MXPA06007245A - Genes encoding proteins with pesticidal activity - Google Patents
Genes encoding proteins with pesticidal activityInfo
- Publication number
- MXPA06007245A MXPA06007245A MXPA/A/2006/007245A MXPA06007245A MXPA06007245A MX PA06007245 A MXPA06007245 A MX PA06007245A MX PA06007245 A MXPA06007245 A MX PA06007245A MX PA06007245 A MXPA06007245 A MX PA06007245A
- Authority
- MX
- Mexico
- Prior art keywords
- plant
- amino acid
- pesticidal
- sequence
- proteolytic
- Prior art date
Links
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Abstract
Compositions and methods for protecting a plant from an insect pest are provided. The invention provides mutagenized nucleic acids that have been engineered to encode pesticidal polypeptides having increased resistance to proteolytic degradation by a plant protease. In particular, nucleic acid sequences encoding pesticidal polypeptides modified to comprise a proteolytic protection site that confers resistance to degradation or proteolytic inactivation by a plant protease are provided. Particular embodiments of the invention provide pesticidal polypeptide compositions and formulations, expression cassettes, and transformed plants, plant cells, and seeds. These compositions find use in methods for controlling pests, especially plant pests. Novel plant proteases, sequences encoding these proteases, and methods for their use are also provided.
Description
GENES CODING PROTEINS WITH PESTICIDE ACTIVITY FIELD OF THE INVENTION The present invention relates to the fields of plant molecular biology and plant pest control. More specifically, the invention relates to modified pesticidal polypeptides and the nucleic acid sequences encoding them. In some embodiments, the pesticidal polypeptides are mutated Cry toxins from Bacillus thuringiensis.
The compositions and methods of the invention utilize the disclosed nucleic acids and their pesticidal polypeptides encoded to control plant pests. BACKGROUND OF THE INVENTION Insect pests are a major factor in the loss of agricultural crops in the world. For example, damage by feeding the corn rootworm or damage by the cotton weevil can be economically devastating for farmers. The loss of the crop related to insect pests of the corn rootworm alone has reached a trillion dollars a year. Traditionally, the primary methods to impact populations of insect pests, such as populations of corn rootworms, are crop rotation and the application of broad spectrum synthetic chemical pesticides. However, consumers and government regulators in common are becoming increasingly concerned with the environmental hazards associated with the production and use of synthetic chemical pesticides. Due to such concerns, regulators have banned or limited the use of some of the most dangerous pesticides. Thus, there is substantial interest in developing alternatives to traditional chemical pesticides that present a lower risk of contamination and environmental hazards and provide a greater objective specificity that is characteristic of traditional broad-spectrum chemical insecticides. Biological control of insect pests of importance to agriculture using a microbial agent, such as fungi, bacteria or other insect species provides an environmentally friendly and commercially attractive alternative. Generally speaking, the use of biopesticides presents a lower risk of contamination and environmental hazards, and provides a larger target specificity that is characteristic of traditional broad-spectrum chemical insecticides. In addition, biopesticides often cost less to produce and thus improve economic performance for a wide variety of crops. Certain species of microorganisms of the genus Bacillus are known to possess pesticidal activity against a wide range of insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillus thuringiensis and Bacillus papilliae are among the most successful biocontrol agents discovered to date. The pathogenicity of the insect has been attributed to strains of: B. larvae, B. lentimorbus, B. papilliae, B. sphaericus, B. thuringiensis (Harwook, ed. (1989) Bacillus (Plenum Press), page 306) and B. cereus (International Publication No. WO 96/10083). The pesticidal activity is shown to be concentrated in the inclusions of parasporal crystalline protein, although the pesticidal proteins have also been isolated from the stage of vegetative growth of Bacillus. Several genes encoding these pesticidal proteins have been isolated and characterized (see, for example, U.S. Patent Nos. 5,366,892 and 5,840,868). Microbial pesticides, particularly those obtained from Bacillus strains, have played an important role in agriculture as alternatives for the chemical control of pests. Recently, agricultural scientists have developed crop plants with increased resistance to insects by genetically engineering crop plants to produce Bacillus pesticide proteins. For example, corn and cotton plants genetically engineered to produce pesticidal proteins, isolated from strains of B. thuringiensis, known as d-endotoxins or Cry toxins (see, for example, Aronson (2002) Cell Mol. Life Sci. 59 (3): 417-425; Schnepf et al., (1998) Microbiol Mol Biol. Rev. 62 (3): 775-806) are now widely used in American agriculture and have provided the farmer with an environmentally friendly alternative to traditional insect control methods. In addition, potatoes genetically engineered to contain Cry toxins pesticides have been sold to the American farmer. However, while they have been proven to be very commercially successful, these genetically engineered insect-resistant crop plants provide resistance to only a small range of economically important insect pests. Some insects, such as the West Corn Rootworm, have been proven to be recalcitrant. Accordingly, efforts have been made to understand the mechanism of action of Bt toxins and to design toxins with improved properties. It has been shown that proteases, for example, insect gut proteases, can affect the impact of Cry toxins from Bacillus thuringiensis and other pesticidal proteins on the insect. Some proteases activate Cry proteins by processing them from a form of "protoxin" in a toxic form, or "toxin". See, Oppert (1999) Arch. Insect Biochem .. Phys. 42: 1-12 and Carrol et al., (1997) J.
Invertebrate Pathology 70: 41-49. This activation of the toxin may include the removal of the N- and C-terminal peptides from the protein and may also include internal segmentation of the protein. Other proteases can degrade pesticidal proteins. See Oppert, ibid; see also U.S. Patent Nos. 6,057,491 and 6,339,491. Researchers have determined that plants express a variety of proteases, including serine and cysteine proteases. The specificity of these proteases for particular proteolytic sites has also been characterized. See, for example, Goodfellow et al., (1993) Plant Physiol. 101: 415-419; Pechan et al., (1999) Plant Mol. Biol. 40: 111-119; Lid et al., (2002) Proc. Nat. Acad. Sci. 99: 5460-5465. While researchers have previously genetically engineered plants, particularly crop plants, to contain biologically active Cry toxins (ie, pesticides), these foreign proteins can be degraded and inactivated by the proteases present in these transgenic plants. A greater understanding of endogenous plant proteases and proteolytic sites sensitive to segmentation by these proteases is needed. Thus, nucleic acid molecules encoding pesticidal polypeptides not susceptible to degradation or inactivation by plant proteases are desired for use in pest management strategies. BRIEF DESCRIPTION OF THE INVENTION Compositions and methods are provided for protecting a pesticidal polypeptide from proteolytic inactivation in a plant and for protecting a plant from a pest. The compositions and methods of the invention find use in agriculture to control pests of many crop plants. Such pests include, but are not limited to, agriculturally significant pests, such as: Western corn rootworms, for example, Diabrotica virgifera virgifera; Northern corn rootworm, Diabrotica longicornis barberi; southern corn rootworm; Diabrotica undecimpunctata howardi; centipede, Melanotus spp. and Aeolus spp; cotton weevil, for example, Anthonomus grandis; Colorado potato beetle, Leptinotarsa decemlineata; and alfalfa weevil, Hypera nigrirostris. The invention provides nucleic acids and variants and fragments thereof, which encode pesticidal polypeptides comprising sites that are designed to be resistant to degradation or inactivation by a plant protease. Pesticidal polypeptides include, for example, the Cry toxin from Bacillus thuringiensis and pentin-1. In some embodiments, a proteolytic site within a pesticidal polypeptide that is susceptible to cleavage by a plant protease is mutated to comprise a site that is not sensitive to the plant protease. In a particular embodiment, the mutation of a proteolytic site within the pesticidal polypeptide protects the protein from proteolytic inactivation by a plant protease, thereby increasing the stability of the active toxin in a transgenic plant and improving the resistance properties of the transgenic plant. associated pests. Methods for using these nucleic acid molecules to protect a pesticidal polypeptide from proteolytic inactivation in a plant and to protect a plant from a pest are provided. Also provided are isolated pesticidal polypeptides, and variants and fragments thereof, encoded by the nucleic acid molecules of the present invention. The nucleic acids of the invention can also be used to produce transgenic (eg, transformed) plants that are characterized by genomes comprising at least one stably incorporated polynucleotide construct comprising a coding sequence of the invention operably linked to a promoter. which induces expression of the pesticide polypeptide encoded in a plant. Therefore, plant cells are also provided, plant tissues, plants and seeds transformed thereof. In a particular embodiment, a transformed plant of the invention can be produced using a nucleic acid that has been optimized for increased expression in a host plant. For example, one of the pesticidal polypeptides of the invention can be further translated to produce a nucleic acid comprising codons optimized for expression in a particular host, for example, a crop plant such as a Zea mays plant. Expression of a nucleotide sequence of the invention by such a transformed plant (eg, dicot or monotone) will result in the production of a pesticidal polypeptide that has increased resistance to proteolytic degradation by a plant protease and can confer increased resistance plagues the plant. In some embodiments, the invention provides transgenic plants that express modified pesticidal polypeptides that find use in methods to protect the plant from a pest and to protect a pesticidal polypeptide from proteolytic inactivation by a protease in a plant. Also provided are nucleic acid molecules, and variants and fragments thereof, which encode novel plant proteases. In one embodiment, the isolated nucleic acids of the invention encode a plant protease similar to novel cathepsin B from corn. In another embodiment, a nucleotide sequence encoding a plant protease that was identified in the corn root tissue and homologous to the cysteine mir2 protease is provided. Also provided are isolated polypeptides (ie, plant proteases) and variants and fragments thereof, encoded by the nucleic acids of the invention. The plant protease nucleic acid molecules and the corresponding polypeptides can be used to identify the specificity of the cleavage site of these proteases. The plant proteases of the invention also find use in the determination of whether a polypeptide or pesticide of the invention is sensitive to cleavage by these proteases. In a particular embodiment, a polypeptide or pesticide that is sensitive to a novel plant protease of the invention is designed to comprise a site that protects the protein from degradation or inactivation by that plant protease. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Probit analysis of the mutant similar to Cry8 1218 (M6) against the Colorado potato beetle (see Example 9). The log (concentration) of the toxin is plotted on the horizontal axis, while the larval mortality is plotted on the vertical axis. The results of the probit analysis were: the LC50 was 0.259 mg / ml; 95% of fiducial limits were 0.171 mg / ml and 0.370 mg / ml. The observed mortality data points are presented by full points, while the predicted mortality is represented by empty squares. 95% of the upper and lower limits are indicated by dashed lines. Figure 2: Effect of Wild Type 1218-1 on Larval Mortality of the Colorado Potato Beetle. The application rate of wild-type endotoxin in micrograms per square centimeter is arranged on the horizontal axis and the percent mortality is shown on the vertical axis. Two replicas of the experiment are shown (bars with vertical stripes = replica 1, bars with diagonal stripes = replica 2). Figure 3: Effect of Mutant K03 similar to Cryd 1218 on Larval Mortality of the Colorado Potato Beetle. The rate of application of wild type endotoxin in micrograms per square centimeter is arranged on the horizontal axis and the percent mortality is shown on the vertical axis. Two replicas of the experiment are shown (bars with diagonal stripes = replica 1, bars with horizontal stripes = replica 2). Figure 4: Effect of Mutant K34 similar to CryS
1218 in the Larval Mortality of the Colorado Potato Beetle. The rate of application of wild type endotoxin in mirams per square centimeter is arranged on the horizontal axis and the percent mortality is shown on the vertical axis. Two replicas of the experiment are shown (bars with diagonal stripes = replica 1, bars with vertical stripes = replica 2). Figure 5: Larval Tests with the Cotton Weevil. This figure shows results of the larval tests with the cotton weevil, as described in Experimental Example 13 and Table 9. The doses are arranged on the horizontal axis, while the combined larval weight in milligrams is shown on the vertical axis . The mutant K03 data is shown by the vertically scratched bars; the data of the M6 mutant are shown by empty bars; the data of 1218-1 (wild type) are shown by dotted bars and the data of the control of the regulatory solution are shown by diagonally striped bars. Figure 6: Probit Analysis of the 1218-1 Type
Wild against the Colorado Potato Beetle (see Example 6). The log (concentration) of the toxin is plotted on the horizontal axis, while the larval mortality is plotted on the vertical axis. The results of the probit analysis were: in probability 0.50, the concentration was 1.1098 mg / ml; 95% of fiducial limits were 0.6859 and 2.4485. The observed mortality data points are represented by full points while the predicted mortality is represented by empty squares. 95% of the upper and lower limits have been indicated by dashed lines. Figure 7: Probit analysis of the Mutant similar to Cry8 1218 (K03) against the Colorado Potato Beetle (see Example 6). The log (concentration) of the toxin is plotted on the horizontal axis, while the larval mortality is plotted on the vertical axis. The results of the probit analysis were: in probability 0.50, the concentration was 0.00808 mg / ml; 95% of fiducial limits were 0.00467 and 0.01184. The observed mortality data points are represented by full points, while the predicted mortality is represented by empty squares. 95% of the upper and lower limits are indicated by dashed lines. Figure 8: Distribution Analysis of Corn Coding Regions (see Example 14). Corn cDNAs with full-length coding regions were analyzed for GC content and plotted as a function of their GC content (see upper panel, "ORFs"). A "UniGene" data set based on EST containing 84,085 sequences was also analyzed ("UniGenes", shown in the lower panel). DETAILED DESCRIPTION OF THE INVENTION Compositions and methods are provided for protecting a pesticidal polypeptide from proteolytic inactivation in a plant and for protecting a plant from a pest. More specifically, the isolated nucleic acids of the invention, and fragments and variants thereof, comprise nucleotide sequences encoding pesticidal polypeptides (e.g., proteins) that have been designed to have increased resistance to proteolysis by plant proteases. The disclosed pesticidal proteins are biologically active (eg, pesticide) against insect pests such as insect pests. Coleoptera order. Insect pests of interest include, but are not limited to: western corn rootworm, eg, Diabrotica virgifera virgifera; Northern corn rootworm, for example, Diabrotica longicornis barberi and southern corn rootworm, for example, Diabrotica undec impune tat a howardi. Additional pests include: centipedes, Melanotus, Eleodes, Conoderus and Aeolus spp.; Japanese beetle, Popillia japonica; white larva, Phyllophaga crinita; corn beetle, Chaetocnema pulicaria; sunflower stem weevil, Cylindrocupturus adspersus; gray sunflower seed weevil, Smicronyx sordidus; sunflower beetle, Zygogramma exclamationis; cotton weevil, for example, Anthonomus grandis; alfalfa weevil, Hypera 'nigrirostris; scarajuelo de crucifera, Phyllotreta cruciferae; Colorado potato beetle, Lepyinotarsa decemlineata; striped beetle, Phyllotreta striolata; striped turnip beetle, Phyllotreta nemorum; and rapeseed beetle, Meligethes aeneus. Accordingly, the present invention provides novel methods for controlling plant pests that do not depend on the use of synthetic, traditional chemical pesticides. Researchers have previously genetically engineered plants to contain biologically active pesticide polypeptides, for example, Cry toxins from Bacillus thuringiensis, in order to confer increased pest resistance on these plants. It is recognized that the pesticidal polypeptides expressed in these transgenic plants may be susceptible to cleavage by proteases of endogenous plants, as demonstrated in Examples 22 and 23 hereinafter. Segmentation of a pesticidal polypeptide by a plant protease in a transgenic plant can lead to the proteolytic inactivation of the toxin, thereby reducing the resistance of the plague achieved by genetically engineering the plant to express the pesticidal protein. For example, a mutant CrydBbl toxin expressed in corn is shown to be segmented in the plant. In addition, the transgenic plant did not exhibit resistance to WCRW as would be anticipated with the expression of a pesticidal polypeptide (see Example 22 below). A variety of proteases have been identified in plants, and these proteases can proteolytically inactivate a pesticidal polypeptide expressed in a transgenic plant. See, for example, Goodfellow et al., (1993) Plant Physiol. 101: 415-419; Pechan et al., (1999) Plant Mol. Biol. 40: 111-119; Lid et al., (2002) Proc. Nat. Acad. Sci. 99: 5460-546B. As used herein, "proteolytic inactivation" means segmentation of the pesticidal polypeptide at a proteolytic site by a plant protease, where segmentation at that site reduces or eliminates the pesticidal activity of the toxin relative to that of the pesticidal polypeptide. not segmented. Compositions and methods are provided for protecting a pesticidal polypeptide from proteolytic inactivation. In a method of the invention, a proteolytic site in a pesticidal polypeptide that is sensitive to a plant protease is altered or mutated to comprise a proteolytic protection site. By "proteolytic protection site", a proteolytic site that has been altered to comprise a site that is not sensitive to a plant protease is proposed. As used herein, "not sensitive to a plant protease" means a site in a pesticidal polypeptide that is not recognized by a plant protease, and, thus, proteolysis at the mutated site is diminished relative to that of the original site Standard techniques for estimating the degree of proteolysis of a particular protein are well known in the art. A proteolytic site can be altered or mutated to form a proteolytic protection site, for example, by making one or more additions, deletions or substitutions of amino acid residues. These sites can be altered by the addition or deletion of any number and class of amino acid residues. In a particular embodiment, the alteration of a proteolytic site to comprise a proteolytic protection site comprises replacing at least one amino acid of the proteolytic site with a different amino acid. Mutations can be made, for example, in or adjacent to a portion of the proteolytic site. In some embodiments, the proteolytic site that is altered is located in a region of inactivation of the toxin. As used in this"inactivation region" refers to a site or region in a pesticidal polypeptide, wherein the cleavage of that site within that region by a protease reduces or eliminates the pesticidal activity of the toxin relative to that of the non-segmented pesticide polypeptide. . Bioassays to estimate the pesticidal activity of a protein are well known in the art. See, for example, Examples 6, 7 and 12 in the. present right away In one embodiment, the proteolytic protection site is inserted in the region between helices 3 and 4 of domain 1 of a Cry toxin.
A number of proteases have been identified in several plant species. In particular, serine and cysteine proteases have been characterized in plants. See, for example, Goodfello et al., (1993) Plant Physiol. 101: 415-419; Pechan et al., (1999) Plant Mol. Biol. 40: 111-119; Lid et al., (2002) Proc. Nat. Acad. Sci. 99: 5460-5465. As used herein, "plant protease" refers to any enzyme that cleaves a polypeptide by hydrolyzing the peptide bonds and is naturally found in any plant of the invention. Any protease plant can be used in the present invention. In some embodiments, the plant protease is a cysteine protease, for example, a protease similar to cathepsin B. In a further embodiment, a method for protecting a plant from a pest is provided. This method comprises introducing into a plant at least one polynucleotide construct comprising a nucleotide sequence encoding a pesticidal polypeptide operably linked to the promoter that induces expression in a plant. The pesticidal polypeptide of this embodiment has at least one proteolytic protection site designed in, for example, a region of inactivation of the toxin. In one embodiment, the pesticidal polypeptide is a Bacillus thuringiensis toxin such as CrydBbl or a variant or fragment thereof. While the invention is not related by any theory of operation, it is believed that the mutation of a plant protease responsive proteolytic site located within an inactivation region to comprise a proteolytic protection site protects the pesticidal polypeptide from proteolytic inactivation by a plant protease, thereby increasing the stability of the active toxin in a transgenic plant and improving the associated pest resistance properties of that plant. The invention further provides isolated pesticide polypeptides comprising at least one designed proteolytic protection site. More specifically, the invention provides pesticidal proteins that are produced from altered nucleic acid designed to introduce particular amino acid sequences (eg, proteolytic protection sites) into polypeptides of the invention. In particular embodiments, the proteolytic protection site is introduced into an inactivation region of the toxin and protects the pesticidal polypeptide from proteolytic inactivation. The isolated pesticidal polypeptides of the invention can be, for example, a mutated Bacillus thuringiensis toxin to comprise a proteolytic protection site. In one embodiment, the Bacillus thuringiensis toxin is CrydBbl or a variant or fragment thereof.
The nucleic acid sequences of the invention
further comprise isolated polynucleotides, and variants and fragments thereof, which encode biologically active pesticidal polypeptides. In some embodiments, the pesticidal polypeptides have at least one designed proteolytic protection site that is not sensitive to a plant protease and protects the pesticidal polypeptide from proteolytic inactivation. Expression cassettes comprising the nucleic acid sequences of the invention operably linked to a promoter that induces expression in a plant are also provided. The nucleic acid molecules and expression cassettes of the present invention can be used to produce transgenic plants comprising at least one stably incorporated polynucleotide construct comprising a nucleotide sequence encoding a pesticidal polypeptide having at least one designed proteolytic protection site. Expression of that nucleotide sequence will result in the production of a pesticidal polypeptide - which has a proteolytic protection site and can increase resistance to plague by protecting the pesticidal polypeptide from proteolytic degradation or inactivation. Accordingly, plant cells, plant tissues, plants and transformed seeds thereof are also provided.
The invention further provides nucleic acid molecules and variants and fragments thereof, which encode novel plant proteases. The nucleotide sequence set forth in SEQ ID NO: 135 encodes a protease similar to cathepsin B that is constitutively expressed in corn. The nucleotide sequence set forth in SEQ ID NO: 137 encodes a cysteine protease identified in maize that is homologous to the cysteine mir2 protease and is preferentially expressed in the corn root tissue (against the leaf). The nucleotide sequences set forth in SEQ ID NOS: 135 and 137 encode the plant protease polypeptide sequences of SEQ ID NOs: 136 and 13d, respectively. The invention further comprises variants and fragments of these polypeptide sequences that possess proteolytic activity. Assays for measuring proteolytic activity are well known in the art and include those assays described hereinafter. The novel plant proteases of the invention find use, for example, in the identification of the proteolytic cleavage site (s) for these proteases. In one embodiment, these proteases are used to determine whether they segment the pesticidal polypeptides of the invention. In a particular embodiment, a pesticidal polypeptide, for example, CrydBbl, is mutated to replace a proteolytic site sensitive to cleavage by a novel plant protease of the invention with a proteolytic protection site. The novel plant proteases and variants and fragments thereof can be used in any embodiment of the present invention. The nucleic acids and nucleotide sequences of the invention can be used to transform any organism to produce the encoded pesticidal proteins or plant proteases. Methods that involve the use of such transformed organisms to impact or control plant pests are also provided. Nucleic acids and nucleotide sequences of the invention that encode pesticidal polypeptides can also be used to transform organelles such as chloroplasts (McBride et al., (1995) Biotechnology 13: 362-365; and Kota et al., (1999) Proc. Nati. Acad. Sel. USA 96: 1840-1845). Nucleic acids of the invention comprise nucleic acids or nucleotide sequences that have been optimized for expression by cells of a particular organism, for example nucleic acid sequences that have been subsequently translated (i.e., reverse translation) using preferred codons of plants based on the amino acid sequence of a polypeptide having pesticidal activity. The invention further provides mutations that confer increased resistance to cleavage by a plant protease in the pesticidal polypeptides comprising them. Mutations of the invention can be used with any background sequence. As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in the form of either single or double strand, and unless otherwise limited, comprises known analogs (e.g. peptide nucleic acids) that have the essential nature of natural nucleotides in that they hybridize single-stranded nucleic acids in a manner similar to the naturally occurring nucleotides. As used herein, the terms "encoded" or "encoded" when used in the context of a specified nucleic acid means that the nucleic acid comprises the information necessary to direct the translation of the nucleotide sequence into a specified protein. The information by which the protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise untranslated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening untranslated sequences (e.g., as a cDNA).
