WO2001036468A2 - Novel proteins having insecticidal activities and method of use - Google Patents

Abstract

The invention relates to isolated nucleotide and amino acid sequences of lipid acyl hydrolases and methods for their use in controlling insect infestation and pathogenic infection in transgenic plants.

Description

NOVEL PROTEINS HAVING INSECTICIDAL ACTIVITIES AND

METHOD OF USE

FIELD OF THE INVENTION The invention relates to isolated nucleotide and amino acid sequences of lipid acyl hydrolases and methods for their use in controlling insect infestation and pathogenic infection in transgenic plants.

BACKGROUND OF THE INVENTION Numerous insects and non-insect pests (e.g., pathogens) pose a serious threat to common agricultural crops such as corn, soybeans, peas, cotton, and similar food and fiber crops. The conventional method of insect control has been through the application of synthetic chemical compounds. However, the widespread use of chemical pesticides poses many problems attributable to both the non-selectivity of the compounds and the insect's development of resistance.

Other approaches to pest control that have been tried include the use of biological organisms that are "natural predators" of the species sought to be controlled or the introduction of exogenous genes encoding proteins that possess a pesticidal activity such as Bacillus thuringiensis endotoxin. Exemplary predator organisms include other insects, fungi, and bacteria such as Bacillus thuringiensis. Transgenic plants that are resistant to specific insect pests have been produced using genes encoding Bacillus thuringiensis (Bt) endotoxins, which have been shown to be effective for the control of both lepidopteran and coleopteran insect pests. However, it is recognized that although the introduction and expression of an exogenous Bt endotoxin gene may initially confer insect resistance to a cultivar expressing the gene, a phenotype that is based on the expression of a single gene may eventually be lost due to the insect's development of Bt resistance in response to repeated exposure to the insecticide. Alternatively, large colonies of insect pests have been raised in captivity, sterilized and released into the environment in the hope that mating between the sterilized insects and fecund wild insects will decrease the insect population.

While these approaches have had some success, they entail considerable expense and present several major difficulties. For example, it is difficult both to apply biological organisms to large areas and to cause such living organisms to remain in the treated area or on the treated plant species for an extended time. For example, a predatory insect may migrate away from the treated area and fungi or bacteria could be washed off of a treated plant or removed from the area by rain. Consequently, in practice these methods are of limited utility.

Advances in biotechnology in the last two decades provide new opportunities for pest control through genetic engineering. In particular, advances in plant genetics and mutagenic and recombinogenic technologies combined with the identification of insect growth-factors and naturally occurring plant defensive agents (e.g., endogenous or native proteins) offers the opportunity to create transgenic crop plants capable of producing agents that allow the plants to protect themselves from infestation and/or infection. Consistent with this goal, investigators have recognized the need to isolate and characterize alternative pesticidal proteins, and their corresponding genes. In particular, there is a need to identify naturally occurring (e.g., native) pesticidal proteins. Examples of naturally occurring pesticidal products include alkaloids, terpenes, and numerous native plant proteins such as enzymes, enzyme inhibitors, and lectins. Importantly, although endogenous pesticidal proteins are usually present in plant tissue at low levels, it has been determined that some of these genes can be induced to higher level (e.g., copy number and/or expression level) upon attack by an insect pest or pathogen. Of particular interest are endogenous (e.g., native) proteins that can confer resistance by modulating the plant's normal biomolecular activity. Accordingly, there is a need for the isolation and characterization of additional genes that are capable of conferring pesticidal resistance to plants.

SUMMARY OF THE INVENTION The invention provides nucleic acid and amino acid sequences with similarity to lipid acyl hydrolases (LAH). More specifically the invention provides Pentin-1- like LAH sequences. Lipid acyl hydrolases have serine residues at the active site in a glycine-X amino acid-serine-X amino acid-glycine motif (GXSXG). Lipid acyl hydrolases are enzymes with insecticidal properties, which makes the sequences of the invention useful as a source of sequences that are capable of conferring pesticidal resistance to other organisms into which they are introduced, including for example, transgenic plants. The nucleotide sequences provided herein also provide nucleic acid sources for use in mutagenic and recombinogenic protocols that can be used to produce polypeptides with improved biological activities.

In the context of a transgenic plant, is well known that the expression of high levels of exogenous proteins with pesticidal activity is difficult to achieve but required for maximal biological activity. Thus, it is desirable to produce pesticidal proteins with enhanced potency that serve as effective pesticides at levels that are not detrimental to the transgenic plant, and which are more likely to be efficacious at lower levels of expression. One possibility for producing such improved activity is by the use of a recombinogenic technique such as directed evolution (e.g., DNA shuffling), which can be used to improve both the biological activity and enzymatic properties of an enzyme. For example, parameters of enzymatic activity, including but not limited to K_m, K_cat, substrate specificity, and physical characteristics, can be altered by the modification of active-site catalytic residues or alternatively by a DNA shuffling protocol.

A prerequisite for recombinogenic techniques is a parental nucleic acid sequence comprising a source of nucleotide sequence variability relative to the sequence of a second parental sequence. Accordingly, the pentinl -like sequences described herein provide novel gene nucleotide sequences that are useful in both recombinogenic and mutagenic protocols designed to produce polypeptides with novel pesticidal properties. Polypeptides with improved activities can be selected by means of well-known enzymatic or biological assays. Accordingly, the nucleic acid and amino acid compositions provided herein enable the isolation and production of polypeptides useful for the control of insects and other pests. In one embodiment, DNA sequences encoding the pesticidal proteins can be used to transform plants, bacteria, fungi, yeasts, and other organisms. In an alternative embodiment, the amino acid sequences of the invention provide novel polypeptides having pesticidal activity. Because the compositions of the invention may be used in a variety of systems for controlling insect and non-insect pests, the invention further provides for the use of the disclosed sequences in methods designed to prevent or control insect infestation or pathogenic infection of plants.

BRIEF DESCRIPTION OF THE DRAWINGS Figures IA and IB show the nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO:2) of a lipid acyl hydrolase LAH from an Impatiens plant (e.g., Ids.pk0008.fl0).

Figures 2A and 2B show the nucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO :4) of a LAH from a rice plant (e.g., Rrl .pk080.g20).

Figures 3A and 3B show the nucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO. 6) of a first LAH from a soybean plant (e.g., Sdp2c.pk020.p8).

Figures 4 A and 4B show the nucleotide sequence (SEQ ID NO: 7) and deduced amino acid sequence(SEQ ID NO: 8) of a first LAH from a wheat plant (Wlk8.pk0017.d6).

