins than any other cell sample. Although all the Bt-Rα fragments that bind to CrylAb showed the same binding affinity (Table 8), the concentration of binding sites was higher for Cad11-12 and Cad12 than for Cad7-12 or cadherin full length Additionally, Cad11-12 had an approximately 3-fold higher concentration of binding sites than Cad 12. These results indicate that there may be conformational limitations of full-length Bt-R1 that prevent the maximum binding of CrylAb, and that both CR11 and CR12 contain CrylAb binding epitopes.

TABLE 8 Dissociation constants (Kcnmiy concentration of the receptors (Bmax) calculated from saturation assays of 125l-Cry Ab toxin binding Fragment Cad BT-Ri K8m (nM) ± error ßma? (Fmoles / mg of protein) ± error 7-12 2.05 ± 0.15 505.65 ± 22.09 Complete CAD 3.55 + 1.25 625.36 + 14.76 12 2.87 ± 0.84 1407.09 ± 44.73 11-12 3.52 ± 0.99 3319.54 ± 626.94 It was previously reported that Bt-R1a was a functional receptor for the CrylA toxin and induced cell death when expressed in S2 cells (Hua et al. 2004). To investigate the role of ectodomains CR11 and 12 in toxicity with CrylAb, flow cytometry was used to quantitatively measure the percentage of cytotoxic response induced by CrylAb in S2 cells expressing different truncated fragments. Co-expression with GFP provided a method to monitor the efficiency of transfection, and propidium iodide (PI) was used to detect dead cells. Cytotoxicity was quantified using a formula previously reported (Hua et al. 2004) which refers to both transfection and cytotoxicity and cytotoxicity with background cell death in control cells (mock transfected). CrylAb was cytotoxic for cells expressing Cad7-12, Cad10-12, Cad11-12, Cad12 and full-length Bt-Rαα cadherin. On the other hand, CrylAb was not toxic for S2 cells expressing Cad7, Cad11, Cad12-11 and Cad-MPED.

There were no significant differences between the CrylAb toxicities in S2 cells expressing Cad7-12, Cad10-12, Cad11-12, Cad12, and complete cadherin. These results (summarized in Table 9) are evidence that the CR 12 ectodomain is the functional epitope of the receptor for CrylAb in Bt- TABLE 9 Compilation To confirm the importance of the Cad12-MPED region in the binding of the toxin, Cad12-MPED peptide was used as a competitor in dot-blot assays against 125ICry1Ab. Cad12-MPED was expressed in E. coli and purified using immobilized metal affinity chromatography. The Cad12-MPED peptide competed with the binding of the CrylAb toxin to the full-length cadherin, and the truncated cadherins 7-12, 10-12, 11-12 and 12. This result was further evidence that the CR12 domain is necessary and sufficient for the union of toxins. CR12 contains the fundamental CrylAb binding site on cadherin Bt-R1a.

EXAMPLE 8 Summary of the CR12-MPED Peptide Results that Enhances the Toxicity of Various Proteins C / ylA Against Several Lepidoptera BtR-? A was donated in the insect cell expression vector plZT-V5-His (Invitrogen). A fragment of BtR1a extending from the repetition of cadherin (CR) 12 through the extracellular domain proximal to the membrane (MPED) in pET30a was cloned and expressed in Escherichia coli. The 27 kDa expressed peptide called CR12-MPED was partially purified from inclusion antibodies. Surprisingly, larval food with CR12MPED peptide with Cryl toxin increased the toxicity of CrylA toxins to insect larvae. The CR12-MPED peptide was evaluated in combination with CrylA toxins against Lepidoptera larvae representing a range of susceptibilities to the CrylA toxin. The following insects were evaluated: Manduca sexta (tobacco worm), Heliothis virescens (tobacco worm), Helicoverpa zea (cotton worm, corn worm), Spodoptera frugiperda (army worm), and Pseudoplusia includens (grasshopper of the soy). The toxins Cryl ka, Cry kb and CrylAc were evaluated with CR12-MPED using diet-surface treatments, in the first stage of the larvae and a 7-day bioassay period.