As used herein, "full length sequence" in reference to a specified polynucleotide or its encoded protein means that it has the complete nucleic acid sequence or the complete amino acid sequence of a native sequence. By "native sequence", the endogenous sequence is proposed, that is, a non-designed sequence found in the genome of an organism. A full-length polynucleotide encodes the catalytically active full-length form of the specified protein. The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to polymers of amino acids in which one or more amino acid residues is an artificial chemical analogue of a naturally occurring corresponding amino acid, as well as polymers of naturally occurring amino acids. The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide or peptide (collectively "protein"). The amino acid may be an amino acid that occurs naturally and, unless otherwise limited, may comprise known analogs of natural amino acids that may function in a similar manner as naturally occurring amino acids. The polypeptides of the invention can be produced either from a nucleic acid disclosed herein, such as by the use of standard molecular biology techniques. For example, a truncated protein of the invention can be produced by expressing a recombinant nucleic acid of the invention in an appropriate host cell, or alternatively by a combination of ex vivo procedures, such as digestion and protease purification. As used herein, the terms "isolated" and "purified" are used interchangeably to refer to nucleic acids or polypeptides or biologically active portions thereof that are substantially or essentially free of components that normally accompany or interact with the nucleic acid or polypeptide when it is in its naturally occurring environment. Thus, an isolated or purified nucleic acid or polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemical substances when chemically synthesized. An "isolated" nucleic acid is free of sequences (preferably sequences encoding proteins) that naturally flank the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the organism from from which the nucleic acid is derived. For example, in several embodiments, the isolated nucleic acids may contain less than about 5 kb, 4, kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences that naturally flank the nucleic acids in the DNA of the cell from which the nucleic acid is derived. As used herein, the term
"isolated" or "purified" as used to refer to a polypeptide of the invention means that the isolated protein is substantially free of cellular material and includes protein preparations having less than about 30%, 20%, 10% or % (in dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is produced recombinantly, preferably the culture medium represents less than about 30%, 20%, 10% or 5% (by dry weight) of chemical precursors or non-chemical substances. of interest of the protein. By "protection of an insect pest plant" is proposed the limitation or elimination of damage related to insect pests to a plant, for example, by inhibiting the ability of the insect pest to grow, feed and / or reproduce or by exterminating the insect pest. As used herein, the terms "pesticidal activity" and "insecticidal activity" are used synonymously to refer to the activity of an organism or a substance (such as, for example, a protein) that can be measured by, but not It is limited to plague mortality, plague weight loss, repellency, plague and other behavioral and physical changes of a plague after feeding and exposure for an appropriate duration of time. In this way, the pesticide activity impacts at least one measurable parameter of plague adjustment. Tests to estimate pesticidal activity are well known in the art. See, for example, U.S. Patent Nos. 6,570,005 and 6,339,144. The preferred development stage for the test for pesticidal activity is larvae or immature forms of these insect pests mentioned above. The insects can be bred in total darkness at about 20 ° C to about 30 ° C and about -30% to about 70% relative humidity. The bioassays can be performed as described in Czapla and Lang (1990) J. Econ. Environment 1. 83 (6): 2460-2485. Methods for breeding insect larvae and performing bioassays are well known to one of ordinary skill in the art. A wide variety of bioassay techniques for estimating pesticidal activity is known to one skilled in the art. General procedures include the addition of the experimental compound or organism to the diet source in an enclosed container. Pesticidal activity can be measured, but is not limited to changes in mortality, weight loss, attraction, repellency or other behavioral and physical changes after feeding and exposure for an appropriate length of time. "Pesticidal polypeptide" or "toxin" or "insect toxin" refers to a polypeptide that possesses pesticidal activity. Cry toxins from Bacillus thuringiensis are pesticidal polypeptides. Other examples of pesticidal proteins include, for example, pentin-1 (see U.S. Patent Nos. 6,057,491 and 6,339,144). Any pesticidal polypeptide can be used to practice the present invention. In some embodiments of the invention, the pesticidal polypeptide is a Bacillus thuringiensis toxin
(Bt) By toxin "Bt" or Bacill us thuringiensis ", the broadest class of toxins found in several strains of
Bacillus thuringiensis, which includes such toxins as, for example, Cry8 d-endotoxins or Cryd-like. 'Bt toxins are a family of insecticidal proteins that are synthesized as protoxins and crystallize as parasporal inclusions. When ingested by an insect pest, the microcrystal structure is dissolved by the alkaline pH of the insect's midgut and the protoxin is cleaved by the insect's intestine proteases to generate the active toxin. Activated Bt toxin binds receptors in the epithelium of the insect's intestine, causing membrane damage and associated swelling and lysis of the insect. The death of the insect results from starvation and septicemia. See, for example, Li et al., (1991) Nature 353: 815-821. By "similar to Cryd" it is proposed that the nucleotide or amino acid sequence shares a high degree of sequence identity or similarity to the previously described sequences classified as Cryd, which includes such toxins as, for example, CrydBbl (see Access of Genbank No. CAD57542) and CrydBcl (see Genbank Access No. CAD57543). Similarly, by "pentin-1-like" it is proposed that the nucleotide or amino acid sequence shares a high degree of sequence identity or similarity to the previously described pentin-1 sequences (see U.S. Patent Nos. 6,057,491 and 6,339,144) . In some cases, the pesticidal polypeptides of the invention and the nucleotide sequences that encode them will share a high degree of sequence identity or similarity to the wild-type CrydBbl or CrydBcl sequence. In particular embodiments, the pesticidal polypeptides are the Cryd-like toxins or mutated Cryd-like toxins disclosed in the origin application, co-pending US Patent Application No. 10 / 606,320, filed on June 25, 2003, incorporated in the present by reference. Of particular interest are the pesticidal polypeptides designated in the source application as wild-type CrydBbl (Cryl218-1; SEQ ID NO: 2; Genbank Accession No. CAD57542), wild-type CrydBcl (Cryl218-2; SEQ ID NO: 4; Genbank Access No. CAD57543), CrydBbl K04 (SEO ID NO: 22); Cry8Bbl K0 (SEQ ID NO: 98) and truncated CrydBbl (SEQ ID NO: 6) and encoded by the nucleotide sequences set forth in SEQ ID NOs: 1, 3, 21, 97 and 5, respectively. In particular embodiments of the invention, these nucleic acid molecules are mutated to comprise a proteolytic protection site to protect the pesticidal polypeptide from proteolytic degradation or inactivation by a plant protease. The term "pesticidally effective amount" means an amount of a substance or organism having pesticidal activity when it occurs in the environment of a pest. For each substance or organism, the pesticidally effective amount is determined empirically for each affected pest in a specific environment. Similarly, an "insectidically effective amount" can be used to refer to a "pesticidally effective amount" when the pest is an insect pest. As used herein, the term "recombinantly designed" or "designed" means the use of recombinant DNA technology to introduce (eg, design) a change in the protein structure based on the understanding of the protein-mechanism of action and a consideration of the amino acids that are introduced, deleted or substituted. As used herein the term "mutant nucleotide sequences" or "mutation" or "utagenized nucleotide sequence" means a sequence of nucleotides that have been mutagenized or altered to contain one or more nucleotide residues (e.g. base) that is not present in the corresponding non-mutagenized wild-type sequence. Such mutagenesis or alteration consists of one or more additions, deletions or substitutions or replacements of nucleic acid residues. When mutations are made, for example, a pesticidal polypeptide by adding, removing or replacing an amino acid from a proteolytic site, such addition, removal or replacement may be within or adjacent to a portion of the proteolytic site, while the subject of the mutation is has performed (that is, as long as the on-site proteolysis is changed). By "mimicking" or "mutation" in the context of a protein, a polypeptide or amino acid sequence that has been mutagenized or altered to contain one or more amino acid residues that is not present in the corresponding non-mutagenized wild type sequence is proposed. . Such mutagenesis or alteration consists of one or more additions, deletions or substitutions or replacements of amino acid residues. Thus, by "mutant" or "mutation" it can be proposed that either or both of the nucleotide sequences and the encoded amino acids are mutated. In some embodiments, the mutant nucleotide sequences are placed in a background sequence previously known in the art, such as CrydBbl, to confer increased resistance to a plant protease in the encoded polypeptide. The mutants can be used alone or in any combination compatible with other mutants of the invention or with other mutants. Where more than one mutation is added to a particular nucleic acid or protein, mutations can be added sequentially at the same time; if it is sequential, the mutations can be added in any suitable order. Thus, a sequence of the invention can be a mutagenized nucleotide sequence, or an optimized nucleotide sequence, or a sequence of the invention can be both mutagenized and optimized. As used herein the term "improved insecticidal activity" or "improved pesticidal activity" characterizes a polypeptide or encoded polypeptide endotoxin of the invention having improved Coleopteran pesticidal activity relative to the activity of its corresponding wild-type protein, and / or an endotoxin that is effective against a wider range of insects, and / or an endotoxin having specificity for an insect that is not susceptible to the toxicity of the wild-type protein. A discovery of improved or increased pesticidal activity requires a demonstration of an increase in toxicity of at least 10%, against the target of the insect, more preferably 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 60%, 70%, 100%, 200%, or greater increase in toxicity in relation to the insecticidal activity of the wild-type endotoxin determined against the same insect. For example, an improved pesticidal or insecticidal activity is provided where a wider or smaller range of insects is impacted by the polypeptide relative to the range of insects that is affected by a pesticidal protein such as wild-type Bt toxin. A wider range of impact may be desirable where versatility is desired, whereas a narrower range of impact may be desirable where, for example, beneficial insects could otherwise be impacted by use in the presence of the toxin. While the invention is not related by any particular mechanism of action, an improved pesticidal activity can also be provided by changes in one or more characteristics of a polypeptide; for example, the stability or longevity of a polypeptide in a plant can be increased in relation to the stability or longevity of a corresponding mutagenized wild-type protein. By "proteolytic site" or "cleavage site", an amino acid sequence is proposed that confers sensitivity to a particular class of proteases or protease such that a polypeptide containing the amino acid sequence is digested by the particular class of proteases or proteases. . A proteolytic site is said to be "sensitive" to the protease (s) that recognize that site. It is recognized that the efficiency of digestion will vary, and that a decrease in the efficiency of digestion can lead to an increase in the stability or longevity of the polypeptide in an insect gut. Thus, a proteolytic site may confer sensitivity to more than one protease or class of proteases, but the efficiency of digestion at that site by several proteases may vary. Proteolytic sites include, for example, trypsin sites, chymotrypsin sites, papain sites, cathepsin sites and cathepsin-like sites. Proteolytic sites for particular proteases often comprise "portions" or sequence patterns, which are known to confer sensitivity to a particular protease. Thus, for example, cathepsin site portions include FRR, a cathepsin L protease cleavage site; RR, a site of trypsin and cathepsin B cleavage; LKM, a chymotrypsin and FF site, a cathepsin D site. A putative proteolytic site is a sequence that comprises a portion or comprises a sequence similar to a portion but that has not been shown to be subjected to digestion by the corresponding protease . Plants express a variety of proteases, including cysteine and serine proteases. The invention provides nucleic acid molecules, and variants and fragments thereof, which encode novel plant proteases. Specifically, the invention provides nucleic acid molecules encoding a novel cathepsin B-like protease (SEQ ID NO: 135) and a novel cysteine protease with mir2 protease homology (SEQ ID NO: 137). The nucleotide sequences set forth in SEQ ID NOS: 135 and 137 encode the polypeptide (i.e., protease) sequences of SEQ ID NOs: 136 and 138, respectively. The invention further comprises variants and fragments of those polypeptide sequences that possess proteolytic activity. Assays for measuring proteolytic activity are well known in the art. The novel plant proteases of the invention find use, for example, in identification of the preferred proteolytic cleavage site (s) for these proteases. In another embodiment, plant proteases are used to identify proteolytic cleavage sites within the polypeptide of pesticides, such as CrydBbl and CrydBcl, which are susceptible to these proteases. Knowledge about the preferred proteolytic sites for the plant proteases of the invention can lead to improvements in the stability of pesticidal proteins expressed in transgenic plants. It is recognized that pesticidal polypeptides expressed in a plant may be susceptible to segmentation by plant proteases. Segmentation of an active pesticide polypeptide by a plant protease can lead to proteolytic inactivation of the toxin. In one embodiment, a pesticidal polypeptide is designed to replace a proteolytic site that is sensitive to segmentation by a plant protease with a proteolytic protection site. The replacement of a proteolytic site sensitive to segmentation by a plant protease with a proteolytic protection site protects the toxin from proteolytic inactivation in the plant. Removal of protease sensitive sites can prt the pesticide polypeptide from degradation or rapid inactivation in the plant, allowing the toxin to reach its target intact and more quickly reach an insecticidal dose within the insect pest. In one embodiment, the proteolytic protection site is designed to be insensitive to cleavage by a protease similar to cathepsin B of the invention, ie, the polypeptide of SEQ ID NO: 136 or a variant or fragment thereof. In another embodiment, the proteolytic protection site is designed to be insensitive to cleavage by the protease set forth in the polypeptide sequence of SEQ ID NO: 138 or a variant or fragment thereof. In some embodiments, the pesticidal polypeptide is CrydBbl or CrydBcl. It is well known that naturally occurring Cry toxins are synthesized by the sporulating cells of B. thuringiensis as a crystalline proteinase inclusion protoxin. When ingested by the susceptible insect larvae, the microcrystals dissolve in the midgut, and the protoxin is transformed to a biologically active portion by the proteases characteristic of the digestive enzymes located in the insect's intestine. Activated toxin binds with high affinity to protein receptors in brush border membrane vesicles. The epithelial cells lining the midgut are the primary target of the toxin and are rapidly destroyed with a consequence of the perforation of the membrane resulting from the formation of the selective channels of cation, gateway by the toxin. Mutations of the invention include mutations that protect the pesticidal polypeptide from degradation by plant protease, for example, removing putative proteolytic sites such as putative serine protease sites * and cathepsin recognition sites from different areas of the endotoxin Some or all of such putative sites can be removed or altered so that proteolysis at the location of the original site is diminished. Changes in proteolysis can be estimated by comparing a mutant pesticide polypeptide with the non-mutagenized toxin or by comparing mutant endotoxins that differ in their amino acid sequence. Putative proteolytic sites include, but ho are limited to the following sequences: FRR, a cathepsin L protease cleavage site; RR, a site of trypsin and cathepsin B cleavage; LKM, a chymotrypsin site; and FF, as a site of cathepsin D. These sites can be altered by the addition or deletion of any number of either amino acid residues, while achieving the object of the invention, ie, altering the sensitivity of the pesticide protein to a plant protease. Of particular interest are the optimized nucleotide sequences that encode the pesticidal proteins of the invention. As used herein, the phrase "optimized nucleotide sequences" refers to nucleic acids that are optimized for expression in a particular organism, for example, a plant. The optimized nucleotide sequences include those sequences that have been modified such that the GC content of the nucleotide sequence has been altered. Such a nucleotide sequence may or may not comprise a coding region. Where the nucleotide sequence comprises a coding region, alterations of the GC content can be made in view of other genetic phenomena, such as, for example, the codon preference of a particular organism or a trend of GC content within a coding region. (See particularly Examples 14, 15 and 16 hereinbelow). In some embodiments, where the nucleotide sequence to be optimized comprises a coding region, the alteration in the GC content does not result in a change in the protein encoded by the nucleotide sequence. In other embodiments, the alteration in GC content results in changes to the encoded protein which are conservative amino acid changes and / or which do not materially alter the function of the encoded protein. The GC content of an optimized nucleotide sequence may differ from the first or the native nucleotide sequence by as little as 1%, 2%, 3%, 4%, 5%, 6%, 7%, d%, 9 %, 10% or 11%, 12%, 13%, 14%, 15%, 16%, 1 / -5, 1 or -g, ly-g, 20-5, you 1 -6, 2. - Í1, 23-s, 24-s, 25-s, 26-6, 27-s, 2d-6, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 36%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 46%, 49% or 50% or greater. Thus, the GC content of an optimized nucleotide sequence can be 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 66%, 69%, 70% , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80% or higher. The term "optimized nucleotide sequences" also comprises sequences in which the GC content has been altered and, in addition, other changes have been made in the nucleotide sequence. Such changes are often made to increase the properties of the sequence, such as its versatility in genetic engineering (for example, by adding or removing restriction enzyme recognition sites) and any other property that may be desirable to generate a transgenic organism. , such as the longevity of the mRNA increased in the cell. (See Examples 14, 15 and 16 immediately below). By "derivative of" it is proposed that a sequence is substantially similar to another sequence. Generally, sequences derived from a particular nucleotide sequence will have at least about 40%, 50%,
60%, 65%, 70%, 75%, 80%, 65%, 86%, 67%, 68%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the particular nucleotide sequence as determined by the sequence alignment programs described elsewhere in the present using error parameters. Sequences derived from a particular nucleotide sequence may differ from those sequence by as few as 1-15 nucleotides, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2 or 1 nucleotide Sequences derived from a particular nucleotide sequence can also cross-hybridize to that sequence. The optimized nucleotide sequences can be prepared from any organism of interest using methods known in the art. For example, a nucleotide sequence comprising preferred corn codons can be prepared by reverse translating an amino acid sequence of the invention to comprise preferred codons of corn as described by Murray et al., (1989) Nucleic. Acids Res. 17: 477-498. The optimized nucleotide sequence is in use by increasing the expression of a pesticidal protein in a plant, for example monocotyledonous plants of the Gramineae family (Poaceae) such as, for example, a corn or cereal plant. The invention further provides isolated pesticidal polypeptides (e.g., insecticide) encoded by a modified (e.g., mutagenized, truncated and / or optimized) nucleotide acid of the invention. Fragments and variants of the novel plant pesticide and protease polypeptides of the invention are also encompassed by the present invention. In particular embodiments, the pesticidal proteins of the invention provide full-length pesticidal proteins, fragments of full-length toxins, and variant polypeptides that are produced from mutagenized nucleic acids designed to introduce particular amino acid sequences into the polypeptides of the invention. In particular embodiments, the amino acid sequences that are introduced into the polypeptides comprise a sequence that protects the pesticidal protein in cleavage by a plant protease. One skilled in the art will appreciate that fragments of the disclosed pesticidal proteins and plant proteases are also encompassed by the present invention. By "fragment" a portion of the amino acid sequence of the exemplary proteins disclosed herein is proposed. Fragments of a pesticidal protein may retain the pesticidal activity of the full length protein or may have altered or improved pesticidal activity compared to the full length protein. Likewise, fragments of a plant protease of the invention can retain the proteolytic activity of the full-length protein or can have altered or improved proteolytic activity compared to the full-length protein. Thus, fragments of a protein can vary from at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 210, 220, 230, 240, 250, 260, 270, 2d0, 290, 300 320, 340, 360, 3d0, 400, 420, 440, 460, 460, 500, 520, 540 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780 800, 820, 840, 860, 880, 900, 920, 940, 960, 960 or 1000 or up to the full length sequence of the protein. A biologically active portion, fragment or truncated version of a pesticidal protein or plant protease can be prepared by isolating a portion of one of the nucleotide sequences of the invention, by expressing the encoded portion of the pesticidal protein or plant protease (per example, by means of the recombinant expression in vi tro) and when estimating the activity of the portion of the pesticide protein or plant protease.
In some cases, the mutants disclosed herein were cloned into the pET expression system, expressed in E. coli and tested for pesticidal activity against exemplary insect pests such as southern corn rootworm (SCRW), western corn rootworm (WCRW), Colorado potato beetle (CPB) , for example, Leptinotarsa decemlineata) and cotton weevil (for example, Anthonomus grandis). It is to be understood that the polypeptides of the invention can be produced either by expression of a nucleic acid disclosed herein, or by the use of standard molecular biology techniques. For example, a truncated protein of the invention can be produced by expressing a recombinant nucleic acid of the invention in an appropriate host cell, or alternatively by a combination of ex vivo procedures, such as protease digestion and purification of a 'purified wild-type protein. It is recognized that pesticidal proteins can be oligomeric and will vary in molecular weight, number of residues, component peptides, activity against particular pests and other characteristics. However, by the methods set forth herein, proteins active against a variety of pests can be isolated and characterized. The pesticidal proteins of the invention can be used in combination with Bt endotoxins or other insecticidal proteins to increase the target insect range. In addition, the use of the pesticidal proteins of the present invention in combination with Bt d-endotoxins or other insecticidal principles of a different nature has particular utility for the prevention and / or management of insect resistance. Other insecticidal principles include, but are not limited to, protease inhibitors
(both types of serine and cysteine), lectins, α-amylase and peroxidase. Fragments and variants of the nucleotide and amino acid sequence and the pesticidal and protease polypeptides of plants encoded in this manner are also encompassed by the present invention. As used herein the term "fragment" refers to a portion of a nucleotide sequence of a polynucleotide or a portion of an amino acid sequence of a polypeptide of the invention. Fragments of a nucleotide sequence can encode protein fragments that retain the biological activity of the corresponding native or full-length protein. Accordingly, fragments of the nucleic acid molecule of the invention can encode protein fragments that possess proteolytic pesticidal activity. Thus, it is recognized that some of the polynucleotides or amino acid sequences of the invention can be correctly referred to as either fragments or variants. This is particularly true of truncated sequences that are biologically active. It is to be understood that the term "fragment", as used to refer to nucleic acid sequences of the invention, also comprises sequences that are useful as hybridization probes. This class of nucleotide sequences generally does not encode fragment proteins that retain biological activity. Thus, fragments of a nucleotide sequence may vary from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention. A fragment of a nucleotide sequence encoding a biologically active portion of a pesticidal protein of a plant protease of the invention will encode at least 15, 25, 30, 50, 100, 200, 300, 400, 500, 600, 700 , 800, 900, 1,000, 1,100 or 1,200 contiguous amino acids or up to the total number of amino acids present in a polypeptide of the invention. Fragments of a nucleotide sequence that are useful as hybridization probes or PCR primers do not generally need to encode a biologically active portion of a protein. Thus, a fragment of, for example, a nucleic acid similar to Cry8 pentin-1 can encode a biologically active portion of a pesticidal protein, or it can be a fragment that can be used as a hybridization probe or PCR primer using disclosed methods right away. A biologically active portion of a pesticidal protein or plant protease of the invention can be prepared by isolating a portion of one of the nucleotide sequences of the invention, by expressing the encoded portion of the protein (eg, by recombinant expression in vitro). vi tro) and when estimating the activity of the encoded portion of the pesticide protein or plant protease. Nucleic acids are fragments of a nucleotide sequence of the invention comprising at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 1,000 , 1,200, 1,400, 1,600, 1,800, 2,000, 2,200, 2,400, 2,600, 2,800, 3,000, 3200, 3,400 or 3,600 nucleotides, or up to the number of nucleotides present in a nucleotide sequence disclosed herein. By "variants", substantially similar sequences are proposed. For nucleotide sequences, conservative variants include those sequences which, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the pesticidal polypeptides in plant proteases of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, such as, for example, the polymerase chain reaction (PCR) and the hybridization techniques as with those consumed immediately. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but still encoding a pesticidal protein or plant protease of the invention. Generally, variants of a particular nucleotide sequence of the invention will have at least about 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88% 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by the sequence alignment programs described elsewhere herein using error parameters. A variant of a nucleotide sequence of the invention may differ from that sequence by as few as 1-15 nucleotides, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2 or 1 nucleotide. Variants of a particular nucleotide sequence of the invention can also be evaluated by comparing the percent sequence identity between the polypeptide encoded by a variant nucleotide sequence and the polypeptide encoded by the reference nucleotide sequence. The percent sequence identity between any two polypeptides can be calculated using sequence alignment programs described elsewhere herein using error parameters. Where any given pair of polynucleotides of the invention is evaluated by comparing the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40% , 45%, 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, preferably at least about 90%, 91%, 92%, 93% , 94%, 95%, '96, 97% and more preferably at least about 98%, 99% or more sequence identity. As used herein, the term "variant protein" comprises polypeptides that are derived from a native protein by: deletion (termed truncation) or addition of one or more amino acids to the N-terminal and / or C-terminal end of the protein native; the deletion or addition of one or more amino acids in one or more sites in the native protein or the substitution of one or more amino acids in one or more sites in the native protein. Accordingly, the term variant protein comprises biologically active fragments of a native protein comprising a sufficient number of contiguous amino acid residues to retain the biological activity of the native protein, i.e., the pesticidal or proteolytic activity. Such activity may be different or improved relative to the native protein or this may be unchanged, as long as the biological activity is stopped. The variant proteins comprised by the present invention are biologically active, that is, they continue to exhibit the desired biological activity of the native protein, that is, the pesticidal or proteolytic activity as described herein. Such variants may result from, for example, genetic polymorphism or human manipulation. Biologically active variants of a pesticidal protein or plant protease of the invention will have at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99% or more of sequence identity to the amino acid sequence for the native protein is not determined by the sequence alignment programs described in this part herein using error parameters. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4.3. , 2 or 1 amino acid residue. The invention further comprises a microorganism that is transformed with at least one nucleic acid of the invention, with an expression cassette comprising the nucleic acid, or with a vector comprising the expression cassette. Preferably, the microorganism is one that multiplies in plants. More preferably, the microorganism is a bacterium that colonizes the root. One embodiment of the invention relates to an encapsulated pesticidal protein, comprising a transformed microorganism comprising at least one pesticidal protein of the invention. The invention provides pesticidal compositions comprising a transformed organism of the invention. Preferably the transformed microorganism is present in the pesticidal composition in a pesticidally effective amount, together with a suitable carrier. The invention also comprises pesticidal compositions comprising an isolated protein of the invention, alone or in combination with a transformed organism of the invention and / or an encapsulated pesticidal protein of the invention, in an insecticidally effective amount, together with a suitable carrier. The invention further provides a method for increasing the target range of the insect by using a pesticidal protein of the invention in combination with at least one second pesticidal protein that is different from the pesticidal protein of the invention. Any pesticidal protein known in the art can be employed in the methods of the present invention. Such pesticidal proteins include, but are not limited to, Bt d-endotoxins, protease inhibitors, lectins, α-amylases, lipid acyl hydrolases and peroxidases. The invention also comprises transformed or transgenic plants comprising at least one nucleotide sequence of the invention. Preferably . the plant is stably transformed with a nucleotide construct comprising at least one nucleotide sequence of the invention operably linked to a promoter that induces expression in a plant cell. As used herein, the terms "transformed plant" and "transgenic plant" refer to a plant that comprises within its genome a heterologous polynucleotide. Usually, the heterologous polynucleotide is stably integrated into the genome of a transgenic plant or transformed such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide can be integrated into the genome alone or as part of a cassette of recombinant expression. It is to be understood that as used herein the term "transgenic" includes any cell, cell line, callus, tissue, part of plant or plant, the genotype of which has been altered by the presence of the heterologous nucleic acid including those transgenics initially altered in this way, as well as those created by sexual crossings or asexual propagation of the initial transgenic. The term "transgenic" as used herein does not encompass alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation. As used herein, the term "plant" includes reference to whole plants, plant organs
(for example, leaves, stems, roots, etc.), seeds, plant cells and progeny thereof. The parts of transgenic plants are to be understood within the scope of the invention comprising, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, ovules, stems, fruits, leaves, roots originating in plants Transgenic cells or their progeny previously transformed with a DNA molecule of the invention and which therefore consists at least in part of transgenic cells, are also an object of the present invention.