Figure 5A and 5B shows a partial nucleotide sequence (SEQ ID NO:9) and a deduced amino acid sequence(SEQ ID NO: 10) of a second LAH polypeptide from a wheat plant (Wreln.pk0073.c6). Figures βA and 6B show the nucleotide sequence (SEQ ID NO: 11) and the deduced amino acid sequences for three polypeptides (SEQ ID NOS: 12-14) of a second LAH from a soybean plant (Src3c.pk002.m3). SEQ ID NO: 12 presents the amino acid sequence encoded by nucleotides 43 to 714, SEQ ID NO: 13 presents the amino acid sequence encoded by nucleotides 969 to 998, and SEQ ID NO: 14 presents the amino acid sequence encoded by nucleotides 1002 to 1520 of SEQ ID NO: 11.

Figures 7 A and 7B show a partial nucleotide sequence (SEQ ID NO: 15) and a deduced amino acid sequence (SEQ ID NO: 16) for a LAH peptide from Pentaclethra (e.g., 18 A).

Figures 8A, 8B, and 8C show a gapped alignment of the deduced amino acid sequences of the LAH polypeptides of the invention: IdsflOconfinal (SEQ ID NO:2); Rrlg20confinal (SEQ ID NO:4), Sdp2cp8con3 (SEQ ID NO:6), Wlk8nc6confinal (SEQ ID NO:8), Wrelncόconfinal SEQ ID NO:10, Src3cm3conl(SEQ ID NOS:12- 14, displayed as a contiguous sequence which has been assigned SEQ ID NO: 19) and 18acon (SEQ ID NO: 16) compared to the amino acid sequences of 5C9 (SEQ ID NO: 17) and pentin-1 (SEQ ID NO: 18).

Figure 9 shows a gapped alignment of the amino acid sequence of the Pentaclethra polypeptide 18acon (SEQ ID NO: 16) to the amino acid sequences of 5C9 (SEQ ID NO: 17) and pentin-1 (SEQ ID NO: 18).

DETAILED DESCRIPTION OF THE INVENTION Compositions and methods for controlling pests, particularly plant insect pests, are provided. In particular, novel pesticidal proteins are provided. In different embodiments of the invention the proteins, nucleic acid sequences, variants and fragments thereof either possess, or encode polypeptides that possess, pesticidal activity. In a particular embodiment the sequences possess insecticidal activity, however, it is to be understood that these molecules may also contain activity against other non-insect pests disclosed herein. Thus, it is intended that the term "pesticidal", as used herein, encompasses insects and other pests against which the molecules of the present invention are effective. It is also understood that where the term "insect" is used, the embodiment may also be applied to other non-insect pests, for example, those disclosed herein. The lipid acyl hydrolases of the invention are derived from the following plant species: lmpatiens (SEQ ID NOS:l and 2); rice (SEQ ID NOS:3 and 4); soybean (SEQ ID NOS:5, 6, 11,12, 13 and 14); wheat (SEQ ID NOS:7, 8, 9 and 10); and Pentaclethra (SEQ ID NOS: 15 and 16). Accordingly, nucleic acid (RNA or DNA) can be purified from these plants as a source of the lipid acyl hydrolases to which the invention is directed.

Compositions of the invention include nucleotide and amino acid sequences of Pentin-1-like LAHs (e.g., homologs of Pentin-1) and are related to the Pentin-1 sequences disclosed in U.S. Patent No. 6,057,491, the teachings of which are herein incorporated by reference. More specifically, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, 13, 14, 16, or nucleic acid molecules comprising nucleotide sequences inserted into bacterial hosts that were deposited as Patent Deposit Nos. PTA-410, PTA-411, PTA-412, PTA-413, PTA-414, PTA-415, and PTA-644. Further provided are polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein, for example those set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 15, those deposited in a bacterial host as Patent Deposit Nos. PTA-410, PTA-411, PTA-412, PTA-413, PTA-414, PTA-415, PTA- 644, and fragments and variants thereof.

Plasmids containing the inserted nucleotide sequences (the "nucleotide sequence inserts") of the invention were deposited with the Patent Depository of the American Type Culture Collection (ATCC), Manassas, Virginia, and assigned Patent Deposit Nos.: PTA-410 (SEQ ID NO:7); PTA-411 (SEQ ID NO: 1); PTA-412 (SEQ ID NO:3); PTA-413 (SEQ ID NO:l 1); PTA-414 (SEQ ID NO:9); PTA-415 (SEQ ID NO:5), and PTA-644 (SEQ ID NO:15). The plasmids of PTA-410 through PTA-415 were deposited with the ATCC on July 22, 1999, and PTA-644 was deposited on September 10, 1999. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112.

In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences shown in Figures 1 -7, or the nucleic acid molecules comprising the nucleotide sequences inserted into bacterial hosts deposited as Patent Deposit Nos. PTA-410, PTA-411, PTA-412, PTA-413, PTA-414, PTA-415, and PTA-644, as well as fragments and variants thereof. Further provided are polypeptides having an amino acid sequence encoded by a nucleic acid molecule described herein, for example those set forth in Figures 1-7, and fragments and variants thereof.

The invention encompasses isolated or substantially purified nucleic acid or protein compositions. An "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) 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 which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can 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 acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments (e.g., domains) that retain a biological activity of the native protein and hence have lipid acyl hydrolase activity. These fragments are particularly useful as sequences for use in a mutagenic or recombinogenic procedure, for example, a DNA shuffling protocol as noted elsewhere herein. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range 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 lipid acyl hydrolase nucleotide sequence that encodes a biologically active portion of a lipid acyl hydrolase protein of the invention may encode fragments as follows. For Figure 1 , the nucleic acid fragment will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, or up to 406 contiguous amino acids. For Figure 2, the nucleotide sequence fragment will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, or up to 405 contiguous amino acids. For Figure 3, the nucleotide sequence fragment encodes 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, or up to 407 contiguous amino acids. For Figure 4, the nucleotide sequence fragment encodes at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, or up to 410 contiguous amino acids. For Figure 5, the nucleotide sequence encodes at least 15, 25, 30, 50, 100, 150, 200, 250, 275, 280, 285, or up to 290 contiguous amino acids. For Figure 6, the nucleotide sequence encodes at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, or up to 407 contiguous amino acids. For Figure 7, the nucleotide sequence encodes at least 15, 25, 30, 50, 100, 150, 200, 250, 260, 270, or up to 275 contiguous amino acids. Fragments of a lipid acyl hydrolase nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of a lipid acyl hydrolase protein. Thus, a fragment of a lipid acyl hydrolase nucleotide sequence may encode a biologically active portion of a lipid acyl hydrolase protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a lipid acyl hydrolase protein can be prepared by isolating a portion of one of the lipid acyl hydrolase nucleotide sequences of the invention, expressing the encoded portion of the lipid acyl hydrolase protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the lipid acyl hydrolase protein. Nucleic acid molecules that are fragments of a lipid acyl hydrolase nucleotide sequence comprise at least about 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or more nucleotides present in a full-length lipid acyl hydrolase nucleotide sequence as disclosed herein for nucleotides for Figures 1-7. By "variants" is intended substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the lipid acyl hydrolase polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis, which still encode a Pentin-1 homolog of the invention. Generally, variants of a particular nucleotide sequence of the invention will have at least about 40%, 50%, 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 to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters. By "variant" protein is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N- terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, pesticidal activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a Pentin- 1 -like LAH protein of the invention will have at least about 40%, 50%, 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 to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default 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 even 1 amino acid residue.