EXAMPLE 9 Peptide CR12-MPED Intensifies the Toxicity of Cryl Ab and CrylAc Against Manduca Sexta proteins In bioassays against M. sexta, CR12-MPED increased mortality from 1.0 ± 1.0% for treatments with 2 ng Cry1Ab / cm2 to 26.0 ± 5.5% mortality at a mass ratio of 1: 100 for Cry1Ab: CR12-MPED. As the concentration of the toxin was increased to 4 ng / cm2 of CR12-MPED the mortality increased from 4.2 ± 1.1% to 82.3 ± 6.8% (P <; 0.01). CR12-MPED was inactive only in all bioassays. CR12-MPED also intensified the potency of CrylAc against the larvae of M. sexta. For example, while 2 ng of Cryl Ac / cm2 killed 13.5 ± 6.5% of the larvae, the Cry1Ac: CR12-MPED ratios of 1: 10 and 1: 100 mortality increased the mortality to 63.5 ± 17.8% (P < 0.05) and 93.8 ± 3.1% (P <0.005), respectively.

EXAMPLE 10 Peptide CR12-MPED Intensifies the Toxicity of Cryl Ac Against Heliothis virescens CR12-MPED intensifies the toxicity of CrylAc for H. virescens (tobacco worm). Neonatal larvae were fed with CrylAc with or without CR12-MPED. At a Cryl Ac concentration of 3 ng / cm2 the mortality of the diet was 8.4 ± 2.1% (toxin only); with the inclusion of 300 ng / cm2 of CR12-MPED mortality increased to 83.4 ± 6.3% (P <0.01). At a CrylAc concentration of 6 ng / cm2, the CR12-MPED peptide greatly enhanced the toxicity of CrylAc for H. virescens larvae from 46.7 ± 9.9% (toxin only) to 88.5 ± 5.5% (either in a ratio of 1: 10 or 1: 100 of CR12-MPED) (P <0.05).

EXAMPLE 11 Peptide CR12-MPED Intensifies the Toxicity of CrylAc Proteins Against Helicoverpa zea H. zea (cotton moth worm, corn worm, tomato fruit worm) has a wide range of hosts, which attack many vegetables, fruits and cotton. Transgenic cultures of ß.í. they are not as effective in controlling H. zea as other pests are. This is because CrylA toxins are less effective against H. zea than other white pests. H. zea is not as sensitive to CrylAc as H. virescens or M. sexta. Therefore, this pest was selected to determine whether the CR12-MPED peptide could increase the activity of β. In this experiment, 50 ng / cm2, 60 ng / cm2 and 120 ng / cm2 of CrylAc were used to evaluate CR12-MPED. The CrylAc toxin did not kill the larvae efficiently. At dosages of 50 ng or 60 ng / cm2, only 5.2 ± 2.8% of larvae were killed, compared to 0% (for single toxin). However, the addition of a 1: 10 ratio of CR12-MPED increased mortality to 56.3 ± 8.3% (PO.05) and 42.7 ± 3.8% (PO.01). If the toxin dosage was increased to 120 ng / cm2, it killed 24.0 ± 2.8% of H. zea larvae while an equal proportion of added CR12-MPED increased the mortality to 47.9 ± 5.5% (P <0.05). A ten-fold higher amount of peptide increased the mortality to 85.4 ± 2.8 (P <0.001).

EXAMPLE 12 The CR12-MPED Peptide Intensifies the Toxicity of Cryl b, Cry Ac, and Cry? C Proteins Against Spodoptera frugiperda Spodoptera frugiperda is not susceptible to CrylA toxins (LC50> dosing of 2000 ng / cm2; see, for example, the website "glfc.forestry.ca/bacillus".) However, when CrylAb or CrylAc were combined with CR12-MPED at ratios of 1: 1 and 1:10 (toxin: CR12-MPED) mortality increased, and larvae fed combinations of CR12-MPED and CrylAb or CrylAc were severely attacked in the culture. Most susceptible to Cryl Ca (LC50 1144 (813-3227) ng / cm2) compared to CrylA toxins The Cryl Ca protoxin (GENBANK Accession Number CAA30396), evaluated at 150 and 300 ng / cm2, killed 6% and 19%. % of larvae The aggregate of CR12-MPED increased mortality to 31% and 41% This is an important observation since there is no published report of interaction of CrylCa with the M. sexta cadherin.

EXAMPLE 13 Summary of CR12-MPED Union Studies and Conclusions regarding Toxin Intensification In general, CR12-MPED enhances or potentiates the toxicity of CrylA and CrylC toxins against susceptible and tolerant insects; in vitro, CR12-MPED binds to the toxin that forms large protein gene families. These groups of proteins still bind specifically to the striatum of the midgut.