As used herein the term "plant cell" includes, without limitation, seed suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. The class of plants that can be used in the methods of the invention is generally as broad as the class of higher plants available for transformation techniques, including both monocotyledonous and dicotyledonous plants. Such plants include, for example, Solanum tuberosum and Zea mays. While the invention does not depend on a particular biological mechanism to increase the resistance of a plant to a plant pest, the expression of the nucleotide sequence of the invention in a plant can result in the production of the pesticidal proteins of the plant. invention and an increase in the resistance of the plant to a plant pest. In some embodiments, the pesticidal polypeptides are designed to possess increased resistance to proteolytic degradation or inactivation by a plant protease. The plants of the invention find use in agriculture in methods to protect plants from pests. In certain embodiments of the invention they provide transformed crop plants, such as, for example, corn plants, which find use in methods for impacting insect pests, such as, for example, western, northern, southern corn rootworms. and Mexican. Other embodiments of the invention provide for transformed potato plants, which find use in methods to impact the Colorado potato beetle, transformed cotton plants, which find use in methods to impact the cotton weevil, and transformed grass grasses, which find use in methods to impact the boll weevil of the bluish grass, Sphenophorous parvulus. One skilled in the art will readily recognize that advances in the fields of molecular biology such as site-specific mutagenesis, polymerase chain reaction methodologies, and protein engineering techniques provide extensive collection of suitable protocol tools. for use to alter or design both the amino acid sequence and the intrinsic genetic sequences of proteins of agricultural interest. Thus, the pesticidal proteins and plant proteases of the invention can be altered in various ways including substitutions, deletions, truncations and amino acid insertions. Methods for such manipulations are generally known in the art. For example, the amino acid sequence variant of the pesticidal proteins and plant proteases can be prepared by introducing mutations into a synthetic nucleic acid (e.g., DNA molecule). Methods for mutagenesis and nucleic acid alterations are well known in the art. For example, the designed changes can be introduced using a site-directed mutagenesis technique, mediated by oligonucleotide. See, for example, Kunkel (1985) Proc. Nati Acad. Sci. USES
82: 4dd-492; Kunkel et al., (1987) Methods in Enzymol
154: 367-382; U.S. Patent No. 4,873,192; Walker and
Gaastra, eds. (1983) Techniques in Molecular Biology
(MacMillan Publishing Company, New York) and the references cited therein. The mutagenized nucleotide sequences of the invention can be modified so as to change about 1, 2, 3, 4, 5, 6, 8, 10, 12 or more of the amino acids present in the primary sequence of the encoded polypeptide. Alternatively even more changes of the native sequence can be introduced such that the encoded protein can have at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9% , 10%, 11%, 12% or even approximately 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, 21%, 22%, 23%, 24% or 25% , 30%, 35% or 40% or more of the codons altered, or otherwise modified compared to the corresponding wild-type protein. In the same way, the encoded protein can have at least about 1% or 2% or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12 % or even approximately 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, 21%, 22%, 23%, 24% or 25%, 30%, 35% or 40 % or more additional codons compared to the corresponding wild-type protein. It should be understood that the mutagenized nucleotide sequences of the present invention are proposed to comprise biologically functional peptides, equivalents having biological activity, for example pesticidal or proteolytic activity. Such sequences may arise as a consequence of the codon redundancy and functional equivalence that is known to occur naturally within the nucleic acid sequences and the proteins thus encoded. One skilled in the art would recognize that amino acid additions and / or substitutions are generally based on the relative similarity of amino acid side chain substituents, eg, their hydrophobicity, charge, size and the like. Exemplary substitutions that take several of the above characteristics into consideration are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine and valine, leucine and isoleucine. The guide as to "substitutions of appropriate amino acids that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Nat. Biomed.- Res. Found., Washington, DC), incorporated herein by reference.Conservative substitutions, such as the exchange of an amino acid with another having similar properties, can be made.Such, the genes and nucleotide sequences of the invention include both naturally occurring sequences as well as mutant forms.Similarly, the proteins of the invention comprise naturally occurring proteins as well as variations (e.g., truncated polypeptides) and modified forms (e.g., mutants) thereof. presenting the desired biological activity Obviously, the mutations that will be made in the DNA encoding the variant should not place the sequence outside the reading structure and preferably will not create complementary regions that could produce the secondary mRNA structure. See, patent application publication EP No. 75,444. The deletions, insertions and substitutions of the protein sequences comprised herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution as suppression or insertion in advance, one skilled in the art will appreciate that the effect will be evaluated by routine sorting tests, such as insect feeding tests.
See, for example, Marrone et al., (1985) J. Econ. Entomol 78: 290-293 and Czapla and Lang (1990) J. Econ. Entomol 83: 2460-2485, incorporated herein by reference. Variant nucleotide sequences and proteins also comprise sequences and proteins derived from a mutagenic and recombinogenic process such as DNA intermixing. With such a procedure, one or more different coding sequences can be manipulated to create a new pesticidal protein having the desired properties. In this manner, the libraries of recombinant polynucleotides are generated from a population of polynucleotides of related sequences comprising regions of sequence having substantial sequence identity and can be homologously combined in vitro or in vivo. For example, using this method, full length coding sequences, sequence portions coding for a domain of interest, or any segment of nucleotide sequences of the invention can be intermixed among the nucleotide sequences encoding the pesticidal or protease proteins. of plant of the invention and corresponding portions of other known nucleotide sequences that encode plant proteins or proteases to obtain a new gene coding for a protein with, for example, pesticidal or proteolytic activity.
The invention is not related by any particular intermixing strategy, only that at least one nucleotide sequence of the invention, or part thereof, is involved in such an intermixing strategy. The intermixing may involve only nucleotide sequences disclosed herein or may additionally involve the intermixing of some other nucleotide sequences known in the art including, but not limited to, Genbank Access Nos. U04364, U04365 and 04366. Strategies for intermixing of DNA are known in the art. See, for example, Stemmer (1994) Proc. Nati Acad. Sci. USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al., (1997) Nature Biotech. 15: 436-438; Moore et al., (1997) JX Mol. Biol. 272: 336-347; Zhang et al., (1997) Proc. Nati Acad. Sci. USA 94: 4504-4509; Crameri et al., (1998) Nature 391: 288-291 and U.S. Patent Nos. 5,605,793 and 5,837,458. The nucleotide sequences of the invention can also be used to isolate corresponding sequences from other organisms, particularly other bacteria, and more particularly other Bacillus strains. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. For example, isolated sequences based on their sequence identity to a Cry8-like sequence or insect gut plant protease sequence set forth herein or to fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologous to the disclosed sequences. By "orthologs", genes derived from a common ancestral gene are proposed and found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and / or their encoded protein sequences share substantial identity as defined elsewhere herein. The functions of orthologs are often highly conserved among species. In a PCR procedure, the oligonucleotide primers can be designed for use in PCR reactions to amplify the corresponding DNA sequences from a genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York) , then in the present Sambrook. See also Innis et al., Eds. (1990) PCR Protocols: A Guide to Methods and Applications Academic Press, New York) and; Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using peer primers, spliced primers, individual specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers and the like. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (ie, genomic or library libraries). cDNA) from a selected organism. Hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides and can be labeled with a detectable group such as 32P, or any other detectable label. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the insect intestine protease sequences of the invention. Methods for the preparation of probes for hybridization and for the construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook. For example, the complete Cry8-like sequence disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing to. corresponding corresponding Cry8 sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among the Cry8-like sequences and are at least about 10 nucleotides in length or at least about 20 nucleotides in length. Such probes can be used to amplify corresponding Cry8-like sequences of a selected organism, by PCR. This technique can be used to isolate additional coding sequences from a desired insect pest or as a diagnostic assay to determine the presence of coding sequences in an insect pest. Hybridization techniques include the hybridization classification of D? A libraries placed on plates (either plates or colonies; see, for example, Sambrook). Hybridization of such sequences can be carried out under severe conditions. "Severe conditions" or "severe hybridization conditions" are proposed to mean conditions under which a probe will hybridize its target sequence to a detectably greater extent than other sequences (eg, at least 2-fold over the background) ). Severe conditions are dependent on the sequences and will be different in different circumstances. By controlling the severity of the hybridization and / or washing conditions, the target sequences that are
100% complementary to the probe can be identified
(homologous sounding). Alternatively, severity conditions can be adjusted to allow some sequence mismatches so that lower degrees of similarity are detected (heterologous polling). Generally, a probe is less than about 1000 nucleotides in length or less than 500 nucleotides in length. Typically, severe conditions will be those in which the salt concentration is less than about 1.5 M NA ion, typically approximately 0.1 to 1.0 M Na (or other salts) concentration at pH 7.0 to 8.3 and the temperature is at least about 30 ° C for short probes (for example, 10 to 50 nucleotides) and at least about 60 ° C for long probes (for example, greater than 50 nucleotides). Severe conditions can also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C and a wash at IX to 2X SSC (20X SSC = 3.0 M NaCl / trisodium citrate 3 M) at 50 to 55 ° C. Exemplary moderate severity conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37 ° C, and a 0.5X to IX SSC wash at 55 to 60 ° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 ° C for at least 4 hours, more preferably up to 12 hours or longer and a final wash in 0.1 X SSC at 60 to 65 ° C for at least 20 minutes. The duration of the hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The specificity is typically the function of the post-hybridization washes, the factors. critical which are the ionic strength and the temperature of the final wash solution. For DNA-DNA hybrids, the Tra (thermal melting point) can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 136: 267-284: Tm = 81.5 ° C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) -500 / L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in DNA,% of form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (defined low ionic strength and pH) in which 50% of a complementary hybrid target sequence is a perfectly matched probe. Tm is reduced by approximately 1 ° C for every 1% of unequalization; thus, Tm, hybridization and / or wash conditions can be adjusted to hybridize the sequences of the desired identity. For example, if sequences with 90% identity are searched, the Tm can be decreased to 10 ° C. Generally, severe conditions are selected to be about 5 ° C lower than the Tm for the specific sequence is a complement at a defined ionic strength and pH. However, severely severe conditions may utilize hybridization and / or washing at 1, 2, 3 or 4 ° C less than Tm; moderately severe conditions can use hybridization and / or washing at 6, 7, 8, 9 or 10 ° C lower than Tm; the conditions of low severity can use a hybridization and / or washing at 11, 12, 13, 14, 15 or 20 ° C lower than the Tm. Using the equation, the hybridization and washing compositions, and the desired Tm, those of ordinary skill will understand that variations in the hybridization severity and / or washing solutions are inherently described. If the desired degree of unequalization results in a Tm of less than 45 ° C
(aqueous solution) or 32 ° C (formamide solution), it is preferred to increase the SSC concentration so that it can be used at a higher temperature. An extensive guide for nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part. I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See also Sambrook. Thus, for example, isolated sequences encoding a Cry8-like protein of the invention and hybridizing under severe conditions to Cry8-like sequences disclosed herein, or to fragments thereof, are encompassed by the present invention. Similarly, isolated sequences encoding a plant protease of the invention and hybridizing under severe conditions to the plant protease sequences disclosed herein, or to fragments thereof, are also encompassed by the present invention. The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) ) "percentage of sequence identity" and (e) "substantial identity". (a) As used herein, "reference sequence" is a defined sequence used as a basis for the sequence comparison. A reference sequence can be a subset in the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. (b) As used in the present "comparison window" it refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window can comprise additions and deletions (ie, spaces) compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences. Generally, the comparison window is at least 20 nucleotides contiguous in length, and optionally may be 30, 40, 50, 100 or longer. Those skilled in the art understand that to avoid high similarity of a reference sequence due to the inclusion of spaces in the polynucleotide sequence a space sanction is typically introduced and is subtracted from the number of matches. Methods of sequence alignment for comparison are well known in the art. Thus, the determination of the percent sequence identity between any of the two sequences can be performed using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the global alignment algorithm of Needleman and Wunsch (1970) JX Mol .. Biol. 48: 443-453; the local search alignment method of Pearson and Lip an (1988) Proc. Nati Acad. 85: 2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Nati Acad. USA 872264, as modified by Karlin and Altschul (1993) Proc. Nati USA 90: 5873-5d77. Computer implementations of these mathematical algorithms can be used for sequence comparison to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics GCG Software Package, Version 10
(available from Accelrys Inc., 9685 Scranton Road, San Diego,
California, USA). The registration matrix used in the
Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Nati, Acad. Sci. USA 89: 10915). Alignments using these programs can be performed using the error parameters. The CLUSTAL program is well known by Higgins (1988) Gene 73: 237-244 (1986); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1986) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331. The 7ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A weighted residue table PAM120, a space length penalty of 12 and a space penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST program by Altschul et al. (1990) JX Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, log = 100, word length = 12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed in the TLASTX program, register = 50, word length = 3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain spaced alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When using BLAST, Gapped BLAST and PSI-BLAST, the error parameters of the respective programs (for example, BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See www.ncbi.nlm.nih.gov. The alignment can also be done manually by inspection. Unless stated otherwise, the sequence identity / similarity values provided herein refer to the value obtained using GAP with error parameters, or any equivalent program. By "equivalent program" it is proposed to imply any sequence comparison program that, for either of two sequences in question, generates an alignment that has identical nucleotide or amino acid residue matches and an identical sequence identity percent when compared with the corresponding alignment generated by the preferred program. GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48-443-453, to find the alignment of two complete sequences that maximizes the number of equalizations and minimizes the number of spaces. GAP considers all possible alignments and space positions and creates the alignment with the largest number of equalized bases and the smallest spaces. This allows the provision of a space creation sanction and a space extension penalty in units of equal bases. GAP must make use of the space creation sanction number of the matches for each space it inserts. If a space extension penalty greater than zero is selected, GAP must also make use of each space inserted in the length of space times of the space extension penalty. The error space creation sanction values and Version 10 space extension sanction settings of the Wisconsin Genetics GCG Software package for protein sequences are 8 and 2, respectively. For nucleotide sequences the penalty space creation penalty is 50 while "the error space extension penalty is 3. The space extension space creation penalties may be expressed as a whole number selected from the group of whole numbers consisting of 0 to 200Thus, for example, the penalties of creating space extension space can be 0, 1, 2, 3, 4, 5, 6, 7, d, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or larger. For purposes of the present invention, comparison of the nucleotide or protein sequences for the determination of percent sequence identity to the Cry8-like sequences disclosed herein is preferably made using the GAP program in the software package of Wisconsin Genetics (Version 10 or later) or any equivalent program. For GAP analysis of nucleotide sequences, we used a GAP Weight of 50 and a Length of 3. (c) As used herein, "sequence identity" or "identity" in the context of two sequences of Nucleic or polypeptide acid refers to the residues in the two sequences that are the same when they are aligned for maximum correspondence over a specified comparison window. When the percentage of sequence identity is used in reference to proteins it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where the amino acid residues are replaced by other amino acid residues with similar chemical properties ( example, loading or hydrophobicity) and therefore do not change the functional properties of the molecule. When the sequences differ in conservative substitutions, the percent sequence identity can be adjusted upward to correct the conservative nature of the substitution. Sequences that differ with such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those skilled in the art. Typically, this involves registering a conservative substitution as a partial unequalization before it completes, in order to increase the percentage of sequence identity. Thus, for example, where an identical amino acid is given a record of 1 and a non-conservative substitution is given a record of zero, a conservative substitution is given a record between zero and 1. The record of conservative substitutions is calculated, for example, how it is implemented in the PC / GENE program (Intelligenetics, Mountain View, California). (d) As used herein, "percent sequence identity" means the value determined by comparing two optimally aligned sequences on a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (ie, spaces) as compared to the reference sequence (which does not comprise additions or deletions) for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to produce the number of equalized positions, by dividing the number of equalized positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. (e) (i) The term "substantial identity" of the polynucleotide sequences means that a polynucleotide comprises a sequence having at least 70% sequence identity, preferably at least 60%, more preferably at least 90%, and much more preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One skilled in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of the proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, positioning of the reading structure, and the like. The substantial identity of the amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90% and much more preferably at least 95%. Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under severe conditions. Generally, severe conditions are selected to be about 5 ° C lower than the Tm for the specific sequence at a defined ionic strength and pH. However, severe conditions comprise temperatures in the range of about 1 ° C to about 20 ° C, depending on the desired degree of severity as otherwise qualified herein. Nucleic acids that do not hybridize to each other under severe conditions are still substantially identical if the polypeptides they encode are substantially identical. This can occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy allowed by the genetic code. An indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. (e) (ii) The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85 %, much more preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, the optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch
(1970) JX Mol. Biol. 48: 443-453. An indication that two peptide sequences are substantially identical is that a peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides that are "substantially similar" share sequences as mentioned above except that positions of the residue that are not identical may differ by conservative amino acid changes. The use of the term "nucleotide constructs" herein is not intended to limit the present invention to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be employed in the methods disclosed herein. The nucleotide, nucleic acid and nucleotide sequence constructions of the invention additionally comprise all complementary forms of such constructs as sequence molecules. In addition, the nucleotide constructs, nucleotide molecules and nucleotide sequences of the present invention comprise all nucleotide, molecule and sequence constructions that can be employed in the methods of the present invention to transform plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs. The nucleotide, nucleic acid and nucleotide sequence constructs of the invention also comprise all forms of nucleotide constructions including, but not limited to, single-stranded forms, double-stranded forms, hairpins, trunk and spiral structures, and Similar. A further embodiment of the invention relates to a transformed organism such as an organism selected from the group consisting of plant cells and insects, bacteria, yeast, baculoviruses, protozoa, nematodes and algae. The transformed organism comprises: a DNA molecule of the invention, an expression cassette comprising the DNA molecule, a vector comprising an expression cassette, preferably stably incorporated into the genome of the transformed organism. The sequences of the invention are provided in expression cassettes for expression in the organism of interest, in particular, a plant. The cassette will include 5 'and 3' regulatory sequences operably linked to a sequence of the invention. By "operably linked" a functional link between a promoter and a second sequence is proposed, wherein the promoter sequence initiates and mediates the transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences that are linked are contiguous and, where necessary, they bind two protein coding regions, contiguous in the same reading structure. The cassette may additionally contain at least one additional gene that is cotransformed in the organism. Alternatively, the additional gene (s) may be provided in multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites for the insertion of the sequence that is under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. The expression cassette will include in the 5 'and 3' transcription direction: a transcription and translation initiation region, a DNA sequence of the invention, and a region of transcription termination and functional translation in the organism that serves as a host. The transcription initiation region (i.e., the promoter) may be native or analogous or foreign or heterologous to the host organism. Additionally, the promoter can be the natural sequence or alternatively a synthetic sequence. By "foreign" it is proposed that the transcription initiation region is not found in the native organism in which the transcription initiation region is introduced. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from wild-type expression, which results in an alteration in the phenotype. The termination region may be native to the transcription initiation region, may be native to the operably linked DNA sequence of interest, or may be derived from another source. Suitable termination regions are available from the A. tumefaciens Ti plasmid, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al., (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al., (1991) Genes Dev 5: 141-149; Mogen et al., (1990) Cell Plant 2: 1261-1272; Munroe et al., (1990) Gene 91: 151-158; Bailas et al., (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al., (1987) Nucleic Acid Res. 15: 9627-9639. Where appropriate, a nucleic acid can be optimized for increased expression in the host organism. Thus, where the host organism is a plant, a sequence can be optimized using preferred plant codons for enhanced expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of the use of the host's preferred codon. For example, although the nucleic acid sequences of the present invention can be expressed in both monocotyledonous and dicotyledonous plant species, the sequences can be modified to take into account the specific codon preferences and the GC content preferences of monocots or dicotyledons. since these preferences have been shown to differ (Murray et al., (1989) Nucleic Acids Res. 17: 477-498), thus, the preferred corn codon for a particular amino acid can be derived from the known gene sequences of corn . The use of the corn codon for 2d genes of maize plants is listed in Table 4 of Murray et al., Supra. Methods are available in the art to synthesize preferred plant genes. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391 and Murray et al., (1969) Nucleic Acids Res. 17: 477-498, incorporated herein by reference. In addition, from the alteration of the codons of a sequence according to the codon preference of the organism, the optimization of a sequence can include the modification of the GC content of the sequence. The GC content of the gene is a common metric of the gene structure. The content of GC can vary greatly within and between genes, and between the genes of the same or different organisms. The reasons for this variation are not definitely known, but may include factors such as the organization and function of the chromosome, methylation pressure, presence of repetitive DNA, adaptations for gene expression, and coadapted codon-anticodon deviations. The variety of organisms have gene populations that exhibit a very normal distribution of GC content, but some warm-blooded vertebrates as well as cereal plants, including corn, have a curious bimodal distribution of GC content (eg, Campbell and Gowri (1990), supra; Bernardi (1995) Annual Review of Genetics 29: 445-475; Carels and Bernardi (2000) Genectis 154: 1819-1625). The biological significance of this bimodality remains unknown, but observations concerning distributions of GC content and bimodal trends are mounted, especially with the completion of genome sequencing, for example as in humans and in 'rice' (International Human Genome Sequencing Consortium (2001) Nature 409: 660-921; Yu et al. (2002) Science 296: 79-91; Wong et al. (2002) Genome Research 12: 851-856). Corn and other cereals have distinctly bimodal gene GC content distributions not observed in other taxonomic groups such as dicotyledonous plants, animals, fungi, bacteria and archaea. Using the largest corn gene data set to date, the inventors explored differences in mRNA structure and expression between high and low GC modes. The phenomenon of bimodality is observed in nuclear-coded genes. In corn, the two modes occur in approximately 51% and 67% GC content (which can be referred to as "low (GC) mode" and "high (GC) mode"). Most corn genes are "low" and have GC content at the lower level of about 51%. Most of the GC content variation is in the coding region, particularly in the third position - codon. The content of GC in the third position of codon can reach 100% and in genes of high GC mode, C can predominate over G by a ratio of 1: 3. The GC content analysis also reveals patterns within the genes, particularly within the coding region (also called the "ORF", or Open Reading Structure). For example, if the GC content is evaluated throughout the coding region of a gene, the maize genes have a generally negative GC gradient (ie, the GC content decreases towards the 3 'end of the region). of coding). However, this gradient pattern is not present in most high GC mode genes and approximately half of low GC mode genes. In addition, the coding regions of the remaining low GC mode genes (ie, the other half) show a reversal of the negative GC gradient labeled in a positive gradient towards the end of the coding region. Another pattern of GC content observed in corn is that high GC mode genes are richer in codon-rich GC amino acids, and this variation also occurs in a gradient along the length of the coding sequence. For example, in genes of high GC mode, the amino acid deviation for alanine is larger near the beginning of the coding sequence. While the expression of the gene varies widely, the inventors have determined that the total average expression of the high and low GC mode genes is similar as revealed by the mRNA profiling of both EST and Lynx MPSS (see Brenner et al. (2000). ) Nature Biotechnology 18: 630-634; Brenner et al. (2000) PNAS 97: 1665-1670 for information on Lynx MPSS; see Simmons et al., Maize Coop? ewsletter 2002, on the World Wide Web site at Agron.Missouri.edu/mnl/77/10simmons.html for the comment on high and low GC mode gene expression). However, high GC mode genes were observed to show preferred higher tissue expression, especially in vegetative and non-core reproductive tissues, while low GC mode genes showed higher expression levels in endosperm tissues, pericarp and the nucleus Rl. Additional sequence modifications are known to increase the expression of the gene in a cell host. These include the elimination of sequences encoding spurious polyadenylation signals, signals from exon-intron splice sites, transposon-like repeats and other well-characterized sequences that may be detrimental to gene expression. Also as described herein, particularly in Examples 14, 15 and 16, the GC content in the sequence can be adjusted to average levels for a given cell host, as calculated by reference to known genes expressed in the cell host By "host cell" a cell is proposed which contains a vector and supports the replication and / or expression of the expression vector. A host organism is an organism that contains a host cell. The host cells can be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells. Preferably, the host cells are cells of monocotyledonous or dicotyledonous plants. A particularly preferred monocot host cell is a maize host cell. When possible, the sequence is modified to avoid the predicted secondary hairpin mRNA structures. The expression cassettes can additionally contain 5 'leader sequences in the construction of the expression cassette. Such guide sequences can act to increase translation. Translation guides are known in the art and include: picornavirus guides, eg, EMCV guide (5 'non-coding region of Encephalomyocarditis) (Elroy-Stein et al. (1989) Proc. Nati. Acad. Sci.
USA 86: 6126-6130); Potivirus guides, for example, TEV guidance
(Tobacco Engraving Virus) (Gallie et al. (1995)
Gene 165 (2): 233-238), MDMV guide (Corn Mosaic Virus
Dwarf) (Virology 154: 9-20), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated guide of the mRNA of alfalfa mosaic virus coating protein (AMV R? A 4) (Jobling et al. (1987) Nature 325: 622-625); Guide to Tobacco Mosaic Virus (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,? ew York), pp. 237-256); and corn chlorotic mottled virus guide (MCMV) (Lo mel et al.