Thus, the proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the lipid acyl hydrolase proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 52:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:361-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred.

Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired hydrolase activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequences encompassed 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, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by well known assays for lipid acyl hydrolase activity such as those found for enzyme assays for patatin, the major storage proteins of potato. See for example, Galliard (1971) J. Biochem. 121:319-390 and Racusen (1983) Can. J. Bot. (52:1640-1644, herein incorporated by reference. Assays for improved activity will be bioassays in which insects are fed on artificial diets with the lipid acyl hydrolase incorporated.

Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different lipid acyl hydrolase coding sequences can be manipulated to create a new lipid acyl hydrolase possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the lipid acyl hydrolase gene of the invention and other known lipid acyl hydrolases genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased K_m in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 97:10747-10751; Stemmer (1994) Nature 570:389-391 ; Crameri et al. (1997) Nature Biotech. 75:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-341; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 597:288-291 ; and U.S. Patent Nos. 5,605,793 and 5,837,458.

In accordance with the invention, the pesticidal proteins can be isolated by methods known in the art. Methods for protein isolation include conventional chromatography, including gel-filtration, ion-exchange, and immunoaffinity chromatography, by high-performance liquid chromatography, such as reversed-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, size-exclusion high-performance liquid chromatography, high-performance chromatofocusing and hydrophobic interaction chromatography, etc., by electrophoretic separation, such as one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, etc. See, for example, Current Protocols in Molecular Biology, Vols. 1 and 2, ed. Ausubel et al. (John Wiley & Sons, New York, 1988), herein incorporated by reference.

Using a biochemical assay, the lipid acyl hydrolase activity can be measured. See, for example, Andrews et al. (1988) Biochem. J. 252:199-206; and U.S. Patent No. 5,743,477, both of which are herein incorporated by reference. To validate the insecticidal activity of the proteins, the lipid acyl hydrolase may be added on or into the laboratory diet, or applied to the surface of a plant.

Once purified protein is isolated, the protein, or the polypeptides of which it is comprised, can be characterized and sequenced by standard methods known in the art. For example, the purified protein, or the polypeptides of which it is comprised, may be fragmented as with cyanogen bromide, or with proteases such as papain, chymotrypsin, trypsin, lysyl-C endopeptidase, etc. (Oike et al. (1982) J. Biol. Chem. 257:9751-9758; Liu et al. (1983) Int. J. Pept. Protein Res. 21:209-2X5). The resulting peptides are separated, preferably by HPLC, or by resolution of gels and electroblotting onto PVDF membranes, and subjected to amino acid sequencing. To accomplish this task, the peptides are preferably analyzed by automated sequenators. It is recognized that N-terminal, C-terminal, or internal amino acid sequences can be determined. From the amino acid sequence of the purified protein, a nucleotide sequence can be synthesized which can be used as a probe to aid in the isolation of the gene encoding the pesticidal protein. In the same manner, antibodies raised against partially purified or purified peptides can be used to determine the spatial and temporal distribution of the protein of interest. Thus, the tissue where the protein is most abundant, and possibly more highly expressed, can be determined and expression libraries constructed. Methods for antibody production are known in the art. See, for example Antibodies, A Laboratory Manual, ed. Harlow and Lane (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988), and the references cited therein. See also, Radka et al. (1983) J. Immunol. 128:2804, and Radka et al. (1984) Immunogenetics 79:63. Such antibodies can be used to isolate proteins with similar binding domains and the proteins tested for activity against insect pests of interest. It is recognized that any combination of methods may be utilized to purify proteins having pesticidal properties. As an isolation protocol is being determined, the pesticidal activity can be tested for each fraction of material obtained after each purification step.

Such purification protocols will result in a substantially purified protein fraction. By "substantially purified" or "substantially pure" is intended protein which is substantially free of any compound normally associated with the protein in its natural state. "Substantially pure" preparations of protein can be assessed by the absence of other detectable protein bands following SDS-PAGE as determined visually or by densitometry scanning. Alternatively, the absence of other amino-terminal sequences or N-terminal residues in a purified preparation can indicate the level of purity. Purity can be verified by rechromatography of "pure" preparations showing the absence of other peaks by ion exchange, reverse-phase or capillary electrophoresis. The terms "substantially pure" or "substantially purified" are not meant to exclude artificial or synthetic mixtures of the proteins with other compounds. The terms are also not meant to exclude the presence of minor impurities which do not interfere with the biological activity of the protein, and which may be present, for example, due to incomplete purification. From fragments of the protein, the entire nucleotide sequence encoding the protein can be determined by PCR experiments. Likewise, fragments obtained from PCR experiments can be used to isolate cDNA sequences from expression libraries. See, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2d ed., Vols. 1-3, (Cold Spring Harbor Laboratory Press, Plainview, New York), and the references cited therein.

The invention is drawn to compositions and methods for inducing resistance in a plant to plant pests. Accordingly, the compositions and methods are also useful in protecting plants against fungal pathogens, viruses, nematodes, insects, and the like. By "disease resistance" is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen is minimized or lessened. The methods of the invention can be utilized to protect plants from disease, particularly those diseases that are caused by plant pathogens.