EXAMPLE 14 The CR11-MPED peptide enhances the toxicity of CrylAb against Manduca sexta The CR11-MPED region (SEQ ID NO: 4; SEQ ID NO: 3 is the DNA) of BtRia was cloned into pET30 and expressed in E. coli. The CR11-MPED region is formed by CR11 in front of CR12-MPED. The peptide was solubilized from inclusion bodies and evaluated in bioassays with purified CrylAb toxin. The CR11-MPED peptide fed CrylAb toxin to M. sexta larvae was more toxic to the larvae than the toxin alone (Figure 7). The intensification effect was dose dependent; increasing with higher ratios of CR11 MPED: Cry1Ab.

EXAMPLE 15 The CR11-MPED Peptide Intensifies the Toxicity of CrylAc Against Grasshoppers of Sola As explained in more detail in Example 16, the peptide CR11-MPED also increased the toxicity of CrylAc for grasshoppers of soy (Figure 8).

EXAMPLE 16 Comparison of the capacity of the peptides CR1-3, CR11MPED, and CR12-MPED to intensify CrylAc against the Grasshoppers of Soy The peptide CR1-3 (SEQ ID NO: 6; SEQ ID NO: 5 is DNA) was constructed as a negative control with the expectation that it does not intensify toxicity based on the lack of CrylAb binding site on the peptide. However, it was surprisingly found that CR1-3 is equal to CR12-MPED in the ability to increase the toxicity of CrylAc for the grasshopper of soy (Figure 8). The CR11-MPED and CR1-3 regions of BtRα were cloned into pET30 and expressed in E coli. All peptides were expressed and purified using standard methodology. The purified peptides were run on SDS page using standard methodology. The concentrations of the samples indicated are the following: CR11-MPED (0.279 mg / m); CR12- MPED (2,106 mg / ml); CR1-3 (1809 mg / ml); Ano-Cad (0.50 mg / ml), Ano-PCAP (0.78 mg / ml) and S1yD (0.046 mg / ml). A 12.5 ng / cm2 CrylAc bioassay was carried out with or without different peptides at a ratio of 1: 1, 1:10 and 1: 100. Newborn soybean grasshoppers were placed in trays for bioassay, where each group had 62 larvae. The Ano-Cad, Ano-PCAP, and SlyD peptides are explained in the following Example.

EXAMPLE 17 Preparation of Ano-Cad and Ano-PCAP Peptides of the Mosquito Cadherin Proteins, and Preparation of the SlyD Peptide Mosquitoes are diptera, as opposed to lepidoptera previously evaluated. Given that selected mosquito cadherin proteins have low amino acid similarity to BtR-i, it was expected that peptide fragments of mosquito cadherins would be less likely to intensify the effect of a toxin on Lepidoptera larvae, thus serving as a control of cadherin negative. Additionally, mosquito cadherin fragments could be used to evaluate mosquito toxin enhancing properties. Two full-length cDNAs coding for cadherin-like proteins of the Anopheles gambiae mosquito were cloned and sequenced. The donated cDNA nucleotide sequences correspond to the sequences deposited by the genome sequencing project of Anopheles gambiae at the site of A. gambiae. Fragments of similar size and location were cloned to the CR12-MPED region of BtR-? A in pET30 vector and expressed in E col /. The cDNA fragments, designated, Ano-Cad and Ano-PCAP, donated in pET are identical to the DNA sequences of the place of A. gambiae XM_312086 and XM_321513, respectively. SEQ ID NO: 7 shows the nucleotide sequence of the putative cell adhesion protein of Anopheles gambiae (NCBI LOCUS XM_321513). SEQ ID NO: 8 shows the corresponding amino acid sequence. SEQ ID NO: 9 shows the nucleotide sequence encoding the putative region of the truncated cell adhesion protein of the Anopheles gambiae protein. This truncated peptide is referred to herein as PCAP (putative cell adhesion protein) or Ano-PCAP, which has 213 amino acid residues, and is approximately 24,417 Daltons (theoretical pl = 4.96). SEQ ID NO: 10 shows the truncated PCAP region of the Anopheles gambiae protein. This sequence is for the peptide expressed in the E coli strain BL21 / DE3 / pRIL having the DNA cloned in the pET-30a vector. SEQ ID NO: 11 (Ano-Cad-Sequence (GH) doc) shows the cadherin sequence of full-length cDNA from Anopheles gambiae. A BLAST search with the sequence matches the DNA and protein sequence predicted for a partial Anopheles gambiae cDNA (NCB! Locus X1 \ 4_312086). SEQ ID NO: 12 shows a partial cadherin nucleotide sequence of Anopheles gambiae. SEQ ID NO: 13 shows the sequence of the expressed peptide ("Ano-Cad") of the fragment cloned in the vector pET-30a and expressed in the E coil strain BL21 / DE3 / pRIL. SlyD is a 21-kDa histidine-rich E. co / 7 protein that is often co-eluted with other immobilized metal affinity column proteins (IMAC). Because similar protein sizes were detected in some eluates, SlyD from E coil was also prepared for evaluation.