(1991) Virology 81: 362-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968. Other methods known to increase translation may also be used, for example, introns and the like. In the preparation of the expression cassette, the various DNA fragments can be manipulated to provide the DNA sequences in the proper orientation and, as appropriate, in the appropriate reading structure. Towards this end, adapters or linkers can be used to join DNA fragments or other manipulations can be involved to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, for example, transitions and transversions, may be involved. A number of promoters can be used in the practice of the invention. Promoters can be selected based on the desired effect. The nucleic acids can be combined with constitutive, tissue-preferred, inducible promoters or other promoters for expression in the host organism. Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 4383d and U.S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubicuitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last and collaborators (1991) Theor, Appl. Genet, 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); ALS promoter (U.S. Patent No. 5,659,026), and the like. Other constitutive promoters include, for example, those discussed in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177, 611. Depending on the desired effect, it may be beneficial to express the gene from an inducible promoter. Of particular interest for regulating the expression of the nucleotide sequences of the present invention in plants are the promoters inducible by injury. Such injury-inducible promoters can respond to damage caused by insect feeding, and include the potato proteinase inhibitor gene (pin II) (Ryan (1990) Ann. Rev. Phytopath., 28: 425-449; Duan et al. (1996) Nature Biotechnology 14: 494-498); wunl and wun2, U.S. Patent No. 5,428,148; winl and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215: 200-208); systemin (McGurl et al. (1992) Science 225: 1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76); MPI gene (1994) Plant J. 6 (2): 141-150); and the like, incorporated herein by reference. Additionally, pathogen-inducible promoters can be employed in the methods and nucleotide constructs of the present invention. Such pathogen-inducible promoters include those of proteins related to pathogenesis (PR proteins), which are induced after infection by a pathogen; for example, PR proteins, SAR proteins, beta-1, 3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Neth J. Plant Pathol. 89: 245-254; Uknes et al. (1992) Plant Cell 4: 645-656; and Van Loon (1985) Plant Mol. Virol. 4: 111-116. See also WO 99/43819 incorporated herein by reference. Of interest are the promoters that are expressed locally at or near the site of the pathogen's infection. See, for example, Marineau et al. (1987) Plant Mol. Biol. 9: 335-342; Matton et al. (1989) Molecular Plant-Microbe Interactions 2: 325-331; Somsisch et al. (1986) Proc. Nati Acad. Sci. USA 83: 2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2: 93-98; and Yang (1996) Proc. Nati Acad. Sci. USA 93: 14972-14977. See also, Chen et al. (1996) Plant J. 10: 955-966; Zhang et al. (1994) Proc. Nati Acad. Sci. USA 91: 2507-2511; Warner et al. (1993) Plant J. 3: 191-201; Siebertz et al. (1989) PJant Cell 1: 961-968; U.S. Patent No. 5,750,386 (inducible by nematode); and the references cited therein. Of particular interest is the inducible promoter for the maize PRms gene, whose expression is induced by the pathogen Fusarium monili forme (see, for example, Cordero et al. (1992) Physiol., Mol. Plant Path. 41: 189-200) . Chemically regulated promoters can be used to modulate the expression of a modified insect protoxin sequence in a plant through the application of an exogenous chemical regulator. Depending on the objective, the promoter can be a chemically inducible promoter, where the application of the chemical induces the expression of the gene, or a chemically repressible promoter, where the application of the chemical represses the expression of the gene. Chemically inducible promoters are known in the art and include, but are not limited to, the corn In2-2 promoter, is activated by moderators of benzenesulfonamide herbicide, the corn GST promoter, which is activated by the hydrophobic electrophilic compounds that they are used as pre-emergent herbicides, and the PR-la promoter of tobacco, which is activated by salicylic acid. Other chemically regulated promoters of interest include spheroidal responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al., (1991) Proc. Nati.
Acad. Sci. USA dd: 10421-10425 and McNellis et al.,
(1996) Plant J. 14 (2): 247-257) and the tetracycline-inducible and repressible promoters by tetracycline (see, for example, Gatz et al., (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Patent Nos. 5,814,618 and 5,769,156), incorporated herein by reference. Preferred tissue promoters can be used to direct the expression of increased pesticidal protein within a particular plant tissue. Preferred tissue promoters include those discussed in Yamamoto et al., (1997) Planl J. 12 (2): 255-265; Kawamata et al., (1997) Plant Cell Physiol. 38 (7): 792-d03; Hansen et al., (1997) Mol. Gen Genet. 254 (3); 337-343; Russell et al., (1997) Transgenic Res. 6 (2): 157-168; Rinehart et al., (1996) Plant Physiol. 112 (3): 1331-1341; Van Camp et al., (1996) Plant Physiol. 112 (2): 525-535; Canevascini et al., (1996) Plant Physiol. 112 (2): 513-524; Yamamoto et al., (1994) Plant Cell Physiol. 35 (5): 773-778; The m
(1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23 (6): 1129-1138;
Matsuoka et al. (1993) Proc Nati. Acad. Sci. USA 90 (20): 9586-9590; and Guevara-García et al., (1993) Plant J. (3): 495-505. Such promoters can be modified, if necessary, for weak expression. Preferred leaf promoters are known in the art. See, for example, Yamamoto et al., (1997) Plant J. 12 (2): 255-265; Kwon et al., (1994)
Plant Physiol. 105: 357-67; Yamamoto et al., (1994)
Plant Cell Physiol. 35 (5): 773-778; Gotor and collaborators,
(1993) Plant 3: 509-18; Orozco et al., (1993) Plant
Mol. Biol. 23 (6): 1129-1-138; and Matsuoka et al., (1993) Proc. Nati Acad. Sci. USA 90 (20): 9586-9590. Root-specific promoters are known and can be selected from the many available from the literature or isolated from -novo of several compatible species. See, for example, Hirey collaborators, (1992) Plant Mol. Biol. 20 (2): 207-218 (specific soybean glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3 (10): 1051-1061 (Root specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol. Biol. 14 (3): 433-443 (root specific promoter of the manpina synthase (MAS) gene from Agrobacterium tumefaciens; and Miao et al., (1991) Plant Cell 3 (1): 11-22 (full-length cDNA clone which encodes cytosolic glutamine synthetase (GS), which is expressed in roots and nodules of soybean root.) See also Bogusz et al., (1990) Plant CelJ 2 (7): 633-641, where two root-specific promoters isolated from genes of hemoglobin from the nitrogen-fixing non-legume Parasponia andersonii and the non-legume non-nitrogen related Trema tomentosa are described.The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the non-legume Nicotiana tabacum and the legume Lotus corniculatus, and in both cases the activity of specific root promoter was preserved, Leach and Aoyagi (1991) describe in their analysis the promoters of the highly expressed Agroba rolC and rolD root induction genes. cterium rhizogenes (see Plant Science (Li erick) 79: 69-76). They concluded that the preferred DNA determinants of tissue and of dissociated enhancers in these promoters. Teeri et al. (1989) used the lacZ gene fusion to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the root tip epidermis and that the TR2 'gene is root specific in the plant intact and stimulated by leaf injury and tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8 (2): 343-350). The TRIA gene fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional preferred root promoters include the VÍENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol. Biol, (1995) .29 (4): 759-772); and the rolB promoter (Capana et al., (1994) Plant Mol. Biol. 25 (4): 681-691 See also U.S. Patents Nos. 5,837,876, 5,750,386, 5,633,363, 5,459,252, 5,401,836, 5,110,732, and 5,023,179. "seed preferred" includes both of the "seed-specific" promoters (those promoters active during seed development such as promoters of "seed storage proteins" as well as the "seed germination" promoters (those active promoters during germination of the seeds.) See Thompson et al., (1989) BioEssays 10: 108, incorporated herein by reference, Such preferred seed promoters include, but are not limited to, Ciml (message induced by cytokinin); cC19Bl (19 kDa corn zein) and milps (myo-inositol 1-phosphate synthase) (see WO 00 / 1-1177 and US Patent No. 6,225,529, incorporated herein by reference) .Gamma-zein is a promoto endosperm specific r. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, β-petrol phaseolin, napin, β-conglycinin, soybean lectin, cruciferin and the like. For monocotyledons, seed-specific promoters include, but are not limited to, 15 kDa corn zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where the seed preferred promoters of the endl and end2 genes are disclosed; incorporated herein by reference. A promoter that has "preferred" expression in a particular tissue is expressed in that tissue to a greater degree than in at least one other plant tissue. Some preferred tissue promoters show expression almost exclusively in the particular tissue. Where low level expression is desired, weak promoters will be used. Generally, a "weak promoter" is proposed to imply a promoter that drives the expression of a coding sequence at a low level. By the term "low level", levels of approximately 1/1000 transcripts are proposed to approximately 1 / 100,000 transcripts to approximately 1 / 500,000 transcripts. Alternatively, it is recognized that weak promoters also comprise. promoters that are expressed in only a few cells and not in others to give a total low level of expression. Where a promoter is expressed at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease expression levels. Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99/43836 and US Patent No. 6,072,050), the core 35M CaMV promoter and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,606,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,266,463; and 5,60d, 142 and 6,177,611, incorporated herein by reference. Generally, the expression cassette will comprise an urgen selectable marker for the selection of transformed cells. Selectable marker genes are used for the selection of transformed cells or tissues. Marker genes include genes that encode antibiotic resistance, such as those that encode neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes that confer resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones and 2, 4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992) Curr. Opin. Biotech 3: 506-511; Christopherson et al. (1992) Proc. Nati Acad. Sci. USA 89: 6314-6318; Yao et al (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) CelJ 48: 555-566; Brown et al (1987) Cell 49: 603-612; Figge et al (1988) Cell 52: 713-722; Deuschle et al. (1989) Proc. Nati Acad. Sci. USA 86: 5400-5404; Fuerst and collaborators (1989) Proc. Nati Acad. Sci. USA 86: 2549-2553; Deuschle et al (1990) Science 248: 4d0-483; Gossen (1993) Ph. D. Thesis, University of Heidelberg; Reines and collaborators (1993) Proc. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell. • Biol. 10: 3343-3356; Zambretti et al. (1992) Proc.
Nati Acad. Sci. USA 89: 3952-3956; Baim et al.
(1991) Proc. Nati Acad. Sci. USA 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph. D. Thesis ,. University of Heidelberg; Gossen et al. (1992) Proc. Nati Acad. Sci. USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36: 913'-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,
Vol. 78 (Springer-Verlag, Berlin); Gilí and collaborators
(1988) Nature 334: 721-724; and the American applications
Nos. Series 10 / 004,357 and 10 / 427,692. Such descriptions are incorporated herein by reference. The above list of selectable marker genes is not proposed to be limiting. Any selectable marker gene can be used in the present invention. Transformation protocols as well as protocols for introducing nucleotide sequences in plants can vary depending on the type of plant or plant cell, i.e., monocot or dicot, directed for transformation. The right methods for
- introducing nucleotide sequences into plant cells and the subsequent insertion into the plant genome includes microinjection (Crossway et al. (1986) Biotechniques 4: 320-334), electroporation (Riggs et al. (1986) Proc. Nati. Acad. Sci. USA 83: 5602-5606), Agrobacterium-mediated transformation (U.S. Patent Nos. 5,563,055 and 5,981,840), direct gene transfer
(Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and ballistic particle acceleration (see, for example, U.S. Patent Nos. 4,945,050; 5,879,918;
.8d6.244; 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment", in Plant Cell, Tissue, and Organ Cul ture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al (1986) Biotechnology 6: 923-926); and the Lecl transformation (WO 00/28058). For potato transformation see Tu et al. (1998) Plant
Molecular Biology 37: 829-838 and Chong and co-workers (2000)
Transgenic Research 9: 71-78. Additional transformation procedures can be found in Weissinger et al.
(1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion);
Christou et al. (1988) Plant Physiol. 87: 671-674
(soy); McCabe et al. (1988) Bio / Technology 6: 923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev.
Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet 96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al (1988) Proc. Nati Acad. Sci. USA 85: 4305-4309 (corn); Klein et al (1988) Biotechnology 6: 559-563 (corn); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91: 440-444 (corn); Fromm et al (1990) Biotechnology 8: 833-839 (corn); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311: 763-764; U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Nati Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman and collaborators (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet 84: 560-566 (transformation mediated by Whisker); D'Halluin et al. (1992) Plant Cell 4: 1495-1505
(electroporation); Li et al. (1993) Plant Cell
Reports 12: 250-255 and Christou and Ford (1995) Annals of
Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize via Agrobacterium tumefaciens); all of which are incorporated herein by reference. The cells that have been transformed can be grown in plants according to conventional manners. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants can then be cultured, either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive or inducible expression of the desired, identified phenotypic characteristic. Two or more generations can be cultured to ensure that the expression of the desired phenotypic characteristic is stably maintained and inherited and then the seeds harvested to ensure the expression of the desired phenotypic characteristic that has been achieved. The nucleotide sequences of the invention can be provided to the plant by contacting the plant with a virus or viral nucleic acids. Generally, such methods involve incorporating the nucleotide construct of interest into a viral DNA or RNA molecule. It is recognized that the recombinant proteins of the invention can be initially synthesized as part of a viral polyprotein, the latter can be processed by proteolysis in vivo or in vitro to produce the desired pesticidal protein. It is also recognized that such a viral polyprotein, comprising at least a portion of the amino acid sequence of a pesticidal protein of the invention, may have the desired pesticidal activity. Such viral polyproteins and the nucleotide sequences encoding them are encompassed by the present invention. Methods for providing plants with nucleotide constructs and the production of proteins encoded in plants, which involve viral DNA or RNA molecules are known in the art. See, for example, U.S. Patent Nos. 5,889,191; 5, d 89.190; 5,866.7 d5; 5,5d9,367 and 5,316,931; incorporated herein by reference. The invention further relates to the plant propagation material of a transformed plant of the invention which includes, but is not limited to, seeds, tubers, bulbous stems, bulbs, leaves and cuttings of roots and shoots. The present invention can be used for transformation of any species of plant, including, but not limited to, monocots and dicotyledons. Examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (for example, B. napus, B. rapa, B. j uence), particularly those Brassica species useful as a source of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Sécale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (for example, pearly millet (Pennisetum glaucum), millet proso
(Panicum miliaceum), foxtail millet [Italic Setaria], extended millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Tri ticum aestívum.), Soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hipogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato
(Ipomoea batatus), casava (Manihot esculenta), coffee (Coffea spp.), Coconut (Cocos nucífera), pineapple (Ananas comosus), citrus trees. { Ci trus spp.), Cacao (Theobroma cacaco), tea
(Camellia sinensis), banana (Musa spp.), Avocado (Persea americana), fig [Ficus -casica], 'guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), beets (Beta vulgaris), sugar cane (Sacaarum spp.), oats, barley, vegetables, ornamental plants and conifers. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (for example, Lactuca sativa), green beans (Phaseolus vulgaris), beans (Phaseolus limensis), peas (Lathyrus spp.), And members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantal upensis), and melon (C. meló). Ornamental plants include azalea (Rhododendron spp.), Hydrangea (Macrophylia hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), Tulips (Tulipa spp.), Daffodils (Narcissus spp.), Petunias (Petunia hybrida), carnation (Dianthus caryophyllus), red shepherdess (Euphorbia pulcherrima), and chrysanthemum. The conifers that can be employed in the practice of the present invention include, for example, pines such as the incense pine. { Pinus taeda), pine tree (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine
(Pinus contorta), and Monterey pine. { Pinus radiata); Douglas fir (Pseudotsuga menziesil); western pinabete
(Tsuga canadensis); Sitka fir (Picea glauca); Red wood
(Sequoia sempervirens); typical firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western red cedar (Thuja plicata) and yellow Alaskan cedar (Chamaecyparis nootka tensis). Plants of the present invention include crop plants (e.g., corn, alfalfa, sunflower, brassica, soy, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.), as well as turfgrass. Grass grasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass. { Lolium mul tiflorum); bluish grass of Canada. { Poa compressa); Cannabis Chewings (Festuca rubra); Agrotestida colonial (Agrostis tenuis); Agrostatic climber (Agrostis palustris); Bristle crested grass (Agropyron desertorum);
smoothed brown grass (Agropyron cristatum); hard cane
(Festuca longifolia); -blue grass of Kentucky (Poa pratensis); orchard grass (Dactylis glomerata); perennial ryegrass (Lolium perenne); red canes (Festuca rubra); agrostis alba (Agrostis alba); blueish hairy grass (Poa trivialis); grazing shells (Festuca ovina); smooth bromine
(Bromus iner is); high cañuela (Festuca arundinacea); thin
(Phleum pratense); velvety agrostide (Agrostis canina); weeping grass (Puccinellia distans); western wheat grass (Agropyron simi thii); Bermuda grass (Cynodon spp.); St. Augustine grass. { Stenotaphrum secundatum); herb zoysia (Zoysia spp.); Bay grass (Paspalum notatum); carpeted grass (Axonopus af finís); centipede grass
(Eremochloa ophiuroides); Kikuyu grass (Pennisetum clandesinum); Páspalo de rivera de mar (Paspalum vaginatum); bluish grass (Bouteloua gracilis); prairie grass. { Buchloe dactyloids); slope grass (Bouteloua curtipendula). Plants of interest include grain plants that provide seeds of interest, oilseed plants and leguminous plants. The seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil seed plants include cotton, soybean, safflower, sunflower, Brassica, corn, alfalfa, palm, coconut, flax, castor, olive, etc. Legume plants include beans and peas. The beans include guar, carob, fenegreco, soybeans, garden beans, cowpeas, mung beans, beans, fava beans, lentils, chickpeas, etc. The compositions of the invention find use in protecting plants, seeds and plant products in a variety of ways. For example, the compositions can be used in a method that involves placing an effective amount of the pesticidal composition in the plague environment by a method selected from the group consisting of spraying, sprinkling, scattering or coating or seed. Before the propagation material of plants (fruit, tuber, bulb, bulbous stem, grains, seeds), but especially seed, has been sold as a commercial product, this is usually treated with a protective coating that includes herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with additional carriers, surfactants, or application-promoting adjuvants usually employed in the formulation art to provide protection against damage caused by bacterial, fungal or animals. In order to treat the seed, the protective coating can be applied to the seeds either by impregnating the tubers or grains with a liquid formulation or by coating them with a combined wet or dry formulation. In addition, in special cases, other methods of application to plants are possible, for example, the treatment
directed to shoots or fruit. The plant seed of the invention comprising a DNA molecule comprising a nucleotide sequence encoding a pesticidal protein of the invention can be treated with a protective coating of
. seed comprising a seed treatment compound, such as, for example, captan, carboxin, thiram, metalaxyl, pyrophos-methyl and others which are commonly used in the treatment of seeds. In one embodiment within the scope of the invention, a seed protective coating that
comprises a pesticidal composition of the invention used alone or in combination with one of the protective seed coatings usually used in seed treatment. It is recognized that the genes that encode
pesticide proteins can be used to transform pathogenic insect organisms. Such organisms include Baculoviruses, fungi, protozoa, bacteria and nematodes. A gene encoding a pesticidal protein of the invention can be introduced via a vector
suitable in a microbial host, and the host applied to the environment, or to plants or animals. The term "introduced" in the context of inserting a nucleic acid into a cell means "transfection" or "transformation" or "transduction" and includes the reference of incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid can be be incorporated into the genome of the cell (eg, chromosome, plasmid, plastid or itochronic DNA), converted into an autonomous, or transiently expressed replicon (eg, transfected mRNA). Hosts of microorganisms that are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere and / or rhizoplane) of one or more crops of interest can be selected. These microorganisms are selected in terms of being able to successfully compete in the particular environment with the wild-type microorganism, provide stable maintenance and expression of the gene expressing the pesticidal protein, and desirably, provide improved pesticide protection from degradation and inactivation. environmental. Such microorganisms include bacteria, algae and fungi. Of particular interest are microorganisms such as bacteria, for example, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,
Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungus, particularly yeast, for example, Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interest are such phytospheric bacterial species as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,
Xanthomonas campestris, Rhizobium melioti, Alcaligenes e? Trophus, Clavibacter xyli and Azotobacter vinlandir and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. Odorus, Kluyveromyces veronae and Aureobasidium pollulans. Of particular interest are pigmented microorganisms. A number of ways are available to introduce a gene that expresses the pesticidal protein in the microorganism host under conditions that allow stable maintenance and expression of the gene. For example, expression cassettes including constructs of nucleotides of interest operably linked to transcriptional and translational regulatory signals for the expression of nucleotide constructs, and a nucleotide sequence homologous to a sequence in the host organism can be constructed. , through which the integration will occur, and / or a replication system that is functional in the host, through which integration or stable maintenance will occur. Transcriptional and detranslating regulatory signals include but are not limited to, promoters, transcriptional initiation initiation sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals and the similar ones. See, for example, patentsAmerican Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al. (1992) Molecular Cloning: A
Laboratory Manual, ed. Maniatis and collaborators (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York); Davis and collaborators, eds. (1980) Advanced Bacterial Genetics
(Cold Spring Harbor Laboratory Press), Cold Spring Harbor, New York; and the references cited therein. Suitable host cells, where the cells containing the pesticidal protein will be treated to prolong the activity of the pesticidal proteins in the cell when the treated cell is applied to the environment of the target pest (s), may include either prokaryotes or eukaryotes, usually being limited to those cells that do not produce toxic substances or higher organisms, such as mammals. However, organisms that produce toxic substances or higher organisms could be used, where the toxin is unstable or the level of application sufficiently low to avoid any possibility of toxicity to a mammalian host. As hosts, of particular interest will be prokaryotes and lower eukaryotes, such as fungi. Illustrative prokaryotes, both gram negative and gram positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhízobíceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacxllaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacter aceae and Ni trobacteraceae. Among the eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces and Schizosaccharomyces; and yeast of Basidiomycetes, such as Rhodotorula, Aureobasidium, Sporobolomyces and the like. Characteristics of particular interest in the selection of a host cell for purposes of pesticide protein production include the ease of introduction of the pesticide protein gene into the host, availability of the expression systems, expression efficiency, stability of the protein in the host. the host, and the presence of auxiliary genetic capabilities. The characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such. as the walls of thick cells, pigmentation and intracellular packing or formation of inclusion bodies; affinity of the leaf; lack of toxicity of the mammal; attractiveness to pests for ingestion; ease of extermination and fixation without damage to the toxin; and the similar ones. Other considerations include ease of formulation and handling, economy, storage stability and the like. Host organisms of particular interest include yeast, such as Rhodotorula spp. , Aureobasidium spp. , Saccharomyces spp. , and Sporobolomyces spp. , phylloplane organisms such as Pseudomonas spp. , Erwinia spp. , and Flavobacterium spp. , and other organisms of such class including Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coli, Bacillus subtilis, and the like. The genes encoding the pesticidal proteins of the invention can be introduced into microorganisms that multiply in plants (epiphytes) to deliver pesticidal proteins to potential target pests. Epiphytes, for example, can be gram positive or gram negative bacteria. Root colonization bacteria, for example, can be isolated from the plant of interest by methods known in the art. Specifically, a strain of Bacillus cereus that colonizes the root can be isolated from the roots of a plant (see, for example, Haldelsman et al. (1991) Appl. Environ Microbiol. 56: 713-718). The genes encoding the pesticidal proteins of the invention can be introduced into a Bacillus cereus root colonization by standard methods known in the art. The genes encoding pesticidal proteins can be introduced, for example, into the Bacillus of root colonization by means of electrotransformation. Specifically, the genes encoding the pesticidal proteins can be cloned in a shuttle vector, for example, pHT3101
(Lerecius et al. (1989) .FEMS Microbiol. Letts.