By "antipathogenic compositions" is intended that the compositions of the invention have antipathogenic activity and thus are capable of suppressing, controlling, and/or killing the invading pathogenic organism. An antipathogenic composition of the invention will reduce the disease symptoms resulting from pathogen challenge by at least about 5% to about 50%, at least about 10% to about 60%, at least about 30% to about 70%, at least about 40% to about 80%, or at least about 50%) to about 90% or greater. Hence, the methods of the invention can be utilized to protect plants from disease, particularly those diseases that are caused by plant pathogens. Assays that measure antipathogenic activity are commonly known in the art, as are methods to quantitate disease resistance in plants following pathogen infection. See, for example, U.S. Patent No. 5,614,395, herein incorporated by reference. Such techniques include, measuring over time, the average lesion diameter, the pathogen biomass, and the overall percentage of decayed plant tissues. For example, a plant either expressing an antipathogenic polypeptide or having an antipathogenic composition applied to its surface shows a decrease in tissue necrosis (i.e., lesion diameter) or a decrease in plant death following pathogen challenge when compared to a control plant that was not exposed to the antipathogenic composition. Alternatively, antipathogenic activity can be measured by a decrease in pathogen biomass. For example, a plant expressing an antipathogenic polypeptide or exposed to an antipathogenic composition is challenged with a pathogen of interest. Over time, tissue samples from the pathogen-inoculated tissues are obtained and RNA is extracted. The percent of a specific pathogen RNA transcript relative to the level of a plant specific transcript allows the level of pathogen biomass to be determined. See, for example, Thomma et al. (1998) Plant Biology 95:15107-15111, herein incorporated by reference. Furthermore, in vitro antipathogenic assays include, for example, the addition of varying concentrations of the antipathogenic composition to paper disks and placing the disks on agar containing a suspension of the pathogen of interest. Following incubation, clear inhibition zones develop around the discs that contain an effective concentration of the antipathogenic polypeptide (Liu et al. (1994) Plant Biology 91 : X 888-1892, herein incorporated by reference). Additionally, micro spectrophotometrical analysis can be used to measure the in vitro antipathogenic properties of a composition (Hu et al. (1997) Plant Mol. Biol. 5 :949-959 and Cammue et al. (1992) J. Biol. Chem. 267: 2228-2233, both of which are herein incorporated by reference). Pathogens of the invention include, but are not limited to, viruses or viroids, bacteria, insects, nematodes, fungi, and the like. Viruses include any plant virus, for example, tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc. Specific fungal and viral pathogens for the major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum,

Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli,

Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibater michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium, Xanthomonas campestris p.v. alfalfae, Aphanomyces euteiches,

Stemphylium herbarum, Stemphylium alfalfae; Wheat: Pseudomonas syringae p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v. translucens, Pseudomonas syringae p.v. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia recondita f.sp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, Soil Borne Wheat Mosaic Virus, Wheat Streak Mosaic Virus, Wheat Spindle Streak Virus, American Wheat Striate Virus, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, High Plains Virus, European wheat striate virus; Sunflower:

Plasmophora halstedii, Sclerotinia sclerotiorum, Aster Yellows, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium,

Phytophthora cryptogea, Albugo tragopogonis; Corn: Fusarium moniliforme var. subglutinans, Erwinia stewartii, Fusarium moniliforme, Gibberella zeae (Fusarium graminearum), Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. nebraskense, Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv. zea, Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis , Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis,

Cephalosporium acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus, Maize Streak Virus, Maize Stripe Virus, Maize Rough Dwarf Virus; Sorghum: Exserohilum turcicum, Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum

(Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum, Pythium arrhenomanes, Pythium graminicola, etc.

Nematodes include parasitic nematodes such as root-knot, cyst, and lesion nematodes, including Heterodera and Globodera spp; particularly Globodera rostochiensis and globodera pailida (potato cyst nematodes); Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); and Heterodera avenae (cereal cyst nematode).

Thus, nucleotide sequences encoding the proteins can be isolated which are inhibitory or toxic to particular insect species. Such proteins and nucleotide sequences of the invention can be utilized to protect plants from pests, including insects, fungi, bacteria, nematodes, viruses or viroids, and the like, particularly insect pests.

As used herein the term "insect pests" include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera. In particular, proteins and nucleotide sequences which are inhibitory or toxic to insects of the order Coleoptera can be obtained. Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern com borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western com rootworm; Diabrotica barberi, northern com rootworm; Diabrotica undecimpunctata howardi, spotted cucumber beetle, Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popilliajaponica, Japanese beetle; Chaetocnema pulicaria, com flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, com leaf aphid; Anuraphis maidiradicis, com root aphid; Blissus leucopterus, chinch bug; Melanoplus femur rubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Delia platura, seedcom maggot; Agromyza parvicornis, corn blotch leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, twospotted spider mite; Busseola fusca, African Maize Stem Borer (AMB); Sesamia calamistis, African Pink Borer (APB); Eldana sacchharina, African Sugarcane Borer (ASB); Chilopartellus, Sorghum Stem Borer (SSB);

Ostrinia furnacalis, Oriental Com Borer (OCB); Sesamia nonagrioides, Corn borer in Europe/N. Africa; Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, com earworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; com leaf aphid; Siphaβava, yellow sugarcane aphid; Blissus leucopterus, chinch bug; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted spider mite; Schizaphis graminum, Greenbug (aphid); Wheat: Pseudaletia unipunctata, army worm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, plae western cutworm; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabrotica undecimpunctata howardi, spotted cucumber beetle; Russian wheat aphid; Schizaphis graminum, greenbug; Sitobion avenae, English grain aphid; Melanoplus femur rubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Eriophyes tulipae, wheat curl mite; Sunflower: Suleima helianthana, sunflower bud moth; Homeosoma ellectellum, sunflower head moth; Zygoramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cochylis hospes, banded sunflower moth; Rachiplusia nu, agentina looper; Smicronyxfulvus, red sunflower seed weevil; Cylindrocopturus adspersus, spotted sunflower stem weevil; Cotton: Heliothis virescens, tobacco budworm; Helicoverpa zea, bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femur rubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper; Blissus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European com borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, cotton boll worm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoascafabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femur rubrum, redlegged grasshopper; Melanoplus differ entialis, differential grasshopper; Delia platura, seedcom maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, twospotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; Blissus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Delia platura, seedcom maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbage aphid; Flea beetle, Phyllotreta spp. ; Bertha Armyworm; Mamestra configurata; Diamondback Moth; Plutella xylostella; Alfalfa: alfalfa looper, Autographa californica; alfalfa snout beetle, Otiorhynchus ligusticii; alfalfa caterpillar, Colias eurytheme; alfalfa blotch leafrunner, Agronyza frontella; Egyptian alfalfa weevil, Hypera brunneipeonis; meadow spittlebug, Philaerius spumarius; spotted alfalfa aphid, Theriophis meculata; clover leaf weevil, Hypera punctata; pea aphid, Acyrthosiphon pisum; blue alfalfa aphid, Acyrthosiphor kondoi; green cloverworm, Plathypena scabia; clover root curculio, Sitona hispidulus; alfalfa seed chalcid, Brachophagus roddi; tamished plantbug, Lygus lineolaris; Say stink bug, Chlorochroa sayi; velvetbean caterpillar, Anticarsia friegiper da, alfalfa weevil, Hypera postica; fall armyworm, Spodoptera; potato leafhopper, Empoascafabae; soybean looper, Psuedolusia includens; Three cornered alfalfa hopper, Spissistilus festinus; See, for example, Manya (1989) Common Names of Insects & Related Organisms, (Entomological Society of America), herein incorporated by reference. The nucleotide sequences of the invention can be used to isolate other homologous sequences in other plant species, particularly the species from which the disclosed lipid acyl hydrolases are derived. 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. Sequences isolated based on their sequence identity to the entire lipid acyl hydrolase sequences set forth herein or to fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. By "orthologs" is intended genes derived from a common ancestral gene and which are 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. Functions of orthologs are often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant or other 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 (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). See also Innis et al, eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); 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 paired primers, nested primers, single 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 (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as ³²P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the lipid acyl hydrolase sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