EXAMPLE 18 Comparison of the ability of Peptides Ano-PCAP, Ano-Cad, and SlyD to intensify C / ylAc Against Grasshoppers of soybean As shown in Figure 8, Ano-PCAP (SEQ ID NO: 10) induced some increase in toxicity, while the peptide Ano-Cad (SEQ ID NO: 13) did not. SlyD had no intensifying effect.

EXAMPLE 19 Comparison of the ability of Peptides Ano-PCAP. CR12MPED, and CR1-3 to intensify Cry2Aa Against the Grasshoppers of soybean The protoxin Crylka (not truncated) (GENBANK Number of Access M31738) was fed to soybean grasshopper larvae (newborns) with CR12-MPED, CR1-3, or Ano-PCAP (SEQ ID NO: 10). Both CR12-MPED and AnoPCAP increased the toxicity of Cry2Aa for the iarvas. See Figure 9. The toxin: sample ratios are indicated in the graph. CR1-3 and CR12-MPED were eluted on a Ni-NTA column containing 0.25 M imidazole, while AnoPCAP was purified by ion exchange chromatography (Q sepharose). The Cry2Aa protein was expressed in E. coli and purified by ion exchange chromatography (Q sepharose). A single asterisk (*) denotes 0.05 < P < 0.1 while two asterisks (**) denotes P < 0.05 in the statistical calculation of Qui square comparing the treatment only with toxin and treatment with sample. CR1-3 did not intensify the toxicity of Cry2Aa. Both CR12-MPED and Ano-PCAP were able to significantly enhance the toxicity of Crylka.

EXAMPLE 20 Peptides CR11-MPED, CR12-MPED, CR1-3, and Ano-Cad have Activity by themselves Against Rootworms, C lAa surprisingly has activity against rootworms (Coleoptera) A CrylAa protein (GENBANK Accession Number AAA22353), which is a toxin produced in Bacillus thuringiensis (Bt), was evaluated as forms treated with protoxin and trypsin to determine its level of activity against rootworms (Diabrotica spp., Coleoptera). ) in case there is one. The expectation was that this toxin would not be active against this coleopter, since Cryl toxins (including CrylAa) are known to be "active lep" toxins (toxins with prolonged activity against caterpillars or lepidoptera). See for example Hofte et al. (1989). It was surprisingly found that this protein has activity against rootworms. Thus, methods of using CrylAa to control rootworms are an aspect of the present ntion. Surprisingly, in the course of this experimentation, it was further found that CR11-MPED and CR12-MPED peptide have activity by themselves against rootworms. Other peptides similar to cadherin, for example, were also evaluated. CR1-3 and Ano-Cad and were found to have significant toxicity against rootworms, despite the low toxicity (CR11-MPED CR12-MPED> CR1-3 «Ano-Cad). As the evaluation and the data included herein are not exhaustive, the present ntion thus includes the use of peptides of the present ntion, alone, to control insects. This methodology is even another aspect of the present ntion.