60: 211-218). The shuttle vector pHT3101 containing the coding sequence for the particular pesticidal protein gene can be, for example, transformed into the Bacillus root colonization by means of electroporation (Lerecius et al. (1989) FEMS Microbiol. Letts. 211-218). The expression systems can be designed so that the pesticidal proteins are secreted out of the cytoplasm of the gram negative bacteria, E. coli, for example. The advantages of having secreted pesticidal proteins are: (1) to avoid potential cytotoxic effects of the expressed pesticidal protein and (2) improvement in the efficiency of the purification of the pesticidal protein, including, but not limited to, increased efficiency in the recovery and purification of the protein by volume of cellular broth and time reduced and / or recovery costs and purification by unitary protein. Pesticidal proteins can be made to be secreted in E. coli, for example, by fusion of an appropriate E. coli signal peptide to the amino-terminal end of the pesticidal protein. Signal peptides recognized by E. coli can be found in known proteins that are secreted in E. coli, for example the OmpA protein (Ghrayeb et al. (1984) EMBO J. 3: 2437-2442). OmpA is a major protein of the outer membrane of E. coli, and in this way its signal peptide is thought to be efficient in the translocation process. Also, the OmpA signal peptide. it does not need to be modified before processing as might be the case for other signal peptides, for example, the lipoprotein signal peptide (Duffaud et al. (1987) Meth., Enzymol, 153: 492). The pesticidal proteins of the invention can be fermented in a bacterial host and the resulting bacteria processed and used as a microbial spray in the same manner as Bacillus thuringiensis strains have been used as insecticidal sprays. In the case of a pesticide protein (s) that is secreted from Bacillus, the secretion signal is removed or mutated using procedures known in the art. Such mutations and / or deletions prevent the secretion of the pesticide protein (s) in the growth medium during the fermentation process. The pesticidal proteins are retained within the cell, and the cells are then processed to produce the encapsulated pesticidal proteins. Any suitable microorganism can be used for this purpose. Pseudomonas have been used to express Bacillus thuringiensis endotoxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide (Gaertner et al. (1993) in: Advanced Engineered Pesticides, ed. Kim). Alternatively, pesticidal proteins are produced by introducing a heterologous gene into a cell host. The expression of the heterologous gene results, directly or indirectly, in intracellular production and maintenance of the pesticide. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell, when the cell is applied to the environment of the target plague (s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticidal proteins can then be formulated according to conventional techniques for application to the environment that host a target pest, for example, soil, water and plant foliage. See, for example, EPA 0192319 and the references cited therein. In the present invention, a transformed microorganism (including whole organisms, cells, spore (s), pesticide protein (s), pesticide component (s), component (s) that impact pests, mutant (s) ) preferably living or dead cells and cell components, including mixtures of living and dead cells and cell components, and including broken cells and cell component) or an isolated pesticidal protein can be formulated with an acceptable carrier in a pesticidal composition (s) that is, for example, a suspension, a solution, an emulsion, a powder sprinkling, a dispersible granule, a wettable powder, and an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste and also encapsulations in, for example, polymeric substances. Such compositions disclosed in the above can be obtained by the addition of an active agent on the surface, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye , a UV protector, a regulatory solution, a flow agent or fertilizers, micronutrient donors or other preparations that influence the growth of the plant. One or more agrochemicals including, but not limited to, herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, acaricides, plant growth regulators, crop assistants and fertilizers can be combined with carriers, surfactants or adjuvants usually employed in the art. of formulation or other components to facilitate the handling of the product of the application for the particular target pests. The suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in the formulation technology, for example, mineral, natural or regenerated substances, solvents, dispersants, wetting agents, glidants, binders or fertilizers. The active ingredients of the present invention are usually applied in the form of compositions the area of culture, plant or seed that is treated can be applied. For example, the compositions of the present invention can be applied to the grain in the preparation or during storage in a grain warehouse or silo, etc. The compositions of the present invention can be applied simultaneously or in succession with other compounds. Methods for applying an active ingredient of the present invention or an agrochemical composition of the present invention containing at least one of the pesticidal proteins produced by the bacterial strains of the present invention include, but are not limited to, foliar application, coating of seed and application to the earth. The number of applications and the proportion of applications will depend on the intensity of the infestation by the corresponding plague. Suitable surface active agents include, but are not limited to, anionic compounds such as carboxylate of, for example, a metal; long chain fatty acid carboxylate; an N-acyl sarcosinate; mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters, fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfate; ethoxylated alkylphenol sulfate; lignin sulfonates; petroleum sulfonates, alkyl aryl sulfonates such as alkylbenzene sulphonates or lower alkylnaphthalene sulfonates, for example, butylated naphthalene sulfonate; salts of sulfonated naphthalene-formaldehyde condensates, salts of sulfonated phenol-formaldehyde condensates; more complex sulfonates such as amide sulfonates, for example, the sulphonated condensation product of oleic acid with N-methyl taurine; or dialkyl sulfosuccinates, for example sodium sulfonate or dioctyl succinate. Agents no. ions include the condensation products of fatty acid esters, fatty alcohols, fatty acid amides or phenols substituted with fatty alkyl or alkenyl with ethylene oxide, fatty esters or polyhydric alcohol esters, for example, sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, for example, polyoxyethylene sorbitan fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2, 4, 7, 9-tetraethyl-5- decin-4, 7-diol, or ethoxylated acetylenic glycols. Examples of an active agent on the cationic surface include, for example, a mono-, di-, or aliphatic polyamine such as an acetate, naphthenate or oleate; oxygen-containing amine such as polyoxyethylene alkylamine amine oxide; an amine linked to amide, prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt. Examples of inert materials include but are not limited to inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates or botanical materials such as cork, powdered corn, peanut pods, rice pods, and walnut shells. The compositions of the present invention have a form suitable for direct application or as a concentrate of the primary composition which requires dilution with a suitable amount of water or other diluent before application. The pesticide concentration will vary depending on the nature of the particular formulation, specifically, if it is concentrated or used directly. The composition contains from 1 to 96% of a solid or liquid inert carrier, and 0 to 50%, preferably 0.1 to 50% of a surfactant. These compositions will be administered in the proportion labeled for the commercial product, preferably approximately 0.1 lb-5.0 Ib. per acre when in dry form or approximately 0.01 pts.-10 pts. per acre when it is in liquid form. In a further embodiment, the compositions, as well as the transformed microorganisms and pesticidal proteins, of the invention. they can be treated prior to formulation to prolong pesticidal activity when applied to the environment of a target pest while the pretreatment is not detrimental to the activity. Such treatment may be by chemical and / or physical means as long as the treatment does not detrimentally affect the properties of the composition (s). Examples of chemical reagents include but are not limited to halogenating agents; aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, such as zephiran chloride; alcohols, such as isopropanol and ethanol; and histological fixatives, such as Bouin's fixative and Helly's fixative (see, for example, Humason (1967) Animal Tissue Techniques (WH Freeman and Co.) In other embodiments of the invention, it may be advantageous to treat the polypeptides with A protease, for example trypsin, for activating the protein prior to the application of a pesticidal protein composition of the invention to the environment of the target pest The methods for the activation of protoxin by a serine protease are well known in the art. . See, for example, Cooksey (1966) Biochem. J. 6: 445-454 and Carrol and Ellar (1989) Biochem J. 261: 99-105, the teachings of which are incorporated herein by reference. For example, a suitable activation protocol includes, but is not limited to, combining a polypeptide that is activated, for example, a purified CrydBbl polypeptide, and trypsin in a 1/100 weight ratio of 1218-1 protein / trypsin in 20 nM NaHC03, pH 8 and by digesting the sample at 36 ° C for 3 hours. The compositions (including the transformed microorganisms and the pesticidal proteins of the invention) can be applied to the environment of an insect pest by, for example, spraying, atomizing, sprinkling, dispersing, coating or casting, introducing into or onto the ground. , introduction into irrigation water, through seed treatment or general application or sprinkling over time when plague has started to appear or before the appearance of pests as a protective measure. For example, the pesticidal protein and / or transformed microorganisms of the invention can be mixed with grain to protect the grain during storage. It is generally important to obtain good control in the pests of the earliest stages of plant growth, since this is the time when the plant can be more severely damaged. The compositions of the invention may conveniently contain another insecticide if this is thought necessary. In one embodiment of the invention, the composition is applied directly on the ground, at the time of planting, in a granular form of a carrier composition and dead cells of a Bacillus strain or transformed microorganisms of the invention. Another embodiment is a granular form of a composition comprising an agrochemical such as, for example, a herbicide, an insecticide, a fertilizer, an inert carrier and dead cells of a Bacillus strain or transformed microorganisms of the invention. The embodiments of the present invention can be effective against a variety of pests. For purposes of the present invention, pests include, but are not limited to, insects, fungi, bacteria, nematodes, acarids, protozoan pathogens, parasitic liver flukes of animals and the like. Pests of particular interest are insect pests, particularly insect pests that cause significant damage to agricultural plants. By "insect pests" it is proposed to imply insects and other similar pests such as, for example, those of the Acari order including, but not limited to, mites and ticks. Insect pests of the present invention include, but are not limited to, insects of the order Lepidoptera, for example Achoroia grisella, Acleris gloveralla, Acleris variana, Adoxophyes orana, Agrotis εilon, Alabama argillacea, Alsophila pometaria, Amyelois trallsitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips sp. , Argyrotaenia sp. , Athetis mindara, Bombix mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp. , Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmia feneralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaría, Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene aerea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina americana, Helicovelpa subflexa, Helicovelpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantia cunea, Keiferia lycopersicella, Lambdina fiscellaria, Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosoma sp. , Mamestra brassicae. Mamestra configurata, Manduca qinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp. , Ostrillia nubilalis, Paleacrita vernata, Papilio cresphontes, Pectinophora gossypiella, Phyganidia calif omica, Phyllonorycter blancardella, Pieris napi, Pieris rapae, Plathypella scabra Platynota flouendana, Platynota stultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp. , Thaurnstopoea pityocampa, Tinsola bísselliella, Trichoplusia hi, Udea rubigalis, Xylomyges curiails and Ypollomeuta padella. Also, the embodiments of the present invention can be effective against a variety of insect pests including insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera, especially Diabrotica virgifera and Lepidoptera. Insect pests of the invention for major crops include, but are not limited to: Corn: Ostrinxa nubilalis, European corn borer; Agrotis Ípsilon, black cutworm; Helicoverpa zea, corn masker worm; Spodoptera frugiperda, devastating autumn worm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, minor corn stem borer; Diatraea saccharalis, sugarcane borer; Diabrotica virgifera virgifera, western corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotus spp., Centipede; Cyclocephala borealis, northern masked bumblebee (white larva); Cyclocephala immaculata, southern masked bumblebee (white larva); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn beetle; Splzenoplzorus maidis, weevil insect of corn; Rhopalosiphum maidis, corn leaf aphid; Anur aphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, bug bugs; Melanoplus femurrubrum, red-legged lobster; Melanoplus sanguinipes, migratory lobster; Hylemya platura, corn worm; Agromyza parvicornis, spotted corn miner; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, ant stealing; Tetranychus urticae, two-spotted red mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, devastating autumn worm; Helicoverpa zea, corn cob worm; Elasmopalpus lignosellus, minor corn stem borer; Underground felting, granulated cutworm; Phyllophaga crinita, white larva; Eleodes, Conoderus and Aeolus spp., Centipedes; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn beetle; Sphenophorus maidis, weevil of corn; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugar cane aphid; Blissus leucopterus leucopterus, bug bugs; Contarinia sorghicola, jej6n de sorgo; Tetranychus cinnabarinus, carmine red mite; Tetranychus urticae, two-spotted red mite; Wheat: Pseudaletia unipunctata, worm devastating; Spodoptera frugiperda, devastating autumn worm; Elasmopalpus lignosellus, minor corn stem borer; Agrotis orthogonia, worm cutter of the west; Elasmopalpus lignosellus, minor corn stem borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, cloverleaf weevil; Diabrotica undecimpuctata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis graminum, green bug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, red-legged lobster; Melanoplus differentialis, differential locust; Melanoplus sanguinipes, migratory lobster; Mayetiola destructor, Hesse fly; Mosellana sitodiplosis, wheat midge; Meromyza americana, wheat stem worm; Hylemya coarctata, wheat bulb fly; Frankliella fusca, tobacco trips; Cephus cinctus, wheat stem sawfly; Tulipae, billowed mire of wheat; Sunflower: Cylindrocupturus adspersus, sunflower stem weevil; Smicronix fulus, red sunflower seed weevil; Smicronix sordidus, gray sunflower seed weevil; Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; zygogramma exclamatíonis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtxana, sunflower seed midge; Cotton: Heliothis virescens, cotton worm; Helicovelpa zea, cotton weevil; Spodoptera exigua, beet devastating worm; Pectinophora gossypiella, pink weevil; Anthonomus granáis, cotton weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, small cotton lobster; Trialeurodes abutilonea, white fly with stripes; Lygus lineolaris, bleached plant bug; Melalloplus femurrubrum, red-legged lobster; Melanoplus diflerentialxs, differential lobster; Thrips tabaci, onion thrips; Franklinkiella filsca, tobacco trips; Tetranychus cinnabarinus, carmine red mite; Tetranychus urticae, two-spotted red mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, devastating autumn worm; Helicoverpa zea, corn cob worm; Colaseis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Si tophilus oryzae, rice weevil; Nephotettix nigropictus, rice grasshopper; Blissus leucopterus leucopterus, bug bugs; Acrosternum hilare, green bug; Soybeans: Pseudoplusia includens, soybean measuring caterpillar; Anticarsia gemmatalis, hay caterpillar; Plathypena scabra, green clover worm; Ostrinia nubilalís, European corn barrel; Agrotis Ípsilon, black cutworm; Spodoptera exigua, beet devastating worm; Heliothis virescens, cotton weevil; Helicoverpa zea, cotton weevil; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato grasshopper; Acrosternum hilare, green forest bug; Melanoplus femurrubrum, red-footed grasshopper; Alelanoplus differentialis, differential grasshopper; Hylemya platura, corn seed larva; Sericothrips variabilis, soybean thrips; Thrips tabad, onion thrips; Tetranychus turkestani, red strawberry mite; Tetranychus urticae, two-spotted red mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis Ípsilon, black cutworm; Schizaphis graminum, green bug; bug bugs, for example Blissus leucopterus leucopterus; Acrosternum hilare, green forest bug; Euschistus servus, coffee bug; Jylemya platura, corn seed larva; Mayetiola destructor, Fly of Hese; Petrobia latens, acaro de trgo coffee; Oilseed rape: Vreviciryne brassicae, cabbage aphid; Phyllotreta cruciferae, cruciferous beetle; Phyllotreta striolata, fringe beetle; Phyllotreta nemorum, turnip beetle with stripes; Aleligethes aeneus, rape beetle; and the pollen beetles; Meligethes rufimanus, Meligethes nigrescens, Meligethes canadianus and Meligethes viridescens; Papa: Leptinotarsa decemlineata, Colorado potato beetle. In addition, the embodiments of the present invention can be effective against Hemiptera such as Lygus hesperus, Lygus lineolaris, Lygus pratensis, Lygus rugullpennis Popp, Lygus pabulinus, Calocoris llorvegicus, Orthops compestris, Plesiocoris rugicollis, Crytopeltis modestlls, Crytopel tis notatus, Spanagonicus albofasdatus, Diaphnocoris clzlorinonis, Labopidicola allii,
Pseudatomoscelis seriatus, Adelphocoris rapidus,
Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae, Nysi usraphanus, Euschistus servus, Nezara viridula,
Eurygaster, Coreidae, Pyrrhocoridae, Tínidae, Blostomatidae, Reduviidae and Cimicidae. Pests of interest also include Araecerus fasciculatus, brown bean weevil;
Acanthoscelides obtectus, bean weevil; Bruchus rufimanus, bean weevil; Bruchus pisorum, pea weevil; Zabrotes subfasciatus, Mexican bean weevil; Diabrotica bal teata, fringe cucumber beetle;
Cerotoma trifurcata, bean leaf beetle; Diabrotica virgifera, Mexican corn rootworm; Epi trix cucumeris, potato beetle; Chaetocnema confinis, sweet potato beetle; Hypera postica, alfalfa weevil; All thonomus quadrigibbus, apple curculio; Sternechus paludatus, bean stem weevil; Hypera brunnipennis, Egyptian lucerne weevil; If tophilus granarles, granary weevil; Craponius inaequalis, grape curculium; Si tophilus zeamais, corn weevil; Conotrachel us nenuphar, plum curculio; Euscepes postfaciatus, sweetpotato weevil from Eastern India; Maladera castano, Asian garden beetle; Rhizotrogus majalis, European bumblebee; Macrodactylus subspinosus, bumblebee of roses; Tribolium confusum, flour beetle; Tenebrio obscurus, dark mealworm; Tribolium castaneum, red flour beetle; Tenebrio moli tor, yellow flour worm. Nematodes include parasitic plant nematodes such as root knot, cyst and nematode lesion, including Heterodera and Globodera spp. such as Globodera rostochiensis and Globodera pailida (potato cyst nematodes); Heterodera glycines (nematode of soybean cyst); Heterodera schachtii (beet cyst nematode); and Heterodera avenae (cereal cyst nematode). Units, prefixes and symbols can be denoted • in their accepted form YES. Unless indicated otherwise, the nucleic acids are written from left to right in the orientation 5 'to 3'; the amino acid sequences are written from left to right in the amino to carboxy orientation, respectively. The numerical ranges are inclusive of the numbers that define the interval. The amino acids can be referred to herein by either their commonly known three letter symbols or by the letter symbols recommended by the IUPA-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes. The terms defined in the above are more fully defined by reference to the specification as a set. The following examples are presented by way of illustration, not by way of limitation. EXPERIMENTAL Example 1: Bioassay for Testing the Pesticide Activity of the B. thuringiensis Strains Against the West Corn Rootworm and Southern Corn Rootworm Insect diets for larvae of Colorado potato beetle (CPB), Southern corn rootworm (SCRW) and western corn rootworm (WCRW) are known in the art. See, for example, Rose and McCabe (1973) JX Econ. Entomol ogy 66: 393, incorporated herein by reference. The insect diet is prepared and emptied into a CD International bioassay tray. Generally 1.5 ml of diet is supplied in each cell with an additional 150 μl of sample preparation applied to the surface of the diet. Bacterial colonies from an original plate of transformants expressing the pesticidal proteins of interest are stained on replica plates and inoculated into 5 ml of 2XYT broth with 500 μl / 1000 ml of kanamycin antibiotic. The tubes are grown overnight. If no growth occurs, the tubes are incubated for an additional 24 hours. After incubation, the tubes are centrifuged at 3500 rpm for 5-8 minutes. The supernatant is discarded and the pellet is resuspended in 1000 μl of PBS. The sample is then transferred to 1.5 ml Eppendorf tubes and incubated on ice until the temperature is 3 to 4 ° C, followed by sonication for 12-15 seconds. Microbial culture broths (150 μl) or other samples (150 μl) are superimposed on artificial diets. The trays are allowed to dry. Rootworm larvae are supplied in the cavities of the bioassay tray. Lids are placed on the bioassay trays and the samples are incubated for 4-7 days at a temperature of 26 ° C. The bioassays are then recorded by counting "live" larvae against "death". Mortality is calculated as the percentage of dead larvae of the total larvae tested.
Example 2: Pesticide activity of Lysates from strain 1218 of B. thuringiensis Samples prepared from cultures of strains 1218 of B. thuringiensis were tested for the presence of pesticidal activity against CPB, WCRW and SCRW as described in Example 1. As a control, the diet was treated with phosphate buffered saline (PBS). To prepare each sample, a single colony of a strain growing on an LB plate was selected and used to inoculate a flask containing 50 ml of TB medium. The flask was incubated overnight at 28 ° C and 250 rpm. After incubation, the culture in the flask was transferred to a tube, and the tube was centrifuged at 4300 x g for 15 minutes. The supernatant was discarded and the pellet was resuspended in 50 ml of sporulation medium. The tube was centrifuged again at 4300 x g for 15 minutes. The second supernatant was discarded, and the second pellet was resuspended in 50 ml of the sporulation medium. The solution of the resuspended culture was transferred into a flask, and the flask was then incubated for 48 hours at 28 ° C and 250 rpm.
After this incubation, the culture in the flask of
• conferred to a tube, and the tube was centrifuged at 4300 x g for 15 minutes. The supernatant was discarded, and the pellet was resuspended in 10 ml of 1 x M9 medium. The sample was then transferred to a 1.5 ml microcentrifuge tube, incubated on ice until the temperature was approximately 3 to 4 ° C, and then sonicated for 12-15 seconds. For the bioassays, 150 μl of a sonicated sample was used. The sporulation medium comprises 200 ml of 5X M9 salt solution, 5 ml of salt solution, 5 ml of
'solution CaCl2 and dH20 to a final volume of 1 liter. The 5X M9 salt solution comprises: 64 g of Na2HP04 • 7H20; 15 g
KH2P04; 2.5 g of NaCl; 5 g of NH 4 Cl; and dH20 to a final volume of 1 liter. The salt solution comprises: 2.46 g of MgSO -7H20: 0.04 g of MnSO4-H0; 0.28 g of ZnS04-7H20; 0.40 g FeS0 -7H20; and dH20 to a final volume of 1 liter. The CaCl 2 solution comprises 3.66 g of CaCl 2"2 H 20 to a final volume of 100 ml. The samples were tested with and without heating to determine if the component (s) responsible for the pesticidal activity is stable to the For the heat treatment, the samples were boiled for 15 minutes before use in the bioassay.The unheated samples prepared from strain 1218 exhibited pesticidal activity against the southern corn rootworm, with less pesticidal activity against Western Corn Rootworm Prepared samples of the lysates of strain 1218 caused moderate atrophy in southern corn rootworm larvae After warming, the samples had greatly reduced pesticidal activity against both species. rootworms The reduction in pesticidal activity after warming indicated that one or more components of the strain 1218 sample that is responsible for the activity pesticide is unstable to heat. Such reduction is consistent with one or more of the components that are a protein. Example 3: Pesticide Activity of Isolated Crystal Proteins of Strain 1218 of B. thuringiensis Using sporulated culture samples of strain 1218 of B. thuringiensis prepared as described in Example 2, the crystal proteins were isolated and then treated with trypsin using methods known in the art. Briefly, after purification (zonal gradient centrifugation, Renografin-76), the purified crystals were dissolved in alkaline buffer (50 mM Na2C03, 10 mM dithiothreitol, pH 10). Before use in the assays, the dissolved crystal proteins were concentrated by filtration with Centriprep® Centrifuge filter units (Millipore Corp.) using a MW cutoff of 10,000. It is recognized that under some experimental conditions, it may be advantageous to treat the Cry8-like polypeptides with a protease, for example trypsin, to activate the protein prior to the determination of the pesticidal activity of a particular sample. Methods for the activation of protoxin by a serine protease are well known in the art. See, for example, Cooksey