For example, an entire Pentin-1 -like LAH sequence disclosed herein, or one or more portions (e.g., fragments) thereof, may be used either as a probe capable of specifically hybridizing to corresponding LAH sequences and messenger RNAs or as a nucleic acid molecule that can be used in a DNA shuffling protocol. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among lipid acyl hydrolase sequences and are preferably at least about 10 nucleotides in length, and most preferably at least about 20 nucleotides in length. Such probes may be used to amplify corresponding lipid acyl hydrolase sequences from a chosen plant or other organism by PCR. This technique may be used to isolate additional coding sequences from a desired plant or other organism or as a diagnostic assay to determine the presence of coding sequences in a plant or other organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may 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 sulphate) at 37°C, and a wash in 1 X to 2X SSC (20X SSC = 3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1 % SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA- DNA hybrids, the T_m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:261-284: T_m = 81.50C + 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 the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T_m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T_m is reduced by about 1°C for each 1% of mismatching; thus, T_m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T_m can be decreased 10°C. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4°C lower than the thermal melting point (T_m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (T_m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower than the thermal melting point (T_m). Using the equation, hybridization and wash compositions, and desired T_m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T_m of less than 45°C (aqueous solution) or 32°C (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids 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 Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

Thus, isolated nucleic acids that encode a pesticidal protein and which hybridize under stringent conditions to the sequence disclosed herein, or to fragments thereof, are encompassed by the present invention. Such sequences will be at least about 40%) to 50% homologous, about 60%, 65%, or 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous with the disclosed sequence [s]. That is, the sequence identity of sequences may range, sharing at least about 40% to 50%, about 60%), 65%, or 70%, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%>, 99%) or more sequence identity.

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 sequence comparison. A reference sequence may be a subset or 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 herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

(c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California). (d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

(e)(i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%o, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1 °C to about 20°C, depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One 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%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman et al (1970) J. Mol. Biol. 48:443. An indication that two peptide sequences are substantially identical is that one 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 noted above except that residue positions that are not identical may differ by conservative amino acid changes.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:X X- 17; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 45:443-453; the search-for-similarity-method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. £5:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5813-5811.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences 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 Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin, USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:231-244 (1988); Higgins et al. (1989) CABIOS 5:151- 153; Covpet et al. (1988) Nucleic Acids Res. 76:10881-90; Huang et al. (1992) CABIOS 5:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:301 '-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized 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 utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity /similarity values provided herein refer to the value obtained using GAP version 10 using the following parameters: % identity using GAP Weight of 50 and Length Weight of 3 ; % similarity using Gap Weight of 12 and Length Weight of 4, or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater. GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89 : 10915).

The nucleotide sequences of the invention can be used in DNA shuffling protocols. DNA shuffling is a process for recursive recombination and mutation, performed by random fragmentation of a pool of related genes, followed by reassembly of the fragments by primerless PCR. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 97:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 75:436-438; Moore et al. (1997) J Mol. Biol. 272:336-341; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 397:288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458. An advantage of DNA shuffling of a rational design is that shuffling can optimize the function of genes without first determining which gene product is rate limiting. The present invention provides methods for sequenced shuffling utilizing polypeptides of the invention, and compositions resulting therefrom.

Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic that can be selected or screened for. Libraries of recombinant polypeptides are generated from a population of related sequence polypeptides that comprise sequenced regions which have substantial sequence identity and can be homologously recombined in vitro or in vivo. The population of sequenced-recombined polynucleotides comprises a subpopulation of polynucleotides that possess desired or advantageous characteristics and can be selected by a suitable selection or screening method. The characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin confirmation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property. In some embodiments, the selected characteristic will be an increased K_m and/or K_cat over the wild-type protein as provided herein. In other embodiments, a protein or polynucleotide generated from sequenced shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide. The increase in such properties can be at least 110%, 120%, 130%, 140% or at least 150% of the wild-type value. The lipid acyl hydrolases disclosed herein are members of a broader gene family of esterases, and more specifically lipid acyl hydrolases as determined by sequence similarity. Gene shuffling is a method that can improve or alter a biological activity of a given gene product. Gene shuffling, in conjunction with a selection strategy, can be used to improve properties such as substrate specificity, solubility, temperature, and pH optima of a protein or enzyme by directed molecular evolution. In the case of lipid acyl hydrolases, toxicity toward insects as determined by the lethal concentrations is a most relevant parameter.

Gene shuffling can be applied to a single gene which introduces mutations within that gene at a given frequency. Combinations of synergistic mutations can then be selected by subsequent generations of gene shuffling from the primary mutant population. This approach can be applied to the proteins of the invention.

Alternatively, different members of gene families that are already encoded by divergent but related sequences can be used for gene shuffling. These could include but not be limited to Pentin-1, from Pentaclethra, or a gene from maize, for example the gene identified by 5C9 which encodes a cDNA that is about 57% identical to Pentin-1 at the nucleotide level. See U.S. Patents Nos. 5,743,477, 5,824,864, and 5,882,668, 5,981,722 (formerly copending patent application 08/938,975) and 6,057,491 (formerly copending patent application 09/074,912) the teachings of each of which are herein incorporated by reference. Both Pentin-1 and 5C9 proteins are known to have insecticidal activity. Concomitantly, mutations will also be introduced by gene shuffling, further contributing to the genetic diversity. Then synergistic combinations of fusions between the members of the gene family and newly introduced mutations can be selected by directed molecular evolution strategies.