. A - Preparation of Cry Aa, CR11-MPED, CR12-MPED, CR1-3, v Ano-Cad and Rootworm bioassays. The CrylAa construct (CrylAa gene in vector pKK223-3) was obtained from Donald H. Dean (The Ohio State University). The toxin was expressed in E. coli and purified by HPLC. The toxin was concentrated and dialysed against distilled water. All peptides were over expressed in E. coli as the inclusion body. The inclusion bodies were extracted from the bacteria and solubilized in 10 mM NaOH. The insoluble materials were removed by centrifugation. The supernatant was applied to a Q-sepharose column (30 mM Na2C03 pH 10.0), and fluid fractions containing CR11-MPED were collected due to CR11-MPED from large aggregates that do not bind to the anion exchange column. The fractions were pooled and centrifuged again. The supernatant was concentrated by filtration (Amicon) and dialyzed against distilled water. Worm eggs were acquired from Lee French roots (French Agricultural Research Inc., Minnesota). Worm diet was acquired from the southern corn roots of Bio-Serv.

The bioassays were carried out in newborn larvae of the Worm of southern corn roots (Diabrotica undecipunctata). The toxins / peptides were diluted in distilled water and applied to the artificial diet in plastic bioassay trays and air-dried. Six larvae were placed in each well, and 24 larvae were evaluated in each dose of toxin in Study 1. In Study 2, four larvae were placed in each well, and 16 larvae were evaluated in each dose of toxin. The bioassays were carried out at room temperature (23 ° C). Mortality was recorded on Day 11 in Study 1 and Day 10 in Study 2.

. B - Toxicity of CrylAa, CR11-MPED. CR12-MPED, CR1-3, and Ano-Cad on D. undecipunctata. In Study 1, 29% mortality was observed at a concentration of 20011 g / cm2 of CrylAa protoxin and 50% mortality was observed at a concentration of 250 μg / cm2. A higher mortality was registered for CrylAa treated with trypsin. A 72% mortality was observed at a concentration of 1001.1 g / cm2 of CrylAa treated with trypsin and 67% mortality was observed at a concentration of 15011 g / cm2. In Study 2, 100% mortality was achieved with CrylAa protoxin at a concentration of 300 μg / cm2. Background mortality was between 6 and 8% in both studies. These results (summarized in Table 10) demonstrated that CrylAa has insecticidal activity against rootworms.

TABLE 10 Results of the bioassay for protoxin C / lAa and trypsin-treated toxin for the larvae of D. undecipunctata The toxicity of the peptides (CR11-MPED, CR12-MPED, CR1-3, and Ano-Cad) towards larvae of the rootworm was unexpected because the initial tests performed on the grasshopper larvae showed no activity toxic when the peptides were applied alone. In addition, a concentration of 100 μg / cm2 of CR12-MPED did not cause any mortality or growth inhibition for H. zea neonates. In the Study 1.25 μg / cm2 of CR11-MPED killed 8%, 50 μg / cm2 of CR11 MPED killed 8%, 100 μgcm2 of CR11-MPED killed 12%, 150 μg / cm2 of CR11-MPED killed the 79 %, 200 μg / cm2 of CR11-MPED killed 83%, and 250 μg / cm2 of CR11-MPED killed 92% of the larvae. In the same study, 25 μg / cm2 of CR12-MPED killed 8%, 50 μg / cm2 of CR12-MPED killed 21%, 100 μg / cm2 of CR12-MPED killed 21%, 150 μg / cm2 of CR12 -MPED killed 96%, 200 μg / cm2 of CR12-MPED killed 92%, and 250 μg / cm2 of CR12MPED killed 83% of the larvae.

In Study 2, 125 μg / cm2 of CR11-MPED killed 87%, 150 μg / cm2 of CR11-MPED killed 75%, 200 μg / cm2 of CR11-MPED killed 88% of the larvae. In the same study, 125 μg / cm2 of CR1-3 killed 6%, 150 μg / cm2 of CR1-3 killed 63%, 200 μg / cm2 of CR1-3 killed 44% of the larvae, while 125 μg / cm2 of Ano-Cad killed 31%, 150 μg / cm2 of Ano-Cad killed 38%, 200 μg / cm2 of Ano-Cad killed 50% of the larvae. Background mortality was between 6 and 8% in both studies. These results (summarized in Table 11) demonstrated the insecticidal activity of these peptides against the rootworm themselves.