(1968) Biochem J. 6: 445-454 and Carrol and Ellar (1989) Biochem J. 261: 99-105; incorporated herein by reference. The isolated crystal proteins were classified for pesticidal activity against western corn rootworm larvae, as described in Example 1. Both a new crystal protein preparation and a previously made preparation ("old preparation") of strain 1218 showed pesticidal activity against western corn rootworms. • Dissolved crystal proteins were stored at -80 ° C for 20 days before use in the assays. An expert person will recognize that there are numerous indicators of pesticide activity and that variables such as the number of dead insects, or the average weight of treated insects can be monitored. For example, the pesticidal activity can be conveniently expressed as percent (%) of mortality, which is the percentage of dead rootworm larvae of the total number of larvae. Example 4: Isolated Nucleotide Sequences of Strain 1218 of B. thuringiensis An effort was made to isolate the nucleotide sequences encoding the crystal proteins of Strain 1218 of B. thuringiensis. Two nucleotide sequences were isolated from 1216 having the nucleotide sequence and amino acid sequence homology to Cry8Bal (GenBank Access No. U04365). Cry8-like coding sequences isolated from strain 1218 have been designated Cryl218-1 (SEQ ID N0: 1), also known as CrydBbl, see Genbank Access No. AX543924 and Cryl218-2 (SEQ ID NO: 3), also known as CrydBcl, see Genbank Access No. AX543926. SEQ ID NO: 17 and SEQ ID NO: 18 provide the nucleic acid sequences of the native genomic clones of Cryl218-1 and Cryl218-2, respectively. To determine whether the proteins encoded by the variant or mutant polynucleotides of the invention encode proteins with pesticidal activity, each of the nucleic acid sequences was expressed in Escherichia coli. For example, to determine whether the polynucleotide sequences 1218-1 or 1218-2 provided herein encode polypeptides with pesticidal activity, truncated nucleotide sequences were prepared. SEQ ID NO: 11 corresponds to nucleotides 1 to 2007 of the nucleotide sequence of Cryl218-1 (SEO ID NO: l). SEQ ID NO: 13 corresponds to nucleotides 1 to 2019 of the nucleotide sequence of Cryl218-2 (SEQ ID NO: 3). SEQ ID NOS: 11 and 13 encode truncated Cry8-like polypeptides having the amino acid sequences set forth in SEQ ID NO: 12 and 14, respectively. Each of the truncated nucleotide sequences (SEQ ID NOS: 11 and 13) was cloned separately into an expression vector pET28a and then used to transform E. colx. The transformed colonies were selected and cultured in the liquid culture as described in Example 1. The truncated Cry8-like, N-terminal His-tagged proteins expressed were isolated from the E. coli lysates by affinity chromatography using a nickel affinity column. Fractions of the column with the protein of interest were dialyzed extensively against 10 mM Tris-HCl (pH 8.5) and then concentrated using Centriprep® centrifuge filter units (Millipore Corp.) with a MW cut-off of 10,000 according to the manufacturer's instructions. Samples of concentrated Cry8-like proteins were tested for the presence of pesticidal activity against the western corn rootworm, as described in Example 1. Bioassays that evaluate the pesticidal activity of purified recombinant Cryd-like proteins. of expressed preparations of E. coli were conducted as described in Example 1 with the aqueous protein samples superimposed on the diet surface of the rootworm. The pesticidal activity of wild-type endotoxin (e.g., native) and mutant were estimated against southern corn rootworms. As expected, it was observed that the pesticidal activity decreased as the concentration of the truncated Cryd-like proteins applied to the diet decreased. Pesticide activity was also estimated by incorporating pesticidal proteins in the rootworm diet, as opposed to the method described above, which involved incorporating a solution containing protein in the diet mixture. For example, sample diets comprising 1000, 500, 400, 300, 200 or 100 ppm of a pesticide polypeptide incorporated in the diet were estimated. Example 5: Preparation of a Preferred Nucleotide Sequence of Plants Encoding a Pesticide Protein Because the codon usage is • diflerent between plants and bacteria, the expression in a plant of a protein encoded by the nucleotide sequence of bacterial origin It can be limited due to the inefficiency of translation in the plant. It is known in the art that expression can be increased in a plant by altering the coding sequence of the protein to contain preferred codons of plants. For optimal expression of a protein in a plant, one. Synthetic nucleotide sequence can be prepared using the amino acid sequence of the protein and backtracking the sequence using preferred plant codons. Using such a procedure, a portion of the amino acid sequence of the protein encoded by Cryl218-1 (SEQ ID NO: 2) was translated backward (ie, inverted translation), using preferred codons of maize. The resulting preferred plant nucleotide sequence is set forth in SEQ ID NO: 5. The nucleotide sequence set forth in SEQ ID NO: 5 encodes a polypeptide (SEQ ID NO: 6) comprising the first 669 amino acids of the sequence of amino acids set forth in SEQ ID NO: 2. Thus, SEQ ID NOS: 6 and 12 encode polypeptides comprising the same amino acid sequence and SEQ ID NO: 11 provides a second polynucleotide that encodes the amino acid sequences set forth in SEQ ID NO: 6. Example 6: Bioassays to Test the Pesticidal Activity of the Cry8-like Polypeptides Mutants against the Colorado Potato Beetle (Leptinotarsa deceml neata) Protocol Briefly, the bioassay parameters were as follows: Bio-Serv diet ( catalog number F9800B, from: BIOSERV, Entomology Division, One 8th Street, Suite 1, Frenchtown, New Jersy 06825) was supplied in a 96-well microtiter plate (catalog number 353918, Bect on Dickinson, Franklin Lakes, NJ 07417.-1866) which has a surface area of 0.33 cm2. Samples similar to Cry8 (1218-1 and K03) were applied topically to the surface of the diet. The amino acid sequence of endotoxin 1218-1 is set forth in SEQ ID NO: 2, while the amino acid sequence of the mutant endotoxin K03 is set forth in SEQ ID NO: 68. Quite a few sample material was provided to provide 8 observations / sample. After the sample was dried, a neonatal colorado potato beetle (CPB) was added to each cavity. Therefore, there was a total of 8 larvae / sample. A Mylar® cap (Clear Lam Packaging, Inc., 1950 Pratt Blvd., Elk Grove Village, II 60007-5993) was attached to each tray. The bioassay trays were placed in an incubator at 25 ° C. The test was recorded for the 7th day mortality after the live infestation. The resulting mortality data were analyzed using a probit model (SAS / STAT Users Guide Version 8 Chapter 54, 1999). The probit analysis of the K03 mutant similar to Cryd and wild-type 1218-1 is shown in Fig. 6 and Fig. 7, respectively. Results The sample marked "I and R" in Table 1 was a control sample consisting of 10 mM carbonate buffer at pH 10. All mutant protein samples similar to Cryd, 1218-1 (AH) and K03 (JQ) were solubilized in 10 mM carbonate buffer at pH 10. Bioassays of 1218-1 and K03 indicated that both
'Protein samples were effective against CPB. The mutant
K03 similar to Cryd was found to be more potent than endotoxin 1218-1 of origin. The LC50 for the K03 mutant similar to Cryd was much lower when compared to the wild-type 1218-1 protein (Table 2). Thus, based on the surface area of the diet, this requires approximately 137 times less protein to achieve an LC50 using the mutant K03 similar to Cryd against 1218-1 (0.61 μg / cm2 for K03 versus 84 μg / cm2 for 1218-1 ). Based on the probit analysis and the LC50 determination (Table 2), the K03 'mutant similar to Cry8 shows significantly better bioactivity against CPB than wild-type 1218-1. TABLE 1. Pesticide Activity of a Cr? 8 -1218 (K03) -like mutant and Wild-type 1218-1 against the Colorado Potato Beetle Protein Samples Code (mg / mL) Mortality Mortality Rep 1 Rep 2 A 1218-1 0.5 * 100% 100% B 1218-1 0.25 75% 100% C 1218-1 0.125 50% 100% D 1218-1 0.0625 25% 63% E 1218-1 0.03125 25% 25% F 1218-1 0.0156 38% 25% G '1218-1 0.0078 13% 38% H 1218-1 0.0039 13% 0% I Solution 13% 13% regulatory J K03 0.5 100% 100% K K03 0.25 100% 100% L K03 0.125 100% 100% M K03 0.0625 100% 100% N K03 0.03125 88% 63% 0 K03 0.0156 75% 75% P K03 0.0078 38% 38% Q K03 0.0039 38% 38% R Solution 25% 13% regulatory * E1 percent mortality was calculated from 8 observations by concentration: TABLE 2. Determination of LC5o of a Mutant Similar to Cry8 1218 (K03) and 1218-1 Wild Type Against Colorado Potato Beetle Sample 1C50 (mg / ml) 95% Fiducial Limits 1218-1 1.1098 0.6859 - 2.4485 K03 0.00808 0.00467 - 0.01184 Example 7: Bioassay for Testing the Pestisid Activity of the Cry8-like Polypeptides Mutants against the Corn Maize of the Southern Corn and Western Corn Rootworm Protocol The test parameters described in the above in Example 6 are modified to allow the evaluation of the pesticidal activity of the additional mutant polypeptides against western corn rootworm (WCRW) and southern corn rootworm (SCRW). Briefly, the Bio-Serv diet (catalog number F9800B, from: BIOSERV, Ento ology Division, One 8-Suite 1, Frenchtown, New Jersey 08825) is supplied in 128-cavity International CD bioassay trays (catalog number BIO- BA-128 from CD International, Pitman, New Jersey 08071). The endotoxin samples are applied topically to the diet. Enough sample material is supplied to provide replicate observations per sample. The trays are allowed to dry. Rootworm larvae are supplied in the cavities of the bioassay trays. The lids are placed on the bioassay trays and the samples are incubated for 4-7 days at a temperature of 26 ° C. For the evaluation of the pesticidal activity against SCRW, the insects are exposed to a solution comprising either regulatory solution (50 mM carbonate buffer (pH 10)) or a mutant polypeptide solution in selected doses, for example, 36 or 3.6 μg / cm2. For evaluation of the pesticidal activity against WCRW, the insects are exposed to a solution comprising either regulatory solution (50 mM carbonate buffer (pH 10)) or a limited number of mutant polypeptides at a particular dose, for example, d8 μg / cm2. Bioassays are then recorded by counting "live" versus "dead" larvae. Mortality is calculated as the percentage of dead larvae among the total larvae tested. Example 8: Construction and Evaluation of Mutant Sequences An experiment was conducted to create and evaluate particular examples of mutant polynucleotide sequences and their encoded mutant proteins. The polynucleotide sequence NGSR1216-1 was cloned into the vector pET28a-c
(+) (Novagen Corporation) as a BamHI-XhoI fragment. This construct (pET28 / NGSR121d-l) was then used as the starting material for the additional genetic modification. A multistage PCR procedure was used to generate the mutants. The mutagenesis primers were first used in combination with two primers designed from the vector pET2d as the forward primer pET (SEO ID NO: 37) and rear primer pET (SEQ ID NO: 38). The mutagenesis primers used to create the M4 mutant were the forward primer M4 (SEO ID NO: 27) and the M4 rear primer
(SEQ ID NO: 28); the mutagenesis primers used to create the M5 mutant were the forward primer M5 (SEQ ID NO: 31) and the rear primer M5 (SEQ ID NO: 32); and the mutagenesis primers used to create the mutant K04 were the forward primer K04 (SEO ID NO: 23) and the back primer K04 (SEQ ID NO: 24). Thus, the amino acid sequence of the mutant endotoxin M4 is set forth in SEQ ID NO: 26; the amino acid sequence of the M5 mutant endotoxin is set forth in SEQ ID NO: 30; and the amino acid sequence of the mutant endotoxin K04 is set forth in SEQ ID NO: 22. After a first round of PCR, the samples were loaded onto a 1% agarose gel, and the expected bands were excised and purified. using the Qiaquick gel extraction equipment (Qiagen). To generate the mutant polynucleotide, a second round of PCR was performed for 7 cycles without primers. This procedure generated the mutant polynucleotide via the superposition of the homologous mutated region. Subsequently, the flanking pET 26 primers (front and rear) were added to generate the mutated polynucleotide sequence. These modified polynucleotide fragments were then used to replace the corresponding fragment in the plasmid pET2d / NGSR1218-l using standard cloning procedures so that the mutated portions of the polynucleotide were replaced by the corresponding portions of the original polynucleotide. Plasmids based on pET28 were used to express the proteins encoded in E. coli. BL21 Star ™ cells (DE3) (Invitrogen) were used as the host of E. coli for the production of protein from plasmids derived from pET2d. Plasmid pET28 provides a "portion" which is a short polypeptide linked to the 3 'end of the polypeptides generated from the plasmid. This portion provides a mechanism by which the protein can be purified from the solution. To produce the protein, bacterial cultures were cultured at a density of approximately OD600 1-0 at 37 ° C. The cultures were then induced with 200 μg / ml of IPTG and incubated overnight at 16 ° C. The culture cells were then harvested and lysed to produce the lice containing the portion-of-interest fusion protein. The fusion proteins were purified using the His Novagen portion purification kit. The concentrations of purified protein were determined using the BCA protein assay (Pierce). • The mutant proteins were used in a bioassay procedure to evaluate the effect of the mutant polypeptides on the pests of interest. Specifically, an experiment was conducted to compare the effects of wild-type (native) and mutant polypeptides on WCRW. Rootworms were cultured on bioassay trays. The insect diet was supplied in each cavity of the bioassay tray. Samples of test protein or control samples were applied topically to the diet. The samples were dried in a laminar flow hood. The test protein samples
^ 5 were used in the bioassays as described in Table 3 to determine which protein concentrations to use in the tests to compare the original protein to the mutant proteins. TABLE 3. Test protein samples used in the 0 bioassays. Western Corn Root Worm Trials:
Concentration Concentration of the Sample Extract Sample in the Diet
2. 5 225 1.25 112.5 0.625 56.25 0.3125 28.13 0.1563 14.06 0.0781 7.03 Colorado Potato Beetle Tests: Concentration of Sample Concentration in Extract Sample in Diet (mg / mL) (μg / cm2) 0.500 38 0.250 19 0.125 9.5 0.0625 4.7 i 0.03125 2.4 0.0156 1.2 0.0078 0.6 0.0039 0.3 Regulatory Solution 0 Four observations were made by concentration of the test protein. Mortality and atrophy were evaluated 5 and 7 days after infestation of the western corn rootworm. The term "atrophy" or "atrophied" means that the WCRW larva is severely retarded in growth and turns pale yellow to brown in coloration, in contrast to the normal larvae of the same stage or crystallized, which are large, round and white creamy in color. Another assay format referred to as the "128 cavity bioassay tray protocol" was also used to evaluate the mutant proteins. Again, the insect diet was added to each cavity of the bioassay tray. Either the test protein sample or the control sample was applied topically to the diet. After the samples had completely dried, the cavities were infested with 10 larvae per cavity. The cavities were then covered with a sealable lid and the trays were incubated at 27 ° C in the dark. Mortality and atrophy were evaluated at 5 and 7 days after infestation, and surviving larvae were weighed (Table 4). Similar tests were conducted for the Colorado potato beetle (CPB). CPB neonates were infested at a rate of 'one per cavity; the test was recorded after 6 days and the percent mortality was calculated for each proportion. The results (shown in Figures 2-4) indicate that CPB larvae are much more susceptible to K03 and K34 mutant endotoxins relative to wild-type endotoxin (1218-1). In addition, survivors who were fed diets treated with K03 and K34 endotoxin were severely atrophied as compared to the controls of the buffer solution, while survivors of CPB from the 1218-1 test were relatively large. TABLE . Initial Results of WCRW Bioassays WCRW Test # 1 5-days 7-days 5-days 7-days Regulator 1218 132 μg / cm2 4/40 4/40 10 10 NGSR 132 μg / cm2 22/40 23/40 55 57 M6 132 μg / cm2 38/40 40/40 95 100 WCRW test # 2 5-days 7-days 5-days 7-days Samples [PROTEIN] REGISTRATION% MORTALITY Solution 4/40 5/40 10 12 Regulator 1218 132 μg / cm2 7/40 7/40 17 17 NGSR 132 μg / cm2 24/40 26/40 62 65 M6 132 μg / cm2 31/40 35/40 78 88
Example 9: Determination of LC50 of Cry8-like Mutants A bioassay experiment was conducted to determine the LC50 of the M6 mutant similar to Cryd for West Corn Rootworm (WCRW) neonates. These bioassays were conducted essentially as set forth in Example 8. Five observations were made per level of treatment
(Table 5). Three WCRW neonates were added to each cavity for a total of 15 larvae / doses. The percent mortality was recorded after incubation at 27 ° C. The PROBIT analysis (SAS / STATU ser Guide Version 8 Chapter 54, 1999) was used to calculate the lethal concentration of the sample in which 50% of the larvae died (ie the LC50) - The summary of the dose response - Mortality of the WCRW neonates for this experiment is shown in Table 6. The Probit analysis was carried out and the result indicated that the LC50 of the mutant M6 protein similar to Cryd was 26 μg / cm2, with fiducial limits of 95% in 17.1 and 37.0. TABLE 5. M6 Protein Samples used in Concentration Concentration Bioassays of the Sample Extract in the Sample Diet (mg / ml) (μg / cm2) 2. 44 244 1. 22 122 0. 610 61 0. 305 30.5 0. 153 15.3 0. 076 7.6 0.038 3.8 TABLE 6. Percent of Mortality of WCRW Larvae in Various Concentrations of M6 Protein
(ND = no data) The Probit analysis of the previous data indicated that the LC50 of the M6 protein corresponded to a concentration of 26 μg / cm2, with fiducial limits of 95% in 17.1 and 37.0. A plot of the proportion of larval mortality as a function of the log of the M6 protein concentration is shown in Figure 1. Example 10: Corn Transformation Through Particle Bombardment and Regeneration of Transgenic Plants Immature corn embryos from donor plants The greenhouse is bombarded with a DNA molecule containing the optimized plant Cryl21ß-1 nucleotide sequence (SEQ ID NO: 5) operably linked to a ubiquitin promoter and the selectable marker gene PAT (Wohlleben et al., (1988) Gene 70 : 25-37), which confers resistance to the Bialafos herbicide. Alternatively, the selectable marker gene is provided in a separate DNA molecule. The transformation is done as follows. The recipes of the media are 'shown right away. Preparation of the Target Tissue The ears are stripped and sterilized on the surface in 30% Clorox ™ bleach plus 0.5% Micro detergent for 20 minutes, and rinsed twice with sterile water. The immature embryos are removed and placed with the side of the embryo shaft down (side of the spider up), 25 embryos per plate, in the 560Y medium for 4 hours and then aligned within the target area of 2.5 cm in the preparation for the bombing. DNA Preparation A plasmid vector comprising a plant-optimized Cryd-like nucleotide sequence (eg, Cryl21 d, SEQ ID NO: 5) operably linked to a ubiquitin promoter. For example, a suitable transformation vector comprises a UBI1 promoter from Zea mays, a 5'UTR from UBI1 and an intron from UBIl, in combination with a Pinll terminator. The vector additionally contains a selectable PAT marker gene induced by a CAMV35S promoter and includes a CAMV35S terminator. Optionally, the selectable marker can reside in a separate plasmid. A DNA molecule comprising a nucleotide sequence similar to Cryd as well as a selectable marker of PAT is precipitated on tungsten pellets of 1.1 μm (mean diameter) using a CaCl 2 precipitation procedure as follows: 100 μl of prepared tungsten particles in water 10 μl (1 μg) of DNA) in Tris EDTA buffer (1 μg of total DNA) 100 μl 2.5 M CaCl2 10 μl of 0.1 M spermidine Each reagent is added sequentially to a suspension of tungsten particles, while maintaining in the multi-tube apex forming apparatus. The final mixture is briefly sonicated and allowed to incubate under constant vortex formation for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, the tubes are centrifuged briefly, the liquid is removed, washed with 500 ml of 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl of 100% ethanol are added to the pellet of final tungsten particles. For bombardment with the particle gun, the tungsten / DNA particles are briefly sonicated and 10 μl is stained on the center of each carrier and allowed to dry approximately 2 minutes before bombardment. Particle Gun Treatment Sample plates are bombarded at level # 4 in particle gun # HE34-1 or # HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles / DNA. Subsequent Treatment After the bombardment, the embryos are conserved in the 560Y medium for 2 days, then transferred to the 560R medium containing 3 mg / liter of Bialafos, and subcultured every 2 weeks. After approximately 10 weeks of selection, callus clones resistant to selection are transferred to medium 268J to initiate plant regeneration. After maturation of the somatic embryo (2-4 weeks), well-developed somatic embryos are transferred to the medium for germination and transferred to the lit culture room. Approximately 7-10 days later, the developing seedlings are transferred to a 272V hormone-free medium in tubes for 7-10 days until the seedlings are well established. The plants are then transferred to inserts in seed boxes (equivalent to 2.5") containing potting soil and grown for 1 week in a growth chamber, subsequently being grown in 1-2 additional weeks in the greenhouse, then transferred to 600 classic pots (1.6 gallons) are grown to maturity.The plants are monitored and recorded for the expression of the Cryl21 d-1 protein by assays known in the art, such as, for example, immunoassays and western blotting. an antibody that binds the Cryl21 d-1 protein Bombardment and Culture Medium The bombardment medium (560Y) comprises 4.0 g / 1 of N6 basal salts (SIGMA C-1416), 1.0 ml / 1 of Erikson's Vitamin Mix (lOOOx SIGMA-1511), 0.5 mg / 1 thiamine HCl 120.0 mg / 1 sucrose, 1.0 mg / 1 2,4-D and 2.88 g / 1 L-proline (brought to volume with H20 di after adjustment at pH 5.8 with KOH); 2.0 g / 1 of Gelrite ™ (it was added after bringing to volume with H20 di); and 8.5 mg / 1 silver nitrate
(added after sterilizing the medium and cooling to room temperature). The selection means (560R) comprises
4. 0 g / 1 of basal salts N6 (SIGMA C-1416), 1.0 ml / 1 of
Mixture of Vitamins by Eriksson (lOOOx SIGMA-1511), 0.5 mg / 1 of thiamine HCl 30.0 g / 1 of sucrose and 2.0 mg / 1 of 2,4-D (brought to volume with H20 di after adjustment to pH 5.8 with KOH); 3.0 g / 1 Gelrite ™ (added after bringing to volume with H20 di); and 0.85 mg / 1 silver nitrate and 3.0 mg / 1 Bialafos (both added after sterilizing the medium and cooling to room temperature). The plant regeneration medium (288J) comprises 4.3 g / 1 of MS salts (GIBCO 11117-074), 5.0 ml / 1 of vitamins MS extract solution (0.100 g of nicotinic acid, 0.02 g / 1 of thiamine, 0.10 g / 1 of pyridoxine HCl and 0.40 g / 1 of glycine brought to volume with H20 di purified) (Murashige and Skoog (1962) Physiol. Plant 15: 473), 100 mg / 1 inositol, 0.5 mg / 1 of zeatin, 60 g / 1 sucrose and 1.0 ml / 1 0.1 mM abscisic acid (brought to volume with H20 di purified after adjusting to pH 5.6); 3.0 g / 1 Gelrite ™ (added after carrying the volume with H20 di); and 1.0 mg / 1 indoleacetic acid and 3.0 mg / 1 Bialafos (added after sterilizing the medium and cooling to 60 ° C.) Hormone-free medium (272V) comprises 4.3 g / 1 of MS salts (GIBCO 11117-074 ), 5.0 ml / 1 of vitamins MS extract solution (0.100 g / 1 of nicotinic acid, 0.02 g / 1 of thiamine, 0.10 g / 1 of pyridoxine HCl and 0.40 g / 1 of glycine brought to volume with H20 di purified (0.1 g / 1 of myo-inositol and 40.0 g / 1 of sucrose (brought to volume with H20 di purified after adjusting the pH 5.6), and 6 g / 1 of Bacto-agar (added after carrying the volume with H20 di purified), sterilized and cooled to 60 ° C. Example 11: Agrobacterial Mediated Transformation of Maize and Regeneration of Transgenic Plants For the Agrobacterium um mediated transformation of maize with an optimized Cryl21 d-1 plant nucleotide sequence (SEQ ID NO: 5), the Zhao method is preferably used (US Patent No. 5,981,840 and Patent Publication of PCT W098 / 32326, the contents of which are incorporated herein by reference). Briefly, in-matured embryos are isolated from maize and the embryos are contacted with a suspension of Agrobacterium um under conditions by which bacteria are capable of transferring the optimized plant Cryl21 d-1 nucleotide sequence (SEQ ID NO: 5) _ to at least one cell of at least one of immature embryos (stage 1: the infection stage). At this stage the immature embryos are preferably submerged in a suspension of Agrobacterxum for the initiation of the inoculation. The embryos are co-cultivated for a time with the Agrobacterium (stage 2: 1st stage of co-culture). Preferably, the immature embryos are cultured in solid medium after the infection stage. After this period of co-culture an optional "resting" stage is contemplated. In this resting stage, the embryos are incubated in the presence of at least one known antibiotic that inhibits the growth of Agrobacterxum without the addition of a selective agent for plant transformants.
(stage 3: resting stage). Preferably the immature embryos are cultured on the solid medium with antibiotic, but without a selection agent, for the removal of Agrobacterxum and for a resting phase for the infected cells. Next, the inoculated embryos are cultured in the medium containing a selective agent and the growth transformed callus is recovered (step 4: the selection step). Preferably, the immature embryos are cultured in the solid medium with a selective agent that results in the selective growth of transformed cells. The callus is then regenerated in plants (step 5: the regeneration stage), and preferably the calluses cultured in the selective medium are grown in the solid medium to regenerate the plants. Example 12: Dose-Response Bioassay for Endotoxins
Mutants against the Cotton Weevil, Anthonomus grandis Treatment: Four endotoxins were tested by dietary incubation for activity against the cotton weevil, Anthonomus grandis, obtained from USDA APHIS PPQ MPPC Insect Production; More Air Bldg. S-6414 Mission, TX: wild type (1218-1); mutant endotoxin K03; M6 mutant endotoxin; and K40 mutant endotoxin. Controls included regulatory solution alone and untreated diet.
Method: Five plates of 24 cavities were adjusted for each treatment, and 200 ml of diet of Bioserv cotton weevil
(# F9247B) was prepared according to the manufacturer's specifications. The diet was kept in a water bath at 40 ° C. A serial dilution of the endotoxin samples was prepared in microfuge tubes using sample aliquots of 3 mg, 1.5 mg, 0.75 mg, 0.37 mg, 0.19 mg. 5 ml of diet was removed from the water bath and placed in a scintillation vial. A protein sample was then added to the diet and mixed thoroughly. After mixing with 5 ml of diet the resulting concentrations were 600, 300, 150, 75 and 37 μg / ml (these proportions were selected to correspond to the topical proportions of 100, 50, 25, 12, 5 and 6.25 μg / cm2 ). 150 microliters of diet were added to four cavities of each of the five plates of 24 cavities. Each plate had the following configuration: TABLE 7: Configuration of the Test Plates
The controls included a single treatment plate with buffer, which was produced with all 24 cavities receiving 500 microliters of buffer. Another control plate was produced without addition to the diet. The amino acid sequence of mutant endotoxin M6 is set forth in SEQ ID NO: 70; the sequence of mutant endotoxin amino acids K03 is set forth in SEQ ID NO: 68; and the K40 mutant endotoxin amino acid sequence is set forth in SEQ ID? O: 94. Results: One week after infestation of the cotton weevil, the larvae of the cotton weevil were retrieved from the diet plugs of all 5 plates that contain the same mutant similar to Cryd and combined. The diet pills were carefully dissected under 4X magnification in order to recover all the larvae. TABLE 8. Results of Bioassay on Cotton Weevil Larvae Concentration of 1218-1 M6 K03 K40 Solution Regulating protein (ug / ml of diet) (500 ul / cavity) 600 5ss 4s 3ss 4 + ls 300 3ss 6s iss 5 + lss 150 2s 7s 3s 3ss 3 + lss 75 3s 3ss 2 + 4s 38 3 11 2s 3ss 4 + ls (s = atrophied, ss = severely atrophied).