Patatin, the nonspecific lipid acyl hydrolase from potato tubers, inhibits the growth of southern cornworm and western com rootworm when the insects are fed patatin on an artificial diet. Patatin is a lipolytic enzyme that catalyzes the nonspecific hydrolysis of a wide variety of acyl lipids including phospholipids, glycolipids, sulfolipids, and mono- and diacylglycerols. Patatin is a bioactive plant enzyme with a novel mode of action distinct from other classes of insect-control polypeptides.

Lipid acyl hydrolases comprise a diverse multigene family that is conserved across many plant species. The enzymes exhibit hydrolyzing activity for many glyco- and phospholipids. Substrates include monogalactosyldiacylglycerol, acylsterylgucoside, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, as well as many other lipid substrates. Similarly membrane composition of various insects as well as plants can vary from species to species and can be affected by diet or growth conditions. Consequently, the activity of a given lipid acyl hydrolase for a given substrate could affect both specificity and potency. Altered substrate specificity could be one parameter for selection of products of gene shuffling.

Solubility and protein stability could also be selected from shuffled gene products. Insecticidal proteins are active in the harsh environment of the insect gut lumen. Their proteins are digested by proteases, and affected by reducing or oxidizing conditions that vary according to the insect species tested. The solubility and stability of lipid acyl hydrolases both in the transgenic plant and in the insect gut lumen could affect biological activity and could be altered through gene shuffling strategies.

Conditions for the enzyme reaction such as pH and temperature optima may also affect the insecticidal activity of the proteins of the invention. The gut pH of com rootworm is 5.5-6.0. Selection of shuffled gene products for enzymatic activity toward lipid substrates in this pH range is another parameter that could affect toxicity. Thus, it is recognized that the sequences disclosed can be used together in shuffling experiments as well as with other insecticidal proteins, particularly other lipid hydrolases, such as patatin, and the like.

The proteins or other component polypeptides described herein may be used alone or in combination with other proteins or agents to control different insect pests. Other insecticidal proteins include those from Bacillus, including δ-endotoxins and vegetative insecticidal proteins, as well as protease inhibitors (both serine and cysteine types), lectins, α-amylases, peroxidases, cholesterol oxidase, and the like.

In one embodiment, expression of the proteins of the invention in a transgenic plant is accompanied by the expression of one or more Bacillus thuringiensis (Bt) δ- endotoxins. This co-expression of more than one insecticidal principle in the same transgenic plant can be achieved by genetically engineering a plant to contain and express all the genes necessary. Alternatively, a plant, Parent 1, can be genetically engineered for the expression of proteins of the invention. A second plant, Parent 2, can be genetically engineered for the expression of other principles, such as a Bt δ- endotoxin. By crossing Parent 1 with Parent 2, progeny plants can be obtained which express all the genes present in both Parents 1 and 2. The present invention also encompasses nucleotide sequences from organisms other than those from which the lipid acyl hydrolases of the invention were derived, where the proteins cross-react with antibodies raised against the proteins of the invention or where the nucleotide sequences are isolatable by hybridization with the nucleotide sequences of the invention. The proteins isolated or those encoded by such nucleotide sequences can be tested for pesticidal activity. The isolated proteins can be assayed for pesticidal activity by the methods disclosed herein or others well-known in the art.

In another embodiment, the proteins of the invention can be used in combination with seed coatings available in the art. In this manner, transformed seed are coated with applications of available insecticide sprays or powders. Such insecticides are known in the art. See, for example, U.S. Patent Nos. 5,696,144; 5,695,763; 5,420,318; 5,405,612; 4,596,206; 4,356,934; 4,886,541 ; herein incorporated by reference.

Once the nucleotide sequences encoding a pesticidal protein encompassed by the invention have been isolated, they can be manipulated and used to express the protein in a variety of hosts including other organisms, including microorganisms and plants.

The proteins of the invention may be used for protecting agricultural crops and products from pests by introduction via a suitable vector into a microbial host, and said host applied to the environment or plants. Microorganism hosts may be selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

The proteins of the invention can be used in expression cassettes for expression in any host of interest. Such expression cassettes will comprise a transcriptional initiation region operably linked to the gene encoding the pesticidal gene of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes suitable for the particular host organism.

The transcriptional initiation region, the promoter, may be native or analogous or foreign or heterologous to the host. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. By foreign is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. As used herein a chimeric gene comprises a coding sequence operably linked to transcription initiation region which is heterologous to the coding sequence.

While any promoter or promoter element capable of driving expression of a coding sequence can be utilized, of particular interest for expression in plants are root promoters (Bevan et al. (1993) in Gene Conservation and Exploitation. Proceedings of the 20th Stadler Genetics Symposium, ed. Gustafson et al. (Plenum Press, New York), pp. 109-129; Brears et al. (1991) Plant J. 7:235-244; Lorenz et al. (1993) Plant J. 4:545-554; U.S. Patent Nos. 5,459,252; 5,608,149; 5,599,670); pith (U.S. Patent Nos. 5,466,785; 5,451,514; 5,391,725); or other tissue-preferred and constitutive promoters (see, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142); herein incorporated by reference.

Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U. S. Patent No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 373:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 72:619-632 and Christensen et al. (1992) Plant Mol. Biol. 75:675-689); pEMU (L st et al. (1991) Theor. Appl. Genet. 57:581- 588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Application Serial No. 08/409,297), and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

Generally, it will be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen; e.g., PR proteins, SAR proteins, beta-l,3-glucanase, chitinase, etc. See, for example, Redolfi et al. (1983) Netb. J. Plant Pathol. 59:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4: 1 11-116. See also the copending applications entitled "Family of Maize PR-1 Genes and

Promoters", U.S. Application Serial No. 09/257,583 filed 2/25/99, herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the site of pathogen 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. Natl Acad. Sci. USA 53:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J. 70:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA 97:2507-2511 ; Warner et l. (1993) Plant J. 3:191-201 ; Siebertz et al. (1989) Plant Cell 7:961-968; U.S. Patent No. 5,750,386 (nematode-inducible); 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 moniliforme (see, for example, Cordero et al. (1992) Physiol Mol. Plant Path. 47:189-200).

Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter may be used in the constructions of the invention. Such wound-inducible promoters include potato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 25:425-449; Duan et al. (1996) Nature Biotechnology 74:494-498); 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 323:13-16); MPI gene (Corderok et al. (1994) Plant J. 6(2): 141 -150); and the like, herein incorporated by reference. Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical -repressible promoter, where application of the chemical represses gene expression. Chemical- inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 55:10421-10425 and McNellis et al. (1998) Plant J. 14(2):241-251) and tetracycline-inducible and tetracycline- repressible promoters (see, for example, Gatz et al. (1991) Mol Gen. Genet. 227:229- 237, and U.S. Patent Nos. 5,814,618 and 5,789,156), herein incorporated by reference. Tissue-preferred promoters can be utilized to target enhanced lipid acyl hydrolase expression within a particular plant tissue. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al. (1997) Plant Cell Physiol. 38 (7) -.192-803; Hansen et α/. (1997) Mol. Gen Genet. 254(3):331-343; Russell et al. (1997) Transgenic Res. 6(2):X51-X68; 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) -.5X3-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5) -.113-118; Lam (1994) Results Probl. Cell Differ. 20:181- 196; Orozco et al. (1993) Plant Mol Biol. 23(6):X 129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression. Leaf-specific 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. 105:351-61; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):113-118; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (X 993) Plant Mol. Biol. 23^:1129-1 138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean root- specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell

3f 0 :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 mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(l):X X-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-64X, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(l):69-16). They concluded that enhancer and tissue- preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1' gene, fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root- preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29 (4) -.159-112); and rolB promoter (Capana et al. (X 994) Plant Mol. Biol. 25(4):68X-69X . See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,1 10,732; and 5,023,179.

"Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seed-germinating" promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 70:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19Bl (maize 19 kDa zein); and celA (cellulose synthase). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc.

Where low level expression is desired, weak promoters will be used. Generally, by "weak promoter" is intended a promoter that drives expression of a coding sequence at a low level. By low level is intended at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Alternatively, it is recognized that weak promoters also encompasses 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 99/43838 and U.S. Patent No. 6,027050) the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also, copending application entitled "Constitutive Maize Promoters", U.S. Application Serial No. 09/257,584 filed 2/25/99, and herein incorporated by reference. The transcriptional cassette will include in 5 '-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al, (1991) Mol. Gen. Genet. 2(52:141-144; Proudfoot (1991) Cell (54:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 97:151-158; Ballas et l. (1989) Nucleic Acids Res. 77:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 75:9627-9639. The nucleotide sequences encoding the proteins or polypeptides of the invention are particularly useful in the genetic manipulation of plants. In this manner, the genes of the invention are provided in expression cassettes for expression in the plant of interest. The cassette will include 5' and 3' regulatory sequences operably linked to the gene of interest. The cassette may additionally contain at least one additional gene to be cotransformed or linked and transformed into the organism. Alternatively, the gene(s) of interest can be provided on another expression cassette. Where appropriate, the gene(s) may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant preferred codons for improved expression. Methods are available in the art for synthesizing plant preferred genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436, 391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Depending upon where the DNA sequence of interest is to be expressed, it may be desirable to synthesize the sequence with plant preferred codons, or alternatively with chloroplast preferred codons. The plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest. See, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 55:3324- 3328; and Murray et al. (1989) Nucleic Acids Research 17:477-498. In this manner, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the gene sequence may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences which may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence may be modified to avoid predicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5' leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) PNAS USA 5(5:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 754:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 57:382-385). See also, Della-Cioppa et al. (X 987) Plant Physiol. 54:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like. The genes of the present invention can be targeted to the chloroplast or amyloplast for expression. In this manner, where the gene of interest is not directly inserted into the chloroplast or amyloplast, the expression cassette will additionally contain a gene encoding a transit peptide to direct the gene of interest to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; della-Cioppa et al. (1987) Plant Physiol. 54:965-968; Romer et al. ( X 993) Biochem. Biophys. Res Commun. 196: X 4 X 4- X 421 ; and Shah et al. (1986) Science 233:478-481. The construct may also include any other necessary regulators such as nuclear localization signals (Kalderon et al. (1984) Cell 39:499-509; and Lassner et al (1991) Plant Molecular Biology 17:229-234); plant translational consensus sequences (Joshi, C.P. (1987) Nucleic Acids Research 75:6643-6653), introns (Luehrsen and Walbot (1991) Mol. Gen. Genet. 225:81-93) and the like, operably linked to the nucleotide sequence of interest. It is recognized that the protein can be expressed comprising the native signal sequence. Alternatively, other signal sequences in the art, for example the barley α-amylase signal sequence, may be utilized.

In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Towards this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, PCR, or the like may be employed, where insertions, deletions or substitutions, e.g. transitions and transversions, may be involved.

The compositions of the present invention can be used to transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 53:5602-5606, Agrobacterium-mediated transformation (Townsend et al., U.S. Pat No. 5,563,055; Zhao et al, U.S. Patent No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al, U.S. Patent No. 4,945,050; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer- Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:21-31 (onion); Christou et al. (1988) Plant Physiol. 57:671-674 (soybean); 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. 96:3X9-324 (soybean); Datta et al. (1990) Biotechnology 5:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 55:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Patent No. 5,240,855; Buising et al, U.S. Patent Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer- Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 97:440-444 (maize); Fromm et al. (1990) Biotechnology 5:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 377:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 54:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (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. 54:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al (1993) Plant Cell Reports 72:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 74:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference. Where desirable, the plant plastid can be transformed directly. Stable transformation of plastids have been reported in higher plants, see, for example, SVAB et al. (1990) Proc. Natl. Acad. Sci. USA 57:8526-8530; SVAB & Maliga (1993) Proc. Natl. Acad. Sci. USA 90:9X3-9X1; Staub & Maliga (1993) Embo J. 72:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-bome transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 97:7301-7305.

The cells which have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting offspring having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved. The proteins will be expressed in the transformed organisms at levels sufficient to be inhibitory to insect growth or toxic to the insects of interest.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL Example 1 : Effect of Lipid Hydrolases on Diabrotica Larvae

Insect diets for southern corn rootworm and western com rootworm larvae are known in the art. See, for example, Rose and McCabe (1973) J Econ. Entomol. 66:393; and Bio-Serv (Frenchtown, New Jersey) catalog pages 6-7, each of which are herein incorporated by reference. Insect diet is prepared and, as discussed above, poured onto a tray. Generally 1.5 ml of diet is dispensed into each cell with an additional 120 μl of sample preparation applied to the diet surface. Colonies from an original plate expressing the lipid acyl hydrolases of interest are spotted on replica plates and inoculated in 5 ml 2x Yt broth with 500 μl/1000 ml kanamycin antibiotic. The tubes are grown overnight. If no growth is present, the tubes are incubated for an additional 24 hours. Following incubation, the tubes are centrifuged at 3500 rpms for 5-8 minutes. The supernatant is discarded and the pellet resuspended in 1000 μl PBS. 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.