TABLE 11 Results of the bioassay for CR11-MPED, CR12-MPED, CR1-3, and Ano-Cad for the larvae of D. undecipunctata EXAMPLE 21 Mortality of the Grasshopper of the soybean (Pseudoplusia includens) before mixtures of CR12-MPED and protoxin CrylAa or CrylAa digested with trypsin The construction of CrylAa (CrylAa gene in vector pKK223-3) was obtained from Donald H. Dean (The Ohio State University). The toxin was expressed in E co // and purified by HPLC. Table 12 shows the bioassay of dietary overlap in the mortality of the grasshopper soybean (Pseudoplusia includens) newly born to CrylAa in the forms of both protoxin and truncated toxin digested with trypsin, and with mixtures of CR12-MPED at ratios of 1: 100 (p / p). CR12-MPED intensifies the toxicity of CrylAa for P. includens. At a CrylAa protoxin concentration of 2 ng / cm2 the quality of the diet was 23% (toxin only); with the inclusion of 200 ng / cm2 of CR12-MPED the mortality increased up to 50%. At a Cry ka protoxin concentration of 5 ng / cm2, 500 ng / cm2 of CR12-MPED peptide greatly enhanced the toxicity of CrylAa protoxin for P. includens larvae from 0% (toxin only) to 88% (P < 0.001). Similar results were obtained using truncated Cry ka digested with trypsin. At a concentration of CrylAa toxin digested with trypsin of 2 ng / cm2, dietary mortality was 6% (toxin alone); with the inclusion of 200 ng / cm2 of CR12-MPED the mortality increased up to 94% (P <0.001). At a Crylka toxin concentration digested with trypsin of 5 ng / cm2, 500 ng / cm2 of CR12-MPED peptide intensified the toxicity of CrylAa protoxin for P. includens larvae from 38% (toxin only) to 100% (PO. 001). Background mortality was 0%. These results demonstrated that the CR12-MPED peptide was able to enhance the protoxin CrylAa activity (Figure 10A) as well as truncated CrylAa digested with trypsin (Figure 10B) against P. includens.

TABLE 12 Results of the bioassay for CrylAa alone and mixtures of Cry1 a: CR12-MPED at a ratio of 1: 100 (w / w) against larvae of Pseudoplusia includens EXAMPLE 22 Mortality of the Grasshopper of soybean (Pseudoplusia includens) to Variable Mixtures of CR12-MPED and CrylAc Figure 11 shows the bioassay of dietary superimposition mortality on the soybean grasshopper (Pseudoplusia includens) newborn to the mixture of CR12-MPED and 2.5 ng / cm2 of CrylAc (p: p) with different toxin ratio : peptides. Mortality was (5.2 ± 1.1)% when soio was applied 2.5 ng / cm2 of CrylAc d. When the same amount of CrylAc was mixed with CR12MPED as a ratio of 1: 1, mortality in newborns was significantly increased to (39.9 ± 7.3)% (P <0.05). Mortality was increased by approximately 50% when the toxin: peptide ratio reached 1: 2 (55.2 ± 8.5)%.

EXAMPLE 23 Mortality of Coles Grasshopper (Trichoplusia ni) to Variable Mixtures of CR12-MPED v CrylAc Figure 12 shows the bioassay of dietary overlap on the mortality of the newborn grasshoppers of the cabbages (Trichoplusia ni) before the mixture of CR12-MPED and 4 ng / cm2 of CrylAc (p: p) with different toxin: peptides ratio. Mortality was (10.4 ± 7.5)% when only 4 ng / cm2 of CrylAc was applied. When the same amount of CrylAc was mixed with CR12-MPED as a ratio of 1: 2, mortality in the neonates was significantly increased to (47.9 ± 13.5)% (P <0.05). Mortality was significantly above 50% when the toxin: peptide ratio reached 1: 8 (57.3 ± 1.0)% (PO.05).

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Sangadala, S., Walters, F., English, L.H.Adang, M.J., 1994. A mixture of Manduca sixth aminopeptidase and alkaline phosphatase enhancers Bacillus thuringiensis insecticidal CrylA (c) toxin binding and 86Rb + - K + leakage in vitro. J. Biol. Chem. 269, 10088-10092. Simpson, R.M.Newcomb, R.D., 2000. Binding of Bacillus thuringiensis ándotoxins Cryl Ac and Cryl Ba to a 120-kDa aminopeptidase-N of Epiphyas postvittana purified from both brush border membrane vesicles and baculovirus-infected Sf9 cells. Insect Biochem. Molec. Biol 30, 10691078. Tabashnik, B., 1992. Evaiuation of synergism arnong Baciiius thuringiensis toxins. Appl. Environ. Microbiol. 58, 3343-3346. Tsuda, Y., Nakatani, F., Hashimoto, K., Ikawa, S., Matsura, C, Fukada, T., Sugimoto, K.Himeno, M., 2003. Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori (silkworm). Biochem. J. 369, 697-703. Vadlamudi, R.K., Ji, T.H. Bulla, L.A., Jr., 1993. A specific binding protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp. Berliner. J. Biol. Chem. 268, 12334-12340. Vadlamudi, R.K., Weber, E., Ji, I., Ji, T.H.Bulla, L, Jr., 1995. Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis. J. Biol. Chem. 270, 5490-5494. Wolfersberger, MG, Luthy, P., Maurer, A., Parenti, P., Sacchi, VF, Giordana, B. Hanozet, GM, 1987. Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp.