Example 13: Second Dose-Response Test for Mutant Endotoxins against Cotton Weevil, Anthonomus grandis An examination of the effect of wild-type endotoxin (1218-1) and two endotoxin mutant proteins (M6 and K03) on the biomass total using a high and low dose of toxin shows that the mutants have increased pesticidal activity relative to wild-type endotoxin. The results are shown in Table 8. The bioassays were conducted as described in Example 12, with the following modifications. Three replicate plates were produced for each sample with four observations per dose per plate. The results were recorded in 96 hours of post-emergence when the larvae recovered from the diet, were counted and weighed. All the larvae of a particular treatment plate were weighed together, this number was divided by the number of individuals to give an average weight. TABLE 9: Effect of Endotoxins on the Larval Weight of the Cotton Weevil Endotoxin Larval Weight Larval Weight (mg) in 600 (mg) in 19 μg / ml of diet μg / ml of diet (These results are also shown graphically in the Figure 5) . Thus, in the highest dose of endotoxin of 600 μg per ml of diet, the treatments with 1218-1 and M6 show a very significant reduction in biomass of 88.6% and 94.9%, respectively. These data represent an increase of 8.80 and 19.4 times in the activity for 1218 and M6, respectively, when compared to the control of regulatory solution. Treatment with the K03 protein produced no survivors in the 600 μg treatment in any of the replicates. In comparison, at the lowest dose of 19 μg per ml of diet, the data indicate a reduction of 50.0%, 63.7%, and 82.6% in the biomass for 1218, M6 and K03, respectively, when compared with the control of regulatory solution. Thus, in a dose that is up to 30 times lower, the K03 mutation in 19 μg per ml of diet exhibits almost equivalent activity (82.6% reduction in biomass) when compared to wild-type endotoxin (1218) in 600 μg per ml of diet (68.6% reduction in biomass). In addition, at a dose of 19 μg per ml of diet, endotoxin K03 shows activity that is 2.08 and 2.87 times better activity than M6 and wild-type endotoxins (1218-1), respectively. Explanation of results: The data indicate a clear reduction in weight for all polypeptide samples when compared to the control buffer solution. Additionally, all mutant endotoxins reduced larval growth below the growth observed for native or wild-type endotoxin (1218-1). The mutants K03, K35 and K40 produced results of few or no larvae recovered at the highest doses and a high degree of atrophy at lower doses. The K40 mutant protein produced an approximately 5-fold reduction in weight gain at the highest doses when comparing wild-type endotoxin. When compared to the control buffer solution, the K40 mutant produced reductions ranging from 46 times at the highest dose to 5 times at the lowest dose based on the comparison of the average larval weights at those doses. Similarly, the results for mutant K03 showed effects ranging from complete mortality at the highest dose to 200 times the reduction in weight at the next dose and 5 times at the reduction in weight at the lowest dose. The K35 mutant showed a pattern similar to that of the K03 mutant. Example 14: Bioassay for Testing the Pesticide Activity of the K03 Polypeptide Similar to Cry8 Mutant against the Corn Scarab (Chaetocnema pulicaria) A bioassay experiment was conducted to determine whether the corn beetle (Chaetocnema pulicaria) is susceptible to the mutant K03 endotoxin ( SEQ ID NO: 68). Since corn beetles are predominantly fed in the upper layer of the leaf cells, a known amount of toxins can be applied to the surface of the leaf or the leaves can be covered with toxin by immersion. The insects are then allowed to feed on the leaves treated with toxin and after a prescribed period of time, the percent mortality can be calculated. For this trial, corn scarabae were collected in the field and presented with leaf discs that were submerged in either K03 or a buffer solution. Leaf discs were evaluated in a CD International 128-well bioassay tray (catalog number BIO-BA-128 from CD International, Pitman, New Jersey 08071) in which each cavity was first filled with 1 ml of agar solution molten. Once the agar solidified, a 1.5 cm filter paper (VWR, catalog number 28309-989) was placed on top of the agar plug and moistened with 25 μl of sterile water. Then, the leaf disks (diameter of 1 cm) were retired from the rolled leaves (harvests of the stage V8 maize plants) and immersed in either a solution of K03 (1 mg / ml) or a buffer solution of 20 mM sodium carbonate (pH 10.5). Both solutions contained 0.01% in Twen 20 to aid in the dispersion of the sample over the entire leaf surface. Once the discs of leaves submerged as wetted dried, they were placed on top of the filter paper in the bioassay tray so that one disc was present per cavity in the 128 cavity bioassay tray. Each cavity was then infested with a corn beetle and covered with sealable lids supplied by International CD, Pitman, New Jersey. The trial was recorded after 5 days and the percent mortality was calculated. The examination of leaf discs after 5 days showed moderate levels of feeding damage as is
- observed by the presence of thin brown strips on both sheets treated with K03 and buffer. It was observed that a larger number of corn beetles died after the leaf discs treated with K03 were fed as compared to those that were fed on leaf discs treated with buffer solution (see Table 10). Table 10. Results of the Corn Beetle Bioassay Example 15: Modification of GC Content to Create Optimized Nucleotide Sequences Analysis of the coding regions of several organisms A data set containing 1831 maize cDNAS with full-length coding regions they were plotted against the GC content of the coding sequence (Figure 8, "ORFs" shown in the upper panel). The graph showed a bimodal distribution with most of the sequences (approximately 2/3) in the low GC mode that reached the maximum in approximately 51% of GC and approximately one third in the high GC mode that reached the maximum in approximately 67% of GC. While this is the largest set of full-length cDNAs of corn so analyzed to date, based on the estimated total gene count of 50,000, this data set can only represent approximately 3.6% of the transcriptome. Consequently, a UniGene assembly sequence data set based on EST is believed to represent the majority of maize genes and contains 84,085 sequences was also analyzed (Figure 8, "UniGenes" shown in the lower panel). As used herein, a Unigene represents a consensual sequence of the assembled Est. The Unigene data set results from an application of the CAP3 assembly algorithm (see Huang and Madan (1999) Genome Research 9: 868-877). The analysis of this data set confirmed the results of previous full-length cDNAs by showing a bimodal distribution with a similar proportion of high and low GC genes. The bimodal distribution for the UniGene data set was concentrated at 45% and 64% GC, slightly less than for the smaller full-length cDNA data set, probably due to the inclusion of non-coding sequences 3'- UTR rich in At not trimmed remaining. GC analysis was performed for other plants. A corresponding study of the coding regions (ie cDNA "ORFs" or Open Reading Structures) revealed very similar bimodal distributions for rice and wheat (2,400 rice sequences and 800 wheat sequences were analyzed). In contrast, the analysis of Arabidopsis (25,700 sequences), Solanaceae ssp. (2,000 sequences), and soybean (G. max, 400 cDNAs, or 49,300 UniGene assemblages), all revealed individual bimodal distributions with peaks or maximums between 42-44% GC contents. In a review of other organisms, a study of warm-blooded mammalian cDNA ORFs all revealed distributions of broad GC content with suggested bi-modality. In this analysis, 19,200 sequences were analyzed from human, 12,000 from mouse (M. musculus), 900 from cattle (B. tauros) and 1,100 from chickens (G. gallus). An examination of the organisms of other major eukaryotic groups showed uni odal distributions with peaks ranging from 38% -56% GC content for C. elegans (16,000 analyzed sequences), D. melanogaster (14,800 secuencais) and S. cereviseae ( 6,300 sequences). Unimodal distributions were also found for sequences of three eubacteria (E. coli, 4,200 sequences, B. sublilus, 4,000 sequences, Synechocyslís sp 3,200 sequences) and four Archaea (T maritima, 1,800 sequences, Tjannaschii, 1,800 sequences, A. fulgidus, 2,400 sequences, H halobium, 2,600 sequences (with very high total GC content.) Thus, a broad study of the GC content distribution showed that, in contrast to most organisms, monocotyledonous cereals have a clearly bimodal GC content distribution. The warm-blooded vertebrates also showed a bimodal tendency, but this was less pronounced than the monocotyledons. MRNA profiling To examine the relationship between gene expression and GC content, mRNA expression of maize genes in high GC mode (centered at approximately 67% of GC content) and low (centered in approximately 51% of GC content) was investigated using both the EST distribution analysis (over 400,000 ESTs) and the Lynx MPSS technology (63.4 million portions of 17-mer) (see Brenner et al., 02000) 'Nature Biotechnology 18: 630-634 , Brenner et al., (2000) PNAS 97: 1665-1670 for information on Lynx MPSS). The data showed that while gene expression varied widely within the high and low GC modes, when average expression levels between 12 different tissue categories were considered key, the total average expression level of high GC genes and low in corn was similar. Example 16: Method to Optimize GC Content of Genes In view of the discoveries about GC content described in the above, it was of interest to develop computerized methods to modify the coding sequences of any gene from any source of organism in a structure compatible with that preferred by corn and other cereals. As discussed in the above, other major cereals such as wheat and rice show bimodal distributions similar to corn, and the preferred high GC codons are the same. Consequently, the methods for sequence optimization described below would be useful not only for the expression of the augmented gene in corn but also in all cereals. These methods allow the coding sequences of various organisms to be optimized for expression in cereals and thus provide improved transgenic plants, for example, a crop plant such as corn. Two exemplary optimization methods are presented below. However, it is recognized that one skilled in the art would be able to optimize a sequence using a variety of methods and would still create a sequence of the invention. Method 1: Content of GC Tuned This method allows the selection and generation of an altered nucleotide sequence containing a specified percentage of GC content (within 0.5%). This method employs the proportional codon usage frequencies and takes into account the tendency of the coding regions to have a GC content gradient from the 5 'to 3' end. The proportional codon usage frequencies are arranged in weighted tables to implement the method. Stage 1. Determine if the selected GC content is theoretically feasible. First, the highest and lowest theoretical GC content are calculated for the sequence of interest. In this step, codon substitutions are made in the original sequence to generate altered sequences with the highest and lowest possible GC content that still encodes the same polypeptide as the original sequence. The original sequence may of course be a predicted coding or polypeptide sequence from any source. Where there are two codons that are equally poor in GC, the codons are substituted in proportion according to the proportional codon tables of low GC mode (see Table 11, Proportional codon table more Rich and more Poor in GC, Columns of frequency) Proportional codon (on the left)). For example, GC poor codons corresponding to alanine include both GCT and GCA. From the low GC mode proportional codon table, the relative frequencies of GCA and GCT are 30.4% and 36.5%, respectively. So, in proportion to their relative frequencies, for the substitution of low GC mode, the substitution frequency of GCA must be 30.5 + 30.4) = 45. % and the substitution frequency of GCT should be 36.5 + 30.4) = 55.6%. These percentages have been calculated and are presented in Table 11, GC Proportional Extreme Columns / Lower GC (on the right). Thus, for low GC mode, GCA must be replaced by 45.4% of the alanine codons and GCT by 55.6% of the alanine codons. Similarly, to determine the highest possible GC content, substitution frequencies are presented in Table 11, Columns of 'GC Proportional Extreme / Higher GC'. Thus, for alanine, the high GC content codons are GCC and GCG, which are found in frequencies of 47.2% and 38.7%, respectively. Thus, in the high GC mode, the GCC codon is replaced with 54.9% alanine codons [47.2 + 38.7) = 54.9%] and the GCT codon is replaced by 45.1% alanine codons [38.7 / 47.2 + 38.7) = 45.1%]. In this way, two new altered nucleotide sequences are created, one with the lowest GC content possible and the other with the highest possible GC content, according to the proportional codon usage of Table 11. These sequences of Altered nucleotides still encode the same polypeptide as the original nucleotide sequence. In a computer program written to implement this algorithm, if the desired GC content is at or outside these high and low GC content values, the program may output the altered nucleotide sequence for the highest GC content. and lower. A feature of this method is that in the altered sequence, the codons for any given amino acid can not be uniformly distributed and could be block stretches of the same codon for a particular amino acid.
Table 11: Richest and Poorest Proportional Codon Table in GC Proportional Codon Frequency Proportional End GC Amino Acid General Codon GC High GC Low GC Higher • GC Lower
GCA 19.88% 5.96% 30.38% 45.43%
GCC Wing 32.00% 47.20% 20.61% 54.93% GCG 22.83% 38172% 12.51% 45.07% GCT 25. 29% 8.13% 36. 49% 54.56% AGA 16., 20% 3.57% 24. 18% 100.00Í AGG 25., 71% 22.04% 26. 57% Arg CGA 7.82% 3.43% 10., 24% CGC 23., 11% 40.18% 13. 28% 61.20% CGG 15,, 94% 25.47% 11., 56% 38.80% CGT 11 .22% 5.31% 14., 17% Asn AAC 60, .68% 92.55% 46., 57% 100.00% AAT 39, .32% 7.45% 53., 43% 100.00%
Asp GAC 55, .30% 90.32% 37. .75% 100.00% GAT 44.70% 9.68% 62, .25% 100.00%
Cys TGC 67.97% 92.08% 54, .31% 100.00% TGT 32 .03% 7.92% 45, .69% 100.00%
Gln CCA 34.97% 9.41% 47.49% 100.00% CAG 65.03% 90.59% 52.51% 100.00% Glu GAA 34 .46% 9.55% 46.37% 100.00% GAG 65.54% 90.45% 53. 63% 100.00% GGA 20 .26% 7.62% 28.39% 48.83% Gly GGC 37.85% 62.57% 23.22% 72.82% GGG 20.48% 23.35% 18.65% 27.18% GGT 21.41% 6.45% 29.74% 51.16%
His CAC 56.40% 87.35% 40.16% 100.00% CAT 43.60% 12.65% 59.84% 100.00% ATA 19.32% 4.90% 24.91% 37.25% lie ATC 48.33% 88.53% 33.13% 100.00% ATT 32.34% 6.57% 41.96% 62.75% CTA 8.04% 2.73% 10.82% CTC 25.61% 44.16% 15.63% 50.06% Leu CTG 27.10% 44.05% 19.29% 49.94% CTI 18.24% 4.61% 24.48% TTA 6.63% 0.54% 10.18% 100.00% TTG 14.37% 3.91% 19.59% Lys AAA 28.98% 7.57% 39.06% 100.00% AAG 71.02% 92.43% 60.94% 100.00% Met ATG 100.00% 100.00% 100.00% 100.00% 100.00%
Phe TTC 64.74% 94.80% 50.08% 100.00% TTT 35.26% 5.20% 49.92% 100.00% Pro CCA 26.66% 10.21% 36.80% 51.94% ccc 22.07% 31.91% 15.40% 40.09% CCG 25.74% 47.67% 13.76% 59.90% CCT 25.53% 10.21% 34.05% 48.05%
TAA 30.64% 24.89% 33.00% 100.00% TAG DETENTION 34.95% 38.33% 33.00% 51.03% TGA 34.41% 36.78% 34.00% 48.97% AGC 21.90% 32.94% 16.65% 37.50% AGT 10.93% 2.56% 15.26% 25.34%
Be TCA 15.95% 4.23% 21.75% 36.12% TCC 20.60% 31.87% 14.46% 36.29% TCG 13.22% 23.02% 8.68% 26.21% TCT 17.40% 5.38% 23.20% 38.53% ACÁ 23.81% 5.61% 34.03% 51.40%
Thr ACC 31.88% 46.40% 22.29% 52.75% ACG 20.74% 41.57% 11.50% 47.25% ACT 23.57% 6.42% 32.18% 48.60%
Trp TGG 100.00% 100.00% 100.00% 100.00% 100.00%
Tyr TAC 63.47% 94.76% 47.77% 100.00% TAT 36.53% 5.24% 52.23% 100.00% GTA 9.86% 2.37% 14.58% 28.73%
Val GTC 29.82% 42.63% 21.73% 45.93% GTG 35.25% 50.19% 27.52% 54.07% GTT 25.07% 4.81% 36.17% 71.27% Stage 2. If the desired GC content is between the highest and lowest possible GC percentage for the original sequence, the sequence can be altered accordingly. The altered sequence from step 1 having GC content closest to the desired GC 'content is selected. This sequence is then further altered according to the codon usage tables so that the GC content is increased or decreased to the desired level. As an initial stage in the changing GC content, only changing the positions of the third codon should be considered. (However, for arginine codons, there could theoretically be changes in the first two codon positions when the preferred low or high GC codon is replaced - see Table 12 below). If the GC content needs to be increased, changes can be made from the N-terminus or 5 'end to the C-terminus or 3' end to preserve and even increase the negative GC gradient in the coding region. Similarly, if the GC content needs to be decreased, changes can be made from the C-terminus or 3 'end to the N-terminus or 5' end to preserve and even increase the negative GC gradient. Not all amino acid codons will be replaced because some codons can be avoided. Among the amino acids and their codons available to change in the method are the following: Table 12: Codon Substitutions to Increase or Decrease GC Content AA to Decrease GC to Increase GC Wing GCT GCC Arg AG CGC Asn AAT AAC Asp GAT GGC Gly GGT GGC His CAT CAC He ATT ATC Leu CTT CTC Pro CCA CCG Be TCT AGC Trh AC ACC Val GTT GTC Output of results Where a computer program implements the method, the output may include a nucleotide sequence which is the sequence altered according to the above method (s). This sequence is then translated into a predicted polypeptide which is compared to the polypeptide encoded or predicted to be encoded by the original nucleotide sequence to ensure that, where desired, the polypeptide sequence has not been changed by alterations in GC content of the nucleotide sequence. Method 2 to Optimize Genes: Stage 1. The first stage is the same as it is described for method 1 except that the appropriate codons are substituted in an alternating pattern, with any excess of one applied at the beginning (ie oriented towards the N- terminal), and the codons that end in G or C are applied first where possible. As in method 1, two altered sequences are generated which represent the highest and lowest GC content for a sequence that is (if desired) still encodes the same polypeptide as the original sequence. If the desired GC content is at or outside these higher and lower theoretical GC content values, the sequence closest to the desired level of GC content is chosen for further alteration. Step 2. If the desired GC content is between the highest and lowest possible GC percentage for the original sequence, the sequence may be altered accordingly. The study of the 1831 maize ORFs described in Example 15 revealed patterns in the GC content and codon content of the maize genes. The coding regions of the maize genes were shown to have a total GC content of 54.5%, with a total GC content at the third codon position of 63%. The GC content of the third position varies as a function of the relative position in the coding region. Thus, for the first 180 nucleotides (first 60 codons, or approximately the first sixth of the coding region), the GC content of the position of the third codon is 70%. For the second 180 nucleotides (seconds 60 codons, or approximately the sixth of the coding region), the GC content of the third codon position is 65%. For the remainder of the coding region, the GC content of the third codon position is approximately 60%. Thus, in approximately the first 60 codons, the GC content of the third codon position is 11% higher than the total GC content; in approximately the second 60 codons, it is 3% higher, and in the rest of the coding region it is 4.8% lower than the total GC content. A scatter plot for the GC content of the third codon position (designated "ORF3GC") against the total GC content (designated "ORFGC") was used to determine the best fit line for this data using the minimum method squares. The resulting equation gives the general relationship between ORF3GC and ORFGC for maize genes, as follows: ORF3GC = 2.03 * ORFGC - 47.2. Changes made to the third position of the codon will generally have an effect on the content of ORFGC in a way according to this equation. 'However, the graph of ORF3GC versus ORFGC is currently slightly curved at the extremes, especially at the high end GC levels, where the slope decreases. This decrease in slope is probably the result of deviations in amino acid composition as well as saturation of GC content in codons that may vary in the GC content of the third position. Thus, unless the above equation is modified, the correct ORF3GC value in relation to ORFGC will generally be underestimated. This is especially true where the percentage of total GC of a sequence is intermediate, a situation in which the alteration of GC content is particularly likely to be desirable. A computer program was designed and implemented to perform the above methods. After using this program (method 2, also known as "10.2") to apply the methods in the equation form and using the previous original linear equation, the empirical observations allowed the correction of the original equation • to one that resulted in better Correlation of ORF3GC with ORFGC. The resulting modified equation is ORF3GC = 2.06 * ORFGC - 44.2. Thus, changing ORF3GC will generally be expected to cause a concomitant change in ORFGC. Given the other previous information that considers the trend towards a gradient of ORF or negative ORF3GC content, the following equation can be developed. Assign L = protein length in amino acids or codons Assign B = level of ORF3GC% base, to which, for example 11% will be added in the first ORF section Assign ORF3GC = or ORF3GC% total ORF Assign ORFGC = or ORFGC% total the ORF Linear equation = 0RF3GC = 2.06 * ORF3GC - 44.2 Thus: Number 3GC nts = Number 3GC nts in the first section ORF + Number 3GC nts in the second section ORF + Number 3GC nts in the rest of the ORF It is the same: L * (ORF3GC / 100) = 60 * (B + ll) / 100 + 60 * (B + 3) / 100 + (L-120) (B-4.8) / 100 Substitute with the linear equation: L * (2.06 * ORFGC - 44.2) / 100 = 60 * (B + 11) / 100 + 60 * (B +
3) / 100 + (L-120) (B-4.8) / 100 Simplify: 2.06 * L * ORFGC - 44.2 * L = 60B + 660 + 60B + 180 + LB -4.8 * L - 120B + 576 2.06 * L * ORFGC-44.2 * L = 1416 + LB -4.8 * L 2.06 * L * ORFGC- 39.4 * L = 1416 + LB Resolver Example: Assign Length = 300 Assign ORFGC = 60 Then: 2.06 * 300 * 60 - 39.4 * 300 = 1416 + 300B 37080 - 11820 = 1416+ 300B 23844 = 300 BB = 79.48 or 79.48% ORF3GC as the base Therefore the objective ORF3GC in the first section will be 90.48, in the second section 82.46 and in the last section approximately 74.68. The target ORF3GC in the last section will be affected by the protein length due to the limitation of the first two sections to 60 codons each, leaving the rest of the ORF to last section. Thus, the number of codons in the last section will vary depending on the length of the protein. As the described methods are applied to proteins of various lengths, the amount of GC adjustments made in the last section will then be affected by the length of this section. Step 3. Creation of a template QRF For the process, a "Template ORF" with coding sequence based on the codon table of general corn is created so that the normal relative proportion of codons is preserved (rounded to the whole number closest complete). Codons that have a G or C in the third position are generally concentrated at the N-terminus or 5 '. Also, codons are distributed such that excess codons are substituted at the 5 'N-terminus of the coding region, followed by an alteration of the codons to disperse their location in the protein. Table 13: Table of General Corn Codons (1831 seqs.) Freq. Amino acid! Codon, of Codon 25 This template ORF is then used to adjust the original coding sequence to conform the GC gradient according to the principles summarized in the above. In this process, the linear equation discussed in the above is used to calculate the base ORF3GC. In addition, the content of OFR3GC is adjusted in view of the increased GC content in the first and second 60-codon regions of the ORF, as described above. Thus, the content of ORF3GC is adjusted by dividing the template ORF into the three sections: the first 60 codons, the second 60 codons and the rest of the ORF. For each section, the ORFGC and ORF3GC are determined and compared and the alterations made to the original sequence are consequently. Thus, for example, the first ORF section of 60 codons is evaluated to determine if the 0RF3GC needs to be raised or decreased. (Frequently the 0RF3GC will need to be raised to agree with the negative GC gradient along the coding sequence). If the 0RF3GC needs to be elevated, then the codon substitutions are made according to Table 11 starting at the N-terminal end of the section. Similarly, if the 0RF3GC needs to be lowered, the corresponding substitutions are made to decrease the GC content according to Table 11 and starting at the 3 'end of the C-terminal region as described in more detail in the foregoing. The codons having a G or C in the third position are used in relative proportions as they occur naturally (as shown in Table 11, columns of extreme GC proportional / GC higher or GC lower, as appropriate). In this way, alterations are made in this section until the desired level of 0RF3GC is reached. If the desired level can not be reached without changing the encoded polypeptide, then changes can be made to bring the GC content as close as possible to the desired level or alternatively the amino acid changes can be considered that will allow the alteration of the content of the GC of the nucleotide sequence but that would not significantly affect the function of the encoded polypeptide. A person skilled in the art is familiar with the genetic code and would be able to make such sequences and perform functional tests to determine if the function has been affected in this way by changing the sequence to transform the undesirable change. This process is then applied to the second section of 60 codons of the same code and then to the rest of the coding region. Again, if the ORF3GC needs to be lowered, which will often be the case in the rest of the coding region, this is done in this way starting from C-terminus and moving to an N-terminus. Once the sequences of these three sections are. have altered as described, the sections are combined to create a second template ORF and the ORFGC and 0RF3GC of this sequence are determined. Due to the changes in this example that were made to 0RF3GC before the ORFGC, the ORFGC may need to be adjusted to the desired level. If the difference between the second template ORFGC and the desired ORFGC is less than an equivalent ucleotide, the sequence need not be changed. However, if the difference is more than one nucleotide equivalent, then the number of necessary changes is determined according to the following equation: Percent difference of ORFGC = desired ORFGC -ORFGC of template 100 * N / L = 100 * (G + C) d / -100 * (G + C) t / L 'N = (G + C) d - (G + C) t A positive number indicates the number of G or C to be added; a negative number indicates the number of G or C to be subtracted. Additional changes are made in the same manner as described above to adjust the content of the GC of the entire coding region. In this way, an altered nucleotide sequence is obtained due to the desired GC content and conforming to other known properties of the coding regions of the desired host organism, as exemplified particularly herein for corn. It will be apparent from the methodologies described herein that any host organism could be studied for GC content patterns and a corresponding substitution pattern designed and implemented to make the alterations of GC content adequate in a sequence of interest. Additional adjustments to sequences Additional changes can be made to an altered sequence to optimize its expression and conformance to the structural standard of the maize gene. For example, it may be desirable to make changes to the Kozak context, which is believed to be involved in the optimization of translation efficiency through the proper coupling of the ribosomal complex. The Kozak context ("ATGGc") occurs around the start codon. Thus, the second amino acid usually starts with a codon that starts with "G", especially "GC", which corresponds to the amino acid alanine. If, on the other hand, the codon after the ATG start codon does not start with a G, then changing e, sa G generally results in a change in the corresponding amino acid (except for arginine). Such a change may not be desirable if it is important that the sequence continue to encode exactly the same polypeptide sequence, but if this first portion of the protein is a transit peptide or is otherwise segmented from the final mature protein, such changes do not they can have an effect on the final polypeptide product. Other adjustments can also be made to the coding region, such as the removal of potential RNA processing sites or degradation sequences, removal of premature polyadenylation sequences, and removal of intron or donor splice sites. The splice sites of
- Potential intron-donors can be identified through publicly available computer programs such as
GeneSeqer (see Usuka et al., (2000) Bioinformatics 16: 203-211). Additional changes can be made to add or subtract restriction enzyme sites or, for example, to interrupt regions of strong palindromic tendency that could result in the formation of the mRNA hairpin spiral. As one skilled in the art will appreciate, such changes are made with consideration of whether the encoded amino acid is also changed. Where possible, sequence changes that replace frequently used codons should be selected on the changes that replace codons less frequently used. Example 17: Optimization of Nucleotide Sequence K04 Similar to Cry8 Mutant The original K04 mutant nucleotide sequence (set forth in SEQ ID NO: 21) was modified for optimal GC content. This modified sequence is set forth in SEQ ID NO: 63 and encodes the original K04 mutant protein (set forth in SEQ ID NO: 22), as demonstrated by the translation of SEQ ID NO: 63 set forth in SEQ ID NO: 64 Additional changes will then be made to improve the expression. These changes to improve the expression of this sequence included the removal of the potential intron-donor splice sites, / ie GT AG), the modification of the potential premature polyadenylation sites, removal of a potential RNA degradation signal and modification of the restriction sites to facilitate cloning without appreciably altering the codon usage of the reconditioned sequence. These changes are shown in Table 14. The sequence containing these additional changes is known as "1218-1K054B" and is set forth in SEQ ID NO: 65 and, as demonstrated by the translation of SEQ ID NO: 65 set out in SEQ ID NO: 66, SEQ ID NO: 65 encodes the original K04 mutant protein as set forth in SEO ID NO: 22. Table 14. Changes made to the K04 sequence in addition to the GC content optimization.
Example 18: Bioassay for Testing the Pesticide Activity of
K04 Polypeptide Similar to Cry8 Mutant against West Corn Worm and Southern Corn Rootworm A bioassay experiment was conducted to determine the efficacy of mutant K04 polypeptide similar to
Cryd against larvae, of western corn rootworm
(WCRW) and southern corn rootworm (SCRW). These bioassays were conducted essentially as set forth in Example 8 except that the individual cavities were infected with eggs instead of neonates. Approximately 25 eggs were added to each bioassay cavity with a total of 7 observations at each dose level. The majority of eggs were incubated within 24 hours. The percent mortality was recorded after 5 days of incubation at 27 ° C. The summary of the mortality data shown in Table 15 indicates that the K04 mutant similar to Cryd killed up to half of the WCRW larvae with dying survivors (dead or near dead). The results shown in Table 16 reveal that SCRW is much more susceptible to mutant K04. It was observed that 80% of SCRW larvae died within 72 hours after feeding at 50 μg / cm2 mutant K04 protein similar to Cryd (data not shown) and by day 5, all SCRW were dead (see Table 16). ). Table 15. Results of WCRW bioassay fed with K0.
regulator * Dying survivors Table 16. SCRW bioassay results fed K04
EXAMPLES 19: In Vivo Study of the Degradation of Protein 1218-1 by the Intestine Proteases of the West Corn Rootworm (WCRW) An in vivo investigation of the degradation pattern of the truncated protein molecule 1218-1 produced by Western corn rootworm gut proteases were carried out in order to identify the proteolytic sites that can cause degradation and loss of insecticidal activity of the 1218-1 protein molecule. The trick 1218-1 protein used for this experiment (SEQ ID NO: 12) was generated from an expression vector pET-28a (Novagen, San Diego, CA). The expressed protein was SHis-Tag purified and treated with thrombin according to the manufacturer's protocol. A small T7 portion was retained with the protein sample 1218-1. 19 additional amino acid residues (1868.01 Da) before the first methionine of the truncated protein 1218-1 were retained after treatment with thrombin. Protocol Larvae WCRW from medium to late 3- actively puffed chickens were sub-fed on agar plates overnight. The sub-ali larvae were fed with a 1218-1 protein solution of 0.5 mg / ml containing blue food dye and sucrose or were fed with solution alone (a control preparation containing sucrose and food dye). The larvae that ingested a sufficient amount of the control test solution
(which stained the food bolus) were allowed to settle at room temperature for one hour. After 1 hour, the larvae were placed on ice for dissection. The middle intestines were carefully removed under a cold carbonate buffer fortified with a protease inhibitor cocktail (Complete ™ protease inhibitor cocktail fortified with 5 mM EDTA, Roche Diagnostics, Mannheim, Germany). After the body of fat and the trachea were removed, each midgut was rinsed with several drops of the same buffer. The medium intestines then recovered from the buffer and the excess buffer was removed with a paper towel. The middle region of the midgut was then cut with a razor blade and 5 μl of buffer was added to the spilled lumenal contents. Therefore, a midgut equivalent was equal to a 5 μl aliquot of the recovered intestine solution / buffer. Western analysis was performed to identify sample 1218-1 and its degraded fragments of the lumenal contents of the intestine. The chemoluminescent Immunodetection kit WestermBreze ™ Invitrogen (Carisbad, CA) was used according to the manufacturer's protocol for the analysis and visualization of samples 1218-1. Results The majority of the 1218-1 protein fed to western corn rootworm larvae is processed in a single predominant band of less than 62 kDa, as observed in a 10 min western blot exposure. Numerous smaller and distinct immunoreactive bands were observed at a 30 minute exposure of the Western blot that were different from the immuno (crossed) reactive protein portions present in the control preparation. The immunoreactive bands in the control preparation were used to discriminate the background of the actual 1218-1 degraded protein fragments shown in the blot.