120 μl of the sample is applied topically to the diet (Pittman Trays). For the screening of western com rootworm, 25 μl of a 0.8 egg agar solution is applied to lids. Trays are allowed to dry under a hood. After drying, lids are placed on trays and stored for 3-4 days at a temperature of 26°C. Trays are then scored counting "live" versus "dead" larvae and tabulating the results. The results are expressed as a percentage of mortality. Any result over 75% is considered a positive result.

Example 2: Western Corn Rootworm Bioassays

Western com rootworm bioassays are performed generally as described above. Microbial culture broths (150 μl) are overlaid onto 1.5 ml artificial diets with a 2.6 cm² surface area. Mortality is calculated as percentage dead larvae out of larvae tested.

Example 3 : Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing a lipid acyl hydrolase operably linked to a ubiquitin promoter plus a plasmid containing the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistance to the herbicide Bialaphos. Transformation is performed as follows. For procedures and media preparations, see, WO 98/09995, herein incorporated by reference.

Preparation of Target Tissue

The ears are surface sterilized in 30%> Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5-cm target zone in preparation for bombardment.

Preparation of DNA

A plasmid vector comprising a lipid acyl hydrolase sequence operably linked to a ubiquitin promoter is made. This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl₂ precipitation procedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in TrisEDTA buffer (1 μg total)

100 μl 2.5 M CaCl₂

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.

Particle Gun Treatment

The 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 often aliquots taken from each tube of prepared particles/DNA. Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection- resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone- free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1 -2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored for insecticidal activity.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/1 N6 basal salts (SIGMA C- 1416), 1.0 ml/1 Eriksson's Vitamin Mix (1000X SIGMA-151 1), 0.5 mg/1 thiamine HC1, 120.0 g/1 sucrose, 1.0 mg/1 2,4-D, and 2.88 g/1 L-proline (brought to volume with D-I H₂0 following adjustment to pH 5.8 with KOH); 2.0 g/1 Gelrite (added after bringing to volume with D-I H₂0); and 8.5 mg/1 silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/1 N6 basal salts (SIGMA C-1416), 1.0 ml/1 Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/1 thiamine HC1, 30.0 g/1 sucrose, and 2.0 mg/1 2,4-D (brought to volume with D-I H₂0 following adjustment to pH 5.8 with KOH); 3.0 g/1 Gelrite (added after bringing to volume with D-I H₂0); and 0.85 mg/1 silver nitrate and 3.0 mg/1 bialaphos(both added after sterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/1 MS salts (GIBCO 111 17- 074), 5.0 ml/1 MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/1 thiamine HCL, 0.10 g/1 pyridoxine HCL, and 0.40 g/1 glycine brought to volume with polished D-I H₂0) (Murashige and Skoog (1962) Physiol. Plant. 75:473), 100 mg/1 myo- inositol, 0.5 mg/1 zeatin, 60 g/1 sucrose, and 1.0 ml/1 of 0.1 mM abscisic acid (brought to volume with polished D-I H₂0 after adjusting to pH 5.6); 3.0 g/1 Gelrite (added after bringing to volume with D-I H₂0); and 1.0 mg/1 indoleacetic acid and 3.0 mg/1 bialaphos (added after sterilizing the medium and cooling to 60°C). Hormone-free medium (272V) comprises 4.3 g/1 MS salts (GIBCO 1 1117-074), 5.0 ml/1 MS vitamins stock solution (0.100 g/1 nicotinic acid, 0.02 g/1 thiamine HCL, 0.10 g/1 pyridoxine HCL, and 0.40 g/1 glycine brought to volume with polished D-I H 0), 0.1 g/1 myo-inositol, and 40.0 g/1 sucrose (brought to volume with polished D-I H₂0 after adjusting pH to 5.6); and 6 g/1 bacto-agar (added after bringing to volume with polished D-I H 0), sterilized and cooled to 60° C.

Example 4: Agrobacterium-mediated Transformation

For Agrobαcterium-mediated transformation of maize with a nucleotide sequence(s) of the invention, preferably the method of Zhao is employed (U.S. Patent No. 5,981,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the nucleotide sequence(s) of interest to at least one cell of at least one of the immature embryos (step 1 : the infection step). In this step the immature embryos are preferably immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the

Agrobacterium (step 2: the co-cultivation step). Preferably the immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional "resting" step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). Preferably the immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). Preferably, the immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and preferably calli grown on selective medium are cultured on solid medium to regenerate the plants.

All publications 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 and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 15; b) a nucleotide sequence consisting of the nucleotide sequence insert deposited with the ATCC in a bacterial host assigned Patent Deposit No. PTA-410, PTA-411, PTA-412, PTA-413, PTA-414, PTA-415, or PTA-644; c) a nucleotide sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, and 16; d) a nucleotide sequence that encodes a polypeptide having pesticidal activity and which differs from a sequence of a) or b) due to the degeneracy of the genetic code; e) a nucleotide sequence that shares at least about 70% sequence identity with a sequence of a), b), c), or d); f) a nucleotide sequence comprising at least about 16 contiguous nucleotides of a sequence of a), b), c), d), or e); g) a nucleotide sequence that hybridizes under stringent conditions to a sequence of a), b), c), d), e), or f).

2. A DNA construct comprising a nucleic acid molecule of claim 1 operably linked to a promoter that is functional in a plant cell.

3. The DNA construct of claim 2, wherein the promoter is a root- preferred promoter.

4. A vector comprising the DNA construct of claim 3.

5. A transformed plant cell comprising the DNA construct of claim 2.

6. A transformed plant comprising the DNA construct of claim 2.

7. A purified polypeptide, the amino acid sequence of which comprises a sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, or 16.

8. The purified polypeptide of claim 7, wherein the polypeptide has pesticidal activity.

9. A purified polypeptide characterized by pesticidal activity the amino sequence of which consists of a sequence having at least about 70% sequence identity to a sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, or 16.

10. A purified polypeptide characterized by pesticidal activity the amino sequence of which consists of a sequence having at least about 80% sequence identity to a sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, or 16.

11. A purified polypeptide characterized by pesticidal activity the amino sequence of which consists of a sequence having at least about 90% sequence identity to a sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, or 16.

12. A purified polypeptide characterized by pesticidal activity the amino sequence of which consists of a sequence having at least about 95% sequence identity to a sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 13, 14, or 16.