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Claims (22)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for inhibiting an insect wherein said method comprises providing a peptide to said insect for ingestion, wherein said peptide has activity as a toxin against an insect, and wherein said peptide is a fragment of an insect cadherin ectodomain.

2. The method according to claim 1, further characterized in that the method comprises, providing a protein toxin to said insect.

3. The method according to claim 2, further characterized in that said peptide and said protein are sprayed on a plant.

4. The method according to claim 2, further characterized in that said peptide and said protein are produced by and are present in a plant.

5. The method according to claim 2, further characterized in that said protein is an insecticidal protein of Bacillus thuringiensis.

6. The method according to claim 2, further characterized in that said protein is a Cry protein of Bacillus thuringiensis.

7. - The method according to claim 2, further characterized in that said protein is a Cry protein of Bacillus thuringiensis selected from the group consisting of Cryl proteins and Cry2 proteins.

8. The method according to claim 1, further characterized in that peptide is 200 to 400 amino acids in length.

9. The method according to claim 1, further characterized in that said cadherin is a receptor for a polypeptide having toxin activity against an insect.

10. The method according to claim 1, further characterized in that said cadherin is a mosquito protein.

11. The method according to claim 1, further characterized in that said cadherin is from an organism selected from the group consisting of Manduca sexta and Anopheles gambiae.

The method according to claim 1, further characterized in that said peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 2 (CR12-MPED), SEQ ID NO: 4 (CR11-MPED), SEC ID NO: 6 (CR1-3), SEQ ID NO: 10 (Ano-PCAP) and residues 1358-1369 of SEQ ID NO: 13 (Ano-Cad).

The method according to claim 1, further characterized in that said insect is selected from the group consisting of lepidoptera and coleoptera.

14. The method according to claim 1, further characterized in that said insect is a root worm.

15. The method according to claim 1, further characterized in that said insect is a lepidopteran selected from the group consisting of Manduca sexta, Heliothis virescens, Helicoverpa sea, Spodoptera frugiperda, and Pseudoplusia includens.

The method according to claim 1, further characterized by comprising the provision of a protein for said insect for ingestion, wherein said peptide and said protein have toxin activity against said insect, and wherein a polynucleotide encoding said peptide hybridizes under stringent conditions with a nucleic acid probe selected from the group consisting of SEQ ID NO: 1 (CR12-MPED), SEQ ID: 3 (CR11-MPED), SEQ ID NO: 5 (CR1-3), SEQ ID NO: 9 (AnoPCAP), SEQ ID NO: 12 (Ano-Cad).

17. The method according to claim 1, further characterized in that said peptide has at least 75% amino acid identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2 (CR12-MPED), SEQ ID NO. : 4 (CR11-MPED), SEQ ID NO: 6 (CR1-3), SEQ ID NO: 10 (Ano-PCAP), and residues 1358-1369 of SEQ ID NO: 13 (Ano-Cad).

18. The method according to claim 16, further characterized in that said protein is selected from the group consisting of CrylAa proteins, CrylAb proteins, CrylAc proteins, CrylC proteins, and Cry2A proteins.

19. A plant cell comprising a first polynucleotide that encodes a peptide, wherein said peptide has toxin activity against an insect, and wherein said peptide is a fragment of an insect cadherin ectodomain.

20. The plant cell according to claim 19, further characterized in that it comprises a second polynucleotide that encodes a protein having toxin activity against an insect pest.

21. A method for inhibiting an insect, wherein said method comprises providing said insect with a plant cell according to claim 19 for ingestion.

22. A method for inhibiting a rootworm wherein said method comprises providing said root worm with a Cry Ac protein for ingestion.