These results indicate that in the western corn rootworm, the 1218-1 protein is first processed into a protein of approximately 62 kDa, and then further degraded by the proteases of the intestine into small protein fragments. Western analysis after in vivo digestion of the 1218-1 protein allowed the identification of proteolytic sites and provided a modification of these sites in order to produce a more effective insecticidal protein. Example 20: SDS-PAGE Analysis of Protein Degradation of Protein 1218-1 An in vitro investigation of the degradation pattern of the truncated protein molecule 1218-1 by proteolytic enzymes was carried out in order to identify the Proteolytic sites in the molecule that may be available for modification. The truncated 1218-1 protein used for this experiment (SEQ ID NO: 12) was generated from a pET28a expression vector (Novagen, San Diego, C?). The expressed protein was purified with His-Tag according to the manufacturer's protocol. Both the His-Tag and a small T7 portion were retained with the 1218-1 protein sample. Western analysis was carried out according to the manufacturer's protocol (chemiluminescent immunodetection equipment; Western Breeze ™ Invitrogen, Carisbad, CA) in order to identify the protein sample 1218-1 and the protein fragments resulting from the digestion. proteolytic For each digestion, 3 μg of protein 1218-1 'and 0.03 μg of enzyme were used. The following enzymes were used for this analysis: chymotrypsin, trypsin and papain. The digested 1218-1 samples, as well as an undigested sample 1218-1, were run on a gel and stained. Results Micrographs were developed and the protein bands were removed from the gel and subjected for N-terminal sequencing. The sequencing results revealed the cleavage sites generated from proteolytic digestion. The residue positions indicated below are relative to the first methionine of the protein sample 1218-1, not the His-Tag methionine. N-terminal sequencing of the approximately 70 kDa band in the chymotrypsin-treated sample indicated cleavage of the 1218-1 protein on the carboxyl side of methionine at position 48. Thus, chymotrypsin removed the first 48 amino acid residues in the N-terminus of the 1218-1 protein. N-terminal sequencing of the approximately 57 kDa band in the trypsin-treated sample indicated segmentation of sample 1218-1 on the carboxyl side of arginine at position 164. In addition, N-terminal sequencing of the band approximately 70 kDa indicated that the 1218-1 protein sample was cleaved by trypsin on the carboxyl side of the lysine at position 47. At least 9 major bands were observed from the papain digestion of the protein sample 1218-1 . When these digested fragments were isolated and sent for N-terminal sequencing, the results of the sequence analysis indicated that 7 of these major bands all contained the same N-terminal sequence at position 49. Thus, these results indicate that there were multiple Segmentations of the 1218-1 protein molecule by means of papain and that these proteolytic sites occur in the C-terminal of the molecule. Example 21: Mutation of Proteolytic Sites in a Modified Pentin-1 Protein Proteolytic Digestion of a Modified Pentin-1 Protein The Pentin-1 protein was modified by removing the putative signal sequence and adding the N-terminal 4 following amino acids; MADV (SEQ ID NO: 124) (see U.S. Patent Nos. 6,057,491 and 6,339,144, incorporated herein by reference). These 4 amino acids were added in order to increase the production of the modified pentin-1 protein in a host cell.
The modified pentin-1 protein (Mod P-19 was produced using the pET30 protein expression system following the manufacturer's protocol (Novagen, Madison, Wl) .The modified pentin-1 protein, purified, at a concentration of 1 mg / ml, was subjected to proteolysis by trypsin chymotrypsin and papain (digestions occurring in 1/50 w / w) After electrophoresis and staining of digested protein samples, selected digestion fragments of modified pentin-1 They cut the trypsin, chymotrypsin and papain locks in the spotting and sent them for N-terminal sequencing.The sequencing results indicated that trypsin, chymotrypsin and papain all segmented the modified pentin-1 protein at the N-terminus. These segmentation sites are designated by the uppercase letters in the following set of contiguous amino acids of the N-terminal of the modified pentin-1 protein: madvaFstQaKaskd (SEQ ID NO: 125) More specifically, chymotrypsin was then segmented 6-F, papain then segmented 9-0 and trypsin then segmented ll-K. Site-Directed Mutagenesis of Modified Pentin-1 The mutagenesis of the modified pentin-1 sequence to remove proteolytic cleavage sites initiated an effort to increase the toxicity of pentin-1 against the western corn rootworm, WCRW. Due to the close proximity of the three N-terminal cleavage sites associated with trypsin, chymotrypsin and papain, all three N-terminal cleavage sites were mutated simultaneously. Mutations were introduced using the Mutagenesis System Directed to the GeneTailor ™ Site following the manufacturer's protocol (Invitrogen, Carisbad, California). The first 30 amino acids of the modified pentin-1 protein (Mod P-l) as well as the first 30 amino acids of the modified pentin-1 mutant sequences called NEZ2 and NEZ3 are shown in the following alignment. Those amino acids that were changed in the mutants are shown in bold. Mod P-l: MADVAFSTQAKASKDGNLVTVLAIDGGGIR (SEQ ID NO: 126)
NEZ 1 MADVAGSTGAGASKDGNLVTVLAIDGGGIR (SEQ ID NO: 127) NEZ 2 MADVAGSTGAHASKDGNLVTVLAIDGGGIR (SEQ ID NO: 128) NEZ 3 MADVAGSTHAHASKDGNLVTVLAIDGGGIR (SEQ ID NO: 129) Primers used to create the mutant sequences NEZ 1, NEZ2 and NEZ3: The rear primer (SEQ ID NO: 130): GCCACATCAGCCATGGCCTTGTCGTCGTCG forward mutation primer for mutant NEZ1 (SEQID NO: 131): GACAAGGCCatggctgatgtggcaggctccacaggtgcgggagcttctaaag atggaaac forward mutation primer for mutant NEZ2 (SEQID NO: 132): GACAAGGCCatggctgatgtggcaggctccacaggtgcgcatgcttctaaag atggaaac forward mutation primer for NEZ3 (SEQID NO: 133): GACAAGGCCatggctgatgtggcaggctccacacac gcgcatgcttctaaagatggaaac The following sequences represent the 5 'end of the modified pentin-1 expression sequence as it exists in the bacterial host cell indicates the start of the modified pentin-1 coding sequence (region of coding in lowercase letters): CGA CGACGACAAGGCCatggctgatgtggc (SEQID NO: 134). Expression and Digestion of Mutants After the mutations were confirmed by DNA sequencing, the mutant genes were placed in pET30 vectors and expressed, and the corresponding mutant proteins were purified. The NEZ3 mutant protein was subsequently subjected to proteolytic digestion using the chymotrypsin, trypsin and papain enzymes and using the protocol described in the above. This mutant protein was not digested by any of the enzymes used. Insect Bioassay Modified pentin-1 protein and modified pentin-1 mutants, NEZ1 and NEZ3, were used in WCRW insect bioassays essentially as described in Example 1. More specifically, 3 neonatal larvae were placed in each cavity (20). cavities per sample), each sample contained protein at a concentration of 1 mg / ml, the volume of the test sample applied topically in each cavity was 50 μl, and the larval mortality was recorded after 5 days after the infestation. The results shown immediately in Table 17 for a first experiment indicate that the pentin-1 mutant called NEZ3 inhibits the growth of the WCRW larvae more than the modified pentin-1 'protein (Mod P-19 .The results shown below in the Table 18 for a second experiment indicate that modified pentin-1 mutants in NEZ1 and NEZ3 inhibit the growth of WCRW larvae more than modified pentin-1 (Mod Pl) Table 17: Modified Pentin-1 WCRW bioassay ( Mod Pl) and its Mutant NEZ3 Sample Mortality (%) Comment Replica 1: NEZ3 29/59 = 49% Moderate-severe atrophy Mod Pl 26/60 = 43% Moderate atrophy Replica 2: NEZ3 34/54 = 62% Moderate atrophy- severe Mod Pl 33/51 = 65% Moderate atrophy Table 18: WCRW Bioassay of Modified Pentin-1 (Mod P-1) and its NEZl and NEZ3 Mutants
Sample Concentration Average Larval Weight (μg) Mod P-l 1 μg / μl 154 Mod P-l 0.67 μg / μl 115 Mod P-l 0.33 μg / μl 137
NEZl 1 μg / μl 109 NEZl 0.67 μg / μl 116 NEZl 0.33 μg / μl 121
NEZ3 1 μg / μl 130 NEZ3 0. 67 μg / μl 122 NEZ3 0. 33 μg / μl 110 Solution ÍS ¡395 regulator Diet 16) 347 Example 22: Creation of Transgenic Maize Plants and
SDS-PAGE Analysis of Proteolytic Segmentation of Toxin K04 Cry8Bbl in Maize Transgenic maize plants expressing the mutant K04 of the Cry8Bbl toxin were produced. Briefly, an expression cassette comprising the nucleotide sequence encoding the K04 toxin CrydBbl (SEQ ID NO: 21) operably linked to a promoter that induces expression to a plant was transferred to a vector suitable for transformation of maize mediated by Agrobacterium um. Transgenic maize plants expressing K04 CrydBbl toxin were generated as described in Example 11. Transgenic plants were tested for resistance to WCRW using standard bioassays. Such assays include, for example, the root or whole plant excision test. See, for example, U.S. Patent Publication No. 2003/0120054 and International Publication No. WO 03/016810. Unexpectedly, the transgenic maize plants expressing the K04 toxin of CrydBbl were not resistant to WCRW. Additional biochemical analysis was carried out to determine if K04 CrydBbl toxin was being degraded and inactivated by a plant protease. Briefly, extracts of the roots and leaves of transgenic corn plants were subjected to SDS-PAGE and Western analysis to identify potential proteolytic fragments of the K04 CrydBbl protein. Western analysis revealed that the K04 CrydBbl protein remained intact in the leaves of the transgenic maize plants. In contrast, the K04 CrydBbl toxin was segmented into the root tissue by the root proteases in at least two major fragments
(data not revealed) . As described below, additional analysis of K04 CrydBbl fragments was performed to identify specific proteolytic cleavage sites. Example 23: Creation of Transgenic Maize Plants and Analysis of Proteolytic Segmentation of Cry? Bbl Toxin Truncated in Maize Transgenic maize plants expressing a truncated CrydBbl protein were produced. Briefly, an expression cassette comprising the nucleotide sequence encoding the truncated CrydBbl toxin (SEQ ID NO: 5) operably linked to a promoter driving expression in a plant was transferred to a vector suitable for transformation mediated by Agrobacterium. Transgenic maize plants expressing the truncated CrydBbl toxin were generated as described in Example 11. Root extracts of a transgenic maize plant expressing the truncated CrydBbl toxin were subjected to SDS-PAGE and Western analysis to determine if the pesticide protein was segmented by the root proteases. Western analysis revealed that the truncated CrydBbl was proteolytically digested in the corn root tissue, resulting in two major CrydBbl protein fragments (data not shown). Identification of Proteolytic Sites Immuno-affinity purification techniques were used to isolate the protein fragments and to identify the site of specific proteolytic cleavage in the truncated Cry8Bbl polypeptide. A CrydBbl affinity column was produced using an AminoLink® kit from Pierce Biotechnology and the CrydBbl antibodies and was used to isolate the truncated CrydBbl fragments. Briefly, to harvest the protein fragments, the roots of corn plants expressing truncated CrydBbl protein were instantly frozen in liquid nitrogen and subsequently ground to a powder. The proteins were extracted from the powder in buffer solution PBS, pH 7.4, which contains protease inhibitors and the resulting supernatant was passed over the CrydBbl affinity column. After several washes, truncated CrydBbl fragments were eluted from the column with low pH buffer and subjected to SDS-PAGE analysis. The SDS-PAGE analysis indicated the presence of two major proteolytic fragments of the truncated CrydBbl protein. The protein band obtained after the electrophoresis was stained on a PVDF membrane for N-terminal peptide sequencing and matrix assisted laser disordered ionization (MALDI) analysis. Results: N-terminal sequencing revealed the same N-terminal for each CrydBbl fragment. Specifically, the N-terminus started with DVRNRFEID (SEQ ID NO: 139), indicating that the truncated CrydBbl protein was processed at the end of the spiral located between helix 3 and 4 in domain 1. In addition, the smaller fragment was shown which is processed in the C-terminal. After a determination that both fragments were not glycosylated in the first plant, the fragments were sent for MALDI analysis. The largest fragment had a mass of 56,330 Kda, while the smaller fragment had a mass of 54,193 kDa. A comparison of these molecular weights with the truncated CrydBbl toxin sequence revealed that the C-terminal cleavage site of the protein was in the last loop of domain 3, which consists of the PNSTLS residues (SEQ ID NO: 140 ). Leucine in this spiral is a putative segmentation site for corn root proteases. Example 24: Identification of Proteolytic Segmentation Sites in Cry B. thuringiensis Toxins Using Root Extracts The proteolytic sites in Bacillus thuringiensis toxins were identified by incubating the root extract with a purified Cry toxin expressed in E. coli. The samples were subjected to SDS-PAGE analysis, and the resulting fragments were stained on a PVDF membrane for N-terminal peptide sequencing. Preparation of the root extract: Complete stage V3 corn plants. to V4 (leaves of 3-4 collars) were collected and the roots were rinsed with water to remove the dirty residues. The plants were then frozen at -dO ° C. To prepare the root tissue for enzyme analysis, approximately 500 mg of primary and secondary root tissue was removed from the plant and transferred to a clean eppendorf tube. The root tissue was homogenized with a disposable plastic latch until the tissue was turned into a paste / fiber tissue homogenate. 500 μl of PBS buffer was then added to the homogenate and the sample mixed briefly. The homogenate was centrifuged at 14,000 rpm, and the resulting supernatant was transferred to a new eppendorf tube for use as a crude root extract. Root Extract Protease Assay The protease assay was performed by incubating 50 μl of the purified Cry toxin from Bacillus thuringiensis (at a concentration of 2 μg / 1) with 10 μl of the root extract prepared in an eppendorf tube at room temperature . The mixtures were incubated at time intervals of 2 hours and 2.5 days. After incubations, 25 μl of each sample was removed and frozen at -20 ° C for subsequent SDS-PAGE and Western blot analysis. Protein Analysis Approximately 10 μg of the digested protein sample described above was loaded per cavity of a polyacrylamide gel for SDS-PAGE analysis. Protein bands obtained after electrophoresis were electro-stained on a PVDF membrane. The PVDF membrane was stained with Coomassie Brillant R250 in 50% methanol and 10% acetic acid for 30 minutes, stained with 50% methanol and 10% acetic acid three times, and then rinsed twice with water. The PVDF membrane was dried with air and the immobilized protein bands were cut for N-terminal peptide sequencing. Results: Mutant K04 CrydBbl: The proteolytic sites in the K04 mutant protein CrydBbl (SEQ ID NO: 22) were analyzed as described above. The largest segmentation site at K04 was determined to be in the glycine residue of the amino acid sequence FRRGFRRG (SEO ID NO: 141) positioned between helix 3 and 4 in domain 1. In addition, further degradation of the protein in the arginine residues located after this site was observed, suggesting that cleavage at the FRRGFRRG site (SEQ ID NO: 141) may expose K04 CrydBbl to additional protease attacks and render it inactive. Mutant KO CrydBbl: The proteolytic sites in the mutant protein KO CrydBbl (SEO ID NO: 98) were analyzed as described above. The C-terminal segmentation site in KO CrydBbl was determined to be in L652. Example 25: Mutation of Protein K04 Cry8Bbl and Analysis of Transgenic Plants Expressing Protein K04 Mutated Cry8Bbl The nucleic acid molecule encoding the K04 protein CrydBbl (SEO ID NO: 21) is mutated to introduce a proteolytic protection site using techniques of molecular biology standards. Specifically, the sequence FRRGFRRGH (SEQ ID NO: 142) in the spiral region between helix 3 and 4 in domain 1 of the K04 protein CrydBbl is replaced by the sequence of proteolytic protection site NGSRNGSR (SEQ ID NO: 143) . An expression cassette comprising the nucleotide sequence K04 mutated CrydBbl operably linked to a promoter driving expression in a plant is transferred to a vector suitable for transformation mediated by Agrobacterium um of corn. Transgenic maize plants expressing the mutated CrydBbl K04 protein is generated as described in Example 11. Transgenic maize plants expressing the mutated CrydBbl K04 protein containing the sequence NGSRNGSR (SEQ ID NO: 143) are analyzed for resistance of insects and for the potential proteolytic degradation of the toxin. Specifically, transgenic plants expressing the mutated Kry4 CrydBbl toxin are stimulated with WCRW and analyzed for resistance to this insect pest using standard bioassays as described in Example 1. Transgenic plants expressing the mutated CrydBbl K04 protein are also analyzed for the potential proteolytic degradation of K04 CrydBbl toxin containing the proteolytic protection site NGSRNGSR (SEQ ID NO: 143). Briefly, as described in Example 22, extracts of the roots and leaves of transgenic plants are subjected to SDS-PAGE and Western analysis to identify potential proteolytic fragments of the mutated CrydBbl K04 protein. Any of the identified proteolytic fragments are analyzed by immuno-affinity purification techniques to identify specific cleavage sites, as summarized in Example 23. All publications, patents and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications, patents and patent applications are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the embodiments.
Claims (6)
- CLAIMS 1. A method for protecting a pesticidal polypeptide from proteolytic inactivation in a plant, the method characterized in that it comprises altering at least one proteolytic site within the pesticidal polypeptide that is sensitive to a plant protease to comprise a proteolytic protection site, wherein the proteolytic protection site is not sensitive to the plant protease and protects the pesticidal polypeptide from proteolytic inactivation in a plant.
- 2. The method of compliance with the claim 1, characterized in that the pesticidal polypeptide is a Bacillus thuringiensis toxin or a variant or fragment thereof, wherein the variant and the fragment have pesticidal activity and the variant has at least 70% sequence identity to an amino acid sequence for the Bacillus thuringiensis toxin.
- 3 . The method in accordance with the claim 2, characterized in that the Bacillus thuringiensis toxin is a CrydBbl toxin or a variant or fragment thereof, wherein the variant and the fragment have pesticidal activity and the variant has at least 70% sequence identity to an amino acid sequence for the CrydBbl toxin.
- 4. A method for protecting a plant from a pest, the method characterized in that it comprises introducing into the plant at least one polynucleotide construct comprising a nucleotide sequence encoding a pesticide polypeptide operably linked to a promoter * that induces expression in the plant, wherein the pesticidal polypeptide comprises at least one designed proteolytic protection site, wherein the proteolytic protection site is not sensitive to a plant protease and protects the pesticidal polypeptide from proteolytic inactivation of the plant, wherein the Expression of the polynucleotide construct produces pesticide polypeptide in the plant, and wherein the pesticide polypeptide protects the plant from the plague. The method according to claim 4, characterized in that the pesticidal polypeptide is a Bacillus thuringiensis toxin or a variant or fragment thereof, wherein the variant and the fragment have pesticidal activity and the variant has at least 70% sequence identity to an amino acid sequence for the Bacillus thuringiensis toxin. The method according to claim 5, characterized in that the Bacxllus thuringiensis toxin is a CrydBbl toxin or a variant or fragment thereof, wherein the variant and the fragment have pesticidal activity and the variant has at least 70% sequence identity to an amino acid sequence for the CrydBbl toxin. 7. The method according to claim 4, characterized in that the plant protease is a cysteine protease. 6. The method of compliance with the claim 4, characterized in that the plague is selected from the group consisting of Colorado potato beetle, western root corn worm, southern root corn worm and cotton weevil. 9. An isolated nucleic acid molecule, characterized in that it comprises a nucleotide sequence encoding a pesticidal polypeptide comprising at least one designed proteolytic protection site, wherein the proteolytic protection site is not sensitive to a plant protease and protects to the pesticidal polypeptide of proteolytic inactivation in a plant. 10. The nucleic acid molecule according to claim 9, characterized in that the pesticidal polypeptide is a CrydBbl toxin or a variant or fragment thereof, wherein the variant and the fragment have pesticidal activity and the variant has at least 70 % sequence identity to an amino acid sequence for the CrydBbl toxin. The nucleic acid molecule according to claim 10, characterized in that the at least one designed proteolytic protection site comprises the amino acid sequence NGSRNGSR (SEO ID NO: 143) and replaces a proteolytic site comprising the amino acid sequence FRRGFRRGH (SEO ID NO: 142). 12. An expression cassette, characterized in that it comprises a nucleotide sequence encoding a pesticidal polypeptide operably linked to a promoter that induces expression in a plant, wherein the pesticidal polypeptide comprises at least one designed proteolytic protection site, wherein the proteolytic protection site is not sensitive to a plant protease and protects the pesticide polypeptide from proteolytic inactivation in the plant. A transformed plant, characterized in that it comprises in its genome at least one stably incorporated polynucleotide construct comprising a nucleotide sequence encoding a pesticide polypeptide operably linked to a promoter that induces expression in a plant, wherein the polypeptide The pesticide comprises at least one designed proteolytic protection site, wherein the proteolytic protection site is not sensitive to a plant protease and protects the pesticidal polypeptide from proteolytic inactivation in the plant. The plant according to claim 13, characterized in that the plant is a monocot. 15. The plant according to claim 13, characterized in that the plant is a dicot. The plant according to claim 13, characterized in that the pesticidal polypeptide is a CrydBbl toxin or a variant or fragment thereof, wherein the variant and the fragment have pesticidal activity and the variant has at least 70% identity of sequence to an amino acid sequence for the CrydBbl toxin. 17. A transformed seed of the plant of claim 13. 16. An isolated pesticidal polypeptide, characterized in that it comprises at least one designed proteolytic protection site, wherein the proteolytic protection site is not sensitive to a plant protease and protects to the pesticidal polypeptide of proteolytic inactivation in a plant. 19. The pesticidal polypeptide according to claim 16, characterized in that the at least one designed proteolytic protection site comprises the amino acid sequence NGSRNGSR (SEQ ID NO: 143) and replaces a proteolytic site comprising the amino acid sequence FRRGFRRGH ( SEQ ID NO: 142). 20. An isolated nucleic acid molecule, characterized in that it comprises a nucleotide sequence encoding a polypeptide having proteolytic activity, wherein the nucleotide sequence is selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 135 or 137; (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 136 or 138; (c) a nucleotide sequence having at least about 70% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 135 or 137; (d) a nucleotide sequence having at least about 75% sequence identity to the amino acid sequence set forth in SEQ ID NO: 135 or 137; (e) a nucleotide sequence having at least about 60% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 135 or 137; (f) a nucleotide sequence having at least about 85% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 135 or 137; (g) a nucleotide sequence having at least about 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 135 or 137; (h) a nucleotide sequence having at least about 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 135 or 137; (i) a nucleotide sequence comprising at least about 25 continuous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 135 or 137; (j) a nucleotide sequence that hybridizes to the complement of the nucleotide sequence set out in the SEQ ID NO: 135 or 137 under severe conditions; (k) a nucleotide sequence encoding a polypeptide having at least about 70% sequence identity to the exposed polypeptide SEQ ID NO: 136 or 138; and (1) a nucleotide sequence complementary to at least one nucleotide sequence set forth in (a) to (j). 21. An isolated polypeptide having proteolytic activity, characterized in that the polypeptide has an amino acid sequence selected from the group consisting of: (a) an amino acid sequence set forth in SEQ ID NO: 136 or 138; (b) an amino acid sequence having at least about 60% sequence identity to the amino acid sequence set forth in SEQ ID NO: 136 or 138; (c) an amino acid sequence having at least about 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 136 or 138; (d) an amino acid sequence having at least about 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 136 or 138; (e) an amino acid sequence having at least about 85% sequence identity with the amino acid sequence set forth in SEQ ID NO: 136 or 138; (f) an amino acid sequence having at least about 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 136 or 138; (g) an amino acid sequence having at least about 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 136 or 138; (h) an amino acid sequence comprising at least about 25 contiguous amino acids of the amino acid sequence set forth in SEQ ID NO: 136 or 138; and (i) an amino acid sequence encoded by a nucleotide sequence according to claim 20.